tag:blogger.com,1999:blog-3853723884821592492024-03-05T00:13:09.730-05:00Emerald BiologyEmeraldBiologyhttp://www.blogger.com/profile/07468813270664433504noreply@blogger.comBlogger18125tag:blogger.com,1999:blog-385372388482159249.post-27529125434145792082023-01-11T21:28:00.003-05:002023-01-24T14:54:01.692-05:00Honey Bee vaccination: good, ugly or bad?<br /><p><table align="center" cellpadding="0" cellspacing="0" class="tr-caption-container" style="margin-left: auto; margin-right: auto;"><tbody><tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjuKtpYsvmuxS__rTkX_tJdENcoE-svl5SYrAk3ExSyptY59De6AVkLl1IG4MAfsLFPPiexpRdTjH9VhTWjQor9OY17-it7_xf083f1DDoFzv28X7nXDMtL-kdXjR-5K-CrW4uVlMzyzP5-PQH5tHW141C8VWezUSOy9oLb2Z-7fLaBUa-gas4Ab9aX/s3446/Bee%20graphic%20v2.jpg" imageanchor="1" style="margin-left: auto; margin-right: auto;"><img border="0" data-original-height="2187" data-original-width="3446" height="203" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjuKtpYsvmuxS__rTkX_tJdENcoE-svl5SYrAk3ExSyptY59De6AVkLl1IG4MAfsLFPPiexpRdTjH9VhTWjQor9OY17-it7_xf083f1DDoFzv28X7nXDMtL-kdXjR-5K-CrW4uVlMzyzP5-PQH5tHW141C8VWezUSOy9oLb2Z-7fLaBUa-gas4Ab9aX/s320/Bee%20graphic%20v2.jpg" width="320" /></a></td></tr><tr><td class="tr-caption" style="text-align: center;">The less than saintly honey bee <span style="font-size: x-small;">(copyright 2023 Emerald Biology)</span></td><td class="tr-caption" style="text-align: center;"><br /></td></tr></tbody></table> </p><p>For years I've been a distant proponent of the honey bee, thinking it a much beloved native species that was essential to plants and putting the natural healthy sweetner on my table. The delightful glow of going to the farmers market and picking up local honey.<br /></p><p>A few months ago, I read that actually honey bees are not native to the United States but are in fact a European import (<a href="https://www.usgs.gov/faqs/are-honey-bees-native-north-america">1</a>). Not only that but they compete with native species for food, spread disease, and because beehives are moved around, the honey bees get an advantage over native species. This article in the Scientific American (<a href="https://www.scientificamerican.com/article/the-problem-with-honey-bees/">2</a>) explains the complexity of the situation admirably. <br /></p><p>I'm all for the elimination of the use of pesticides and antibiotics; a reduction in the addition of chemicals to the environment can only help all species but the introduction of a vaccine (<a href="https://www.npr.org/2023/01/08/1147737268/a-new-vaccine-will-protect-honeybees-from-a-bacterial-disease-affecting-their-la">3</a>) to deal with foulbrood in honey bees seems to be a step that helps commercial interests much more than keeping the native ecosystem in balance. I feel righteous indignation in my pure minded eco warrior stance. BUT I also read that honey bees are essential for agriculture to achieve the level of production need to feed the population at reasonable cost. I've already seen my grocery bill increase. Can I afford my righteous indignation? What would be the consequences of not using the vaccine and looking at ways to promote native pollinators? Can we do without honey?</p><p>It turns out that there are 4000 species of bees (<a href="https://www.thebiglittlegarden.com/native-bees">4</a>) and there are plants they prefer. npr (<a href="https://www.nwf.org/Our-Work/Wildlife-Conservation/Pollinators">5</a>) suggests a number of ways to support native species. Can agriculture do the same and still be commercially viable?<br /></p><br /><br />EmeraldBiologyhttp://www.blogger.com/profile/07468813270664433504noreply@blogger.com0tag:blogger.com,1999:blog-385372388482159249.post-74216765332165803072022-05-10T15:02:00.005-04:002022-05-10T15:02:53.909-04:00Lower and faster<p>Great news about a enzymes that degrade plastic (of a certain type) faster at lower temperatures: see it <a href="https://www.chemistryworld.com/news/ai-engineered-enzyme-eats-entire-plastic-containers/4015620.article?utm_source=Nature+Briefing&utm_campaign=25384cfc13-briefing-dy-20220510&utm_medium=email&utm_term=0_c9dfd39373-25384cfc13-43540113">here</a><br /></p>EmeraldBiologyhttp://www.blogger.com/profile/07468813270664433504noreply@blogger.com0tag:blogger.com,1999:blog-385372388482159249.post-44306284586557203662019-04-07T07:57:00.000-04:002019-04-07T07:57:30.110-04:00Fuels for Biofuels part 6: the cellulosomeIn the last post (yes, its been a while!) I talked about free cellulases. The problem with free cellulases is recovery and that you need several different types of cellulases to work together to accomplish the breakdown of cellulose. What if you could have a scaffold that did that for you? Bacteria and fungi have done just that and its called the cellulosome! Of course, its a complex structure but the basics are illustrated in Figure 1.<br />
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<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgkXEXRgrlNH5ZAOCQPUN_1jO-_siMpfxnyhIVlqt7SyNhV3X2puj07U8XedaGS3JtlBSRWspzDz4-jAScn6TMHcs8QUbq3PICaDG54Ge3vblhQazf9TJ2Pym1so8bvKt2RaN6yWr15Krc/s1600/Basic+cellulosome.jpg" imageanchor="1" style="margin-left: auto; margin-right: auto;"><img border="0" height="540" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgkXEXRgrlNH5ZAOCQPUN_1jO-_siMpfxnyhIVlqt7SyNhV3X2puj07U8XedaGS3JtlBSRWspzDz4-jAScn6TMHcs8QUbq3PICaDG54Ge3vblhQazf9TJ2Pym1so8bvKt2RaN6yWr15Krc/s1600/Basic+cellulosome.jpg" width="640" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;">Figure 1 (taken from ref 1)</td><td class="tr-caption" style="text-align: center;"><br /></td><td class="tr-caption" style="text-align: center;"><br /></td></tr>
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<br />
The cellulosome consists of several different parts. First a protein known as scaffoldin is attached to the bacterial (or fungal) cell. Scaffolding contains domains known as cohesins and carbohydrate binding proteins (binding to cellulose for example) and a domain with surface layer homology of unknown function (2004); cohesins bind a second type of protein called dockerins and dockerins bind the cellulosome enzymes that degrade carbohydrates, cellulose being just one of those carbohydrates. There are different types of cohesins (type I-III in 2004) and they bind different classes of dockerins (types I-III) through a small binding site of four amino acids. Which dockerin binds to which cohesin is highly specific. Figure 1 shows a fairly simple representation of a cellulosome. Other micro-organisms have more complex structures involving 3 different scaffoldin proteins that link together. <br />
<br />
Interestingly, the presence of cellulosome genes in an organisms genonome does not necessarily mean it can degrade cellulose. Developing an efficient and robust cellulosome containing the enzymes that work effectively together is then the job of the lignocellulose biochemist. To this end, researchers have been working on designing minicellulosomes.<br />
<br />
We already know that Saccharomyces cerevisea is a organism of choice in the fermentation of glucose to produce ethanol due to its tolerance for high ethanol concentration (see blog post#). S. cereviseae is also a good choice because its genetics are well studied with many tools available for gene manipulation. Further, yeast is able to "display" proteins on its surface. i.e. it can display a cellulosome on on its surface meaning that not only will it do the expression but it puts the whole system together and presents it on its surface. The scientist, doesn't need to purify the proteins and put them together herself. Because of the genetic tractability, different cohesins from different organisms can be mixed and matched to produce the most efficient cellulosome, unavailable naturally. Tsai et al, 2009, put together cohesins from 3 different bacterial strains and displayed them on the yeast cell surface (figure 2). The antibodies (upside down green and blue and brown Y shapes with yellow or green stars) were used to detect the expression of the different components ofn teh surface of the yeast. <br />
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<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEj0uN8ZK6ujiXdH7KZ9drTRJDbPZwoZ_BtkmN36bNFq5PH54PE9zt_hkMHdv7IPTvK2UiUSIQYpCMVcn9-pvnkSQAEjgZj-_ogPtxcTkYTBY8RukREbNhre0DNKP9Y5JhWIVlrzuRfmlIM/s1600/minicellulosome+Tsai+et+al+oct2014.png" imageanchor="1" style="margin-left: auto; margin-right: auto;"><img border="0" height="436" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEj0uN8ZK6ujiXdH7KZ9drTRJDbPZwoZ_BtkmN36bNFq5PH54PE9zt_hkMHdv7IPTvK2UiUSIQYpCMVcn9-pvnkSQAEjgZj-_ogPtxcTkYTBY8RukREbNhre0DNKP9Y5JhWIVlrzuRfmlIM/s1600/minicellulosome+Tsai+et+al+oct2014.png" width="640" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;"><h2 style="direction: ltr; line-height: 93%; margin-bottom: 0pt; margin-top: 0pt; text-align: left; unicode-bidi: embed; vertical-align: baseline;">
<span style="font-weight: normal;"><span style="font-family: "times" , "times new roman" , serif;"><span style="font-size: small;"><span style="color: black;">Figure 2. Functional assembly of </span><span style="color: black;">minicellulosomes</span><span style="color: black;"> on
the yeast cell surface. A </span><span style="color: black;">trifunctional</span><span style="color: black;"> </span><span style="color: black;">scaffoldin</span><span style="color: black;"> (</span><span style="color: black;">Scaf-ctf</span><span style="color: black;">)
consisting of an internal CBD flanked by three divergent </span><span style="color: black;">cohesin</span><span style="color: black;"> (C)
domains from C. </span><span style="color: black;">thermocellum</span><span style="color: black;"> (t),
C. </span><span style="color: black;">cellulolyticum</span><span style="color: black;"> (c),
and R. </span><span style="color: black;">flavefaciens</span><span style="color: black;"> (f)
was displayed on the yeast cell surface. Three different </span><span style="color: black;">cellulases</span><span style="color: black;"> (E1,
E2, and E3) fused with the corresponding </span><span style="color: black;">dockerin</span><span style="color: black;"> domain (either </span><span style="color: black;">Dt</span><span style="color: black;">, Dc,
or </span><span style="color: black;">Df</span><span style="color: black;">)
were expressed in E. coli. Cell lysates containing these </span><span style="color: black;">cellulases</span><span style="color: black;"> were
mixed with yeast cells displaying </span><span style="color: black;">Scaf-ctf</span><span style="color: black;"> for the functional assembly of the
</span><span style="color: black;">minicellulosome</span><span style="color: black;">.</span></span></span></span></h2>
<span style="font-family: "times" , "times new roman" , serif;"><span style="font-size: small;">
</span></span></td></tr>
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<span style="font-family: "times" , "times new roman" , serif;"><span style="font-size: small;"> The authors showed that this cellulosome was 2.6x more efficient than if the enzymes were added in their soluble form and ethanol production was 95% of its theoretical value! </span></span><br />
<br />
References:<br />
1. <span style="font-size: small;"><b><span style="font-weight: normal;">Roy H. Doi<sup><a href="http://www.nature.com/nrmicro/journal/v2/n7/abs/nrmicro925.html#a1" title="affiliated with ">1</a></sup>
&
Akihiko Kosugi<b>. 2004 </b>Cellulosomes: plant-cell-wall-degrading enzyme complexes </span></b></span><span class="journalname">Nature Reviews Microbiology</span> <span class="journalnumber">2</span>, <span class="cite-pages">541-551</span> <span class="cite-month-year">(July 2004)</span><br />
<br />EmeraldBiologyhttp://www.blogger.com/profile/07468813270664433504noreply@blogger.com0tag:blogger.com,1999:blog-385372388482159249.post-45105749079877955752016-09-07T14:08:00.000-04:002018-08-03T14:25:54.252-04:00Blog direction change...get ready!This blog is changing direction synthetic biology is taking over. Microbiology and Biochemistry can come together!EmeraldBiologyhttp://www.blogger.com/profile/07468813270664433504noreply@blogger.com1tag:blogger.com,1999:blog-385372388482159249.post-91060225110357818272015-03-05T11:46:00.003-05:002015-03-05T11:46:51.922-05:00Aside - the realty of drug use<br />
<div style="clear: left; float: left; margin-bottom: 1em; margin-right: 1em;">
<a href="https://encrypted.google.com/books/content/images/frontcover/xkyLgbIyk4IC?fife=w300" imageanchor="1" style="clear: right; float: right; margin-bottom: 1em; margin-left: 1em;"><img alt="Cover art" border="0" class="cover-image" height="200" itemprop="image" src="https://encrypted.google.com/books/content/images/frontcover/xkyLgbIyk4IC?fife=w300" width="131" /></a>This is an unrelated topic but one I feel strongly enough to make a post.I recently read this book by Carl Hart and can't recommend it highly enough. </div>
<br />
<div class="parseasinTitle ">
<span id="btAsinTitle">High Price: A Neuroscientist's Journey of Self-Discovery That Challenges Everything You Know About Drugs and Society</span></div>
<br />
<br />
Drug abuse is caused by poor education, prejudice and poverty. Drug abuse does usually not lead to this or to crime. Open your mind and get out of the media and political brain washing that is pervasive in our culture about so many things including drug use, race and gender.<br />
<br />
Here is link to Carl Hart website: <a href="http://www.drcarlhart.com/">link</a>EmeraldBiologyhttp://www.blogger.com/profile/07468813270664433504noreply@blogger.com1tag:blogger.com,1999:blog-385372388482159249.post-58909467984878346532014-03-11T21:47:00.000-04:002014-03-11T21:49:09.694-04:00Fuels for Biofuels part 5: free cellulases and cellulose hydrolysis<span style="font-family: Arial,Helvetica,sans-serif;"><span style="font-size: small;">Following pretreatment and prior to fermentation, the broken down components of lignocellulose (lignin, celluose and hemicellulose) are subjected to enzymatic action. Actually, its economically favorable to combine hydrolysis and fermentation, but lets first look at how cellulose is broken down <u>for</u> fermentation? </span></span><br />
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<span style="font-family: Arial,Helvetica,sans-serif;"><span style="font-size: small;"><b>Cellulose hydrolysis - cellulases.</b></span></span><br />
<span style="font-family: Arial,Helvetica,sans-serif;"><span style="font-size: small;">Pretreatment opens up the lignocelluose but not to its monomeric form and before it can be used as an energy source it needs to be broken down much more. How does this occur? Enzymes! In particular, one enzyme, cellulase.</span></span><br />
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<span style="font-family: Arial,Helvetica,sans-serif;"><span style="font-size: small;">Well, actually one class of enzymes; one very large class, grouped together by their ability to hydrolyze the beta 1,4 glycosidic bond (see my last post). One of the reasons for the huge diversity of cellulases is that their substrate, cellulose, comes in many different forms and cellulases have evolved to suit their purpose. If you want to delve into cellulose structure, see this <a href="http://www.yovisto.com/video/19028">video</a> and this <a href="http://www.nrel.gov/biomass/staff_pages/gregg_beckham.html">link</a>. Micro-organisms produce many types of cellulases and they work synergistically. The subject is complex and we'll skim the surface.</span></span><br />
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<span style="font-family: Arial,Helvetica,sans-serif;"><span style="font-size: small;">Cellulases can be classified in a number of different ways depending on what you are interested in: by structure; by sequence; by enzyme mechanism; by substrate etc. One of the most widely used classification systems is the <a href="http://www.cazy.org/Definitions-and-Terminology.html">Carbohydrate-active enzymes database</a>, or CAZymes which is based on sequence similarity. In CAZymes, enzymes are grouped into enzymatic groups through their sequence similarity and cellulases fall into the glycosidic hydrolase (GH) group. </span></span><br />
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<tr><td style="text-align: center;"><span style="font-family: Arial,Helvetica,sans-serif;"><span style="font-size: small;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgkXUsOlqkYSgXpZnuJmy83o02Iect7J6koRjxKDBD9tZJW63QuxsEkTz1PASVdL18VCdgY9EYsLZDlbkbJL4zaRQtzYdMePLWrP_8NvR9dMe5_Iv2UPKx4nqZSvUMPCNetP_jotVHiXE4/s1600/cellulases.jpg" imageanchor="1" style="clear: left; margin-bottom: 1em; margin-left: auto; margin-right: auto;"><img border="0" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgkXUsOlqkYSgXpZnuJmy83o02Iect7J6koRjxKDBD9tZJW63QuxsEkTz1PASVdL18VCdgY9EYsLZDlbkbJL4zaRQtzYdMePLWrP_8NvR9dMe5_Iv2UPKx4nqZSvUMPCNetP_jotVHiXE4/s1600/cellulases.jpg" height="480" width="640" /></a></span></span></td></tr>
<tr><td class="tr-caption" style="text-align: center;"><span style="font-family: Arial,Helvetica,sans-serif;"><span style="font-size: small;">Figure 1. Examples of endo and excoellulase and endogluconasess (<a href="https://lookfordiagnosis.com/mesh_info.php?term=Cellulases&lang=1">link</a>)</span></span></td></tr>
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<tr><td style="text-align: center;"><span style="font-family: Arial,Helvetica,sans-serif;"><span style="font-size: small;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEiFIfMq4Ah2NSMute2AglJ1mR8X31_MzxH2nePsdo7-t03xTi9NVg7oJ-ssA60EsVqgDYqsCYPZv3Ptc7Ropu-Wujj48T4PDMtkrlwiZ7ISIEAjfzjedhv0Y1VYqAWqh7QCWsNXiKUaGT8/s1600/Cellobiose.jpg" imageanchor="1" style="clear: left; margin-bottom: 1em; margin-left: auto; margin-right: auto;"><img border="0" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEiFIfMq4Ah2NSMute2AglJ1mR8X31_MzxH2nePsdo7-t03xTi9NVg7oJ-ssA60EsVqgDYqsCYPZv3Ptc7Ropu-Wujj48T4PDMtkrlwiZ7ISIEAjfzjedhv0Y1VYqAWqh7QCWsNXiKUaGT8/s1600/Cellobiose.jpg" height="153" width="200" /></a></span></span></td></tr>
<tr><td class="tr-caption" style="text-align: center;"><span style="font-family: Arial,Helvetica,sans-serif;"><span style="font-size: small;">Figure 2. Cellobiose</span></span></td></tr>
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<span style="font-family: Arial,Helvetica,sans-serif;"><span style="font-size: small;">Cellulases can be further classified into four subclasses that are commonly used in lignocellulase breakdown.</span></span><br />
<span style="font-family: Arial,Helvetica,sans-serif;"><span style="font-size: small;">1.Endocellulases bind randomly along a cellulose polymer strand and make several cuts before releasing.</span></span><br />
<span style="font-family: Arial,Helvetica,sans-serif;"><span style="font-size: small;">2.Exocellulases (also known as cellobiohydrolases) bind from one end of the cellulose polymer and further bind at either the reducing end or the non reducing end (see my last post on cellulose structure). The polymer strand gets fed into the exocellulose and a D-glucose dimer (cellubiose - figure 2) is cut off, one at a time as the enzyme moves along. This ability - to move along the polymer - is called processivity. </span></span><br />
<span style="font-family: Arial,Helvetica,sans-serif;"><span style="font-size: small;">3.Endoglucanases bind to the cellulose polymer, make a cut like an endocellulase and then moves processively along the strand, releasing cellotetraose (figure 3) rather than cellobiose. </span></span><br />
<span style="font-family: Arial,Helvetica,sans-serif;"><span style="font-size: small;">4.beta glucosidases cellobiose (and cellotetraose?) into the glucose monomers.</span></span><br />
<span style="font-family: Arial,Helvetica,sans-serif;"><span style="font-size: small;"><br /></span></span>
<span style="font-family: Arial,Helvetica,sans-serif;"><span style="font-size: small;">Additionally there are other proteins such as swollenein that insert themselves inbetween the strands of cellulose in crystalline cellulose and help to break it apart. </span></span><br />
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<tr><td style="text-align: center;"><span style="font-family: Arial,Helvetica,sans-serif;"><span style="font-size: small;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhtDOs5zCnW1-A5JgJlssA-khmDogYstxMHRbE6oR5ghJWAxiabl-9-RYH0DOM4iSpRm956bPpf4lv3RAK6dtdRQqCw-QLkAmsSCxp6hcVgQuOo8VIvLzkPyNHkX_6_UJEVVlhL_CtIu0U/s1600/Cellotetraose.jpg" imageanchor="1" style="clear: left; margin-bottom: 1em; margin-left: auto; margin-right: auto;"><img border="0" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhtDOs5zCnW1-A5JgJlssA-khmDogYstxMHRbE6oR5ghJWAxiabl-9-RYH0DOM4iSpRm956bPpf4lv3RAK6dtdRQqCw-QLkAmsSCxp6hcVgQuOo8VIvLzkPyNHkX_6_UJEVVlhL_CtIu0U/s1600/Cellotetraose.jpg" height="320" width="320" /></a></span></span></td></tr>
<tr><td class="tr-caption" style="text-align: center;"><span style="font-family: Arial,Helvetica,sans-serif;"><span style="font-size: small;">Figure 3. Cellotetraose</span></span></td></tr>
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<span style="font-family: Arial,Helvetica,sans-serif;"><span style="font-size: small;">One aspect that makes these enzymes a little different is that their
substrate is generally insoluble. Therefore the
enzymes are often secreted by the micro-organisms that makes them and must
diffuse to the substrate rather than the substrate diffusing to the
enzyme as is common if the reaction is occurring inside a cell. Figure 4 shows a model of an exocellulase shredding up some
fibers from Dr GT Beckhams <a href="http://www.nrel.gov/biomass/staff_pages/gregg_beckham.html">page</a> - (isn't it awesome!?)(1). Unlike the active site of endocellulase which is situated in an open groove, the active site of exocellulase consists of a tunnel with several binding sites for the cellulose polymer. You will notice from Figure 4 that a small light blue domain extends on a thin "string" from the main body that has the polymer (green) passing through it. This is the carbohydrate binding module (CBM) that allows the enzyme to bind to cellulose. Not all cellulases have a CBM and it has been shown that cellulase catalytic activity does not always require the CBM even if its present. </span></span><br />
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<tr><td style="text-align: center;"><span style="font-family: Arial,Helvetica,sans-serif;"><span style="font-size: small;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEipVTuCx3nIrBzO-_-Ce-YeTzCBBepe2D2U18-RJ2zhtNBbhdRmR7sXOriMycb-Hqq0MAvYVxkvo-N9AIzp6jOHSwUtVRC_IGJY2dsYHjqQ7xqbn_uNnC1udUddo3xDeERPbqpGQqQlnII/s1600/beckham_cbh1.jpg" imageanchor="1" style="clear: right; margin-bottom: 1em; margin-left: auto; margin-right: auto;"><img border="0" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEipVTuCx3nIrBzO-_-Ce-YeTzCBBepe2D2U18-RJ2zhtNBbhdRmR7sXOriMycb-Hqq0MAvYVxkvo-N9AIzp6jOHSwUtVRC_IGJY2dsYHjqQ7xqbn_uNnC1udUddo3xDeERPbqpGQqQlnII/s1600/beckham_cbh1.jpg" height="352" width="640" /></a></span></span></td></tr>
<tr><td class="tr-caption" style="text-align: center;"><span style="font-family: Arial,Helvetica,sans-serif;"><span style="font-size: small;">Figure 4. The Family 7 cellobiohydrolase from <i>T. reesei</i> consists of three sub-domains: a small carbohydrate-binding module (CBM); a long, flexible linker decorated with <i>O</i>-linked glycosylation (yellow); and a large catalytic domain (CD) with <i>N-</i>linked
glycosylation (blue) and a 50 Å tunnel for the threading of
cellodextrin for catalytic cleavage. Cellulose (shown here in green
spacefill) is hypothesized to thread into the CD and cleavage occurs at
the end of the tunnel. The catalytic product of this enzyme is a
disaccharide of β1,4-glucose (cellobiose). (<a href="http://www.nrel.gov/biomass/staff_pages/gregg_beckham.html">link</a>)</span></span></td></tr>
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<span style="font-family: Arial,Helvetica,sans-serif;"><span style="font-size: small;"> Two main pathways to cellulose hydrolysis exist: 1. Non-complexed cellusase systems. 2. Complexed cellulase systems (cellulosome). </span></span><br />
<span style="font-family: Arial,Helvetica,sans-serif;"><span style="font-size: small;"><br /></span></span>
<span style="font-family: Arial,Helvetica,sans-serif;"><span style="font-size: small;"> Lets look at <i>Trichoderma reesei </i>and its non-complexed cellulases. <i>T. reesei </i>is a fungus that has been extensively studied because of its high level of excretion of three cellulases, to the tune of 100g/L. <i>T. reesei</i> has relatively few cellulases (2 exocellulases, 8 endoglucanases and so far 7 beta-glucosidases and I think no endocellulases). Its success as a producer of biotechnology enzymes is due to its high expression under cellulase inducing conditions.The exocellulases are some of the most important enzymes. Cel7a of <i>T. reesei</i> (Figure 4) makes up 60% of the cellulases excreted and degrades cellulose from the reducing end. Cel6a makes up 15-20% and degrades cellulose from the non-reducing end. </span></span><br />
<span style="font-family: Arial,Helvetica,sans-serif;"><span style="font-size: small;"><br /></span><span style="font-size: small;">
The production and purification of cellulases at sufficient levels is one of the main costs of lignocellulose utilization for biofuel and a great deal of research is carried out trying to optimize induction of cellulases, the speed with which they breakdown cellulose and into understanding the regulation of expression. For example, the highest producing cellulase strain of <i>T. reesei</i> in the public domain (i.e. not owned by a company) produces 30g/L of cellulases and is known as RUT30C. It was made by mutagenesis of the parent strain by UV irradiation and subsequent sequencing showed several mutations. One of the most important of these mutations was the truncation of the catabolite repressor protein 1 (cre1). Cre1 represses the expression of cellulases in the presence of more easily metabolizable carbohydrates (catabolites) such as glucose. This is because producing large enzymes such as cellulase is an energetic drain on the micro-organism and only performed when necessary for survival. Therefore turning off the repression of cellulase production, even in the presence of glucose, allows an increased yield of cellulases.</span></span><br />
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<span style="font-family: Arial,Helvetica,sans-serif;"><span style="font-size: small;">A second area of improvement is in the post translational modifications of the enzyme. If you look at Figure 4, you will notice some yellow blobs attached to the protein. These are sugar molecules that are added during its expression and modified as it is excreted. They help with the expansion of the linker domain that connects the CBM to the catalytic domain (large light blue structure) and increase the reach of the enzyme as it moves along the polymer. Not all micro-organisms "do" glycosylation. For example, bacteria are a favoured hosts for protein expression because they are easier to break open than fungi, grow quickly and can be made to produce high quantities of protein. However, the way in which they glycosylate (add sugars to) proteins is different from that in fungi and even production of cellulase in other model fungi such as <i>S. cerevisae</i> leads to incorrect glycosylation. Lack of correct glycosylation leads to abberrant or less efficient protein production and function.</span></span><br />
<span style="font-family: Arial,Helvetica,sans-serif;"><span style="font-size: small;"><br /></span></span>
<span style="font-family: Arial,Helvetica,sans-serif;"><span style="font-size: small;">A third area of improvement is to engineer in cellulases from other organisms. <i>T reesei </i></span><span style="font-size: small;">has a rather lower level beta-glucosidases and these are
also inhibited by the end product glucose (2). By engineering the <i>T. reesei</i> to include the beta-glucososidase of another fungus, <i>Aspergillus aculeatus</i>,
which is produced at a higher level and which is less susceptible to
glucose inhibition, the cellulose hydrolysis rate is much improved.</span></span><br />
<span style="font-family: Arial,Helvetica,sans-serif;"><span style="font-size: small;"><br /></span></span>
<span style="font-family: Arial,Helvetica,sans-serif;"><span style="font-size: small;">Thermal stability and rate of cellulose degradation are also aspects that can lead to cost savings in lignocellulose break down. </span></span><br />
<span style="font-family: Arial,Helvetica,sans-serif;"><span style="font-size: small;"><br /></span></span>
<span style="font-family: Arial,Helvetica,sans-serif;"><span style="font-size: small;">These are a few of the aspects to consider when contemplating cellulase and their cellulose hydrolysis function. There are also aspects to improve with respect to fermentation and recovery of function cellulases. While <i>T. reesei</i> has been the most studied and utilised organism for biofuel production, much research is also focused on bacterial cellulases and other fungi. In particular, I am interested in the cellulosome....a large and many faceted protein monster...... and what happens to the lignin and hemicellulose? Until next time...</span></span><br />
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<b><span style="font-family: Arial,Helvetica,sans-serif;"><span style="font-size: small;">References</span></span></b><br />
<span style="font-family: Arial,Helvetica,sans-serif;"><span style="font-size: small;">1.</span><span style="font-size: small;">Beckham GT, J. Ståhlberg, B.C. Knott, M.E. Himmel, M.F. Crowley, M. Sandgren, M. Sørlie, C.M. Payne. Towards a molecular-level theory of carbohydrate processivity in glycoside hydrolases. </span></span><br />
<div class="title">
<span style="font-family: Arial,Helvetica,sans-serif;"><span style="font-size: small;">Current Opinion in Biotechnology Volume 27, June 2014, Pages 96–106.</span></span></div>
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<span style="font-family: Arial,Helvetica,sans-serif;"><span style="font-size: small;">2. Tomohisa Hasunuma, Fumiyoshi Okazaki, Naoko Okai, Kiyotaka Y. Hara, Jun Ishii, Akihiko Kondo, A review of enzymes and microbes for lignocellulosic biorefinery and the possibility of their application to consolidated bioprocessing technology. Bioresource Technology 135 (2013) 513–522.</span></span></div>
<div class="title">
<span style="font-family: Arial,Helvetica,sans-serif;"><span style="font-size: small;">3. Another good reference: </span></span><a href="http://www.intechopen.com/download/get/type/pdfs/id/44414"><span style="font-family: Arial,Helvetica,sans-serif;"><span style="font-size: small;">Chapter 6 Hydrolysis of Biomass Mediated by Cellulases for the</span></span><span style="font-family: Arial,Helvetica,sans-serif;"></span><span style="font-family: Arial,Helvetica,sans-serif;"><span style="font-size: small;"> Production of Sugars</span></span></a><span style="font-family: Arial,Helvetica,sans-serif;"><span style="font-size: small;"><br /></span></span></div>
EmeraldBiologyhttp://www.blogger.com/profile/07468813270664433504noreply@blogger.com0tag:blogger.com,1999:blog-385372388482159249.post-75600411887831670762014-02-03T19:36:00.000-05:002014-02-04T09:30:17.864-05:00A note on cellulose structure<span style="font-family: Arial,Helvetica,sans-serif;">Before we launch into cellulases, I want to cover a little bit on the structure and nomenclature of cellulose. I'll start with glucose and how its drawn and named.</span><br />
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<span style="font-family: Arial,Helvetica,sans-serif;">a) Glucose can be depicted linearly in the "Fisher projection" as shown below. </span><br />
<span style="font-family: Arial,Helvetica,sans-serif;"><span id="goog_890401227"></span><span id="goog_890401228"></span></span><br />
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<span style="font-family: Arial,Helvetica,sans-serif;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEi32G_C7iU5d8BEYfyT-RjgT-gxz2beRL0VB19TvtwAt67SwT5ZnsP7n1Iwvo8NvCqs2hNRComozvtHcf8-oG1X0zygcWkVL6FPwKBvZpiZdTDCJV1yO_21DKrj1dafpQ50o7GhbV7X9yo/s1600/Glucose+fisher.jpg" imageanchor="1" style="margin-left: 1em; margin-right: 1em;"><img border="0" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEi32G_C7iU5d8BEYfyT-RjgT-gxz2beRL0VB19TvtwAt67SwT5ZnsP7n1Iwvo8NvCqs2hNRComozvtHcf8-oG1X0zygcWkVL6FPwKBvZpiZdTDCJV1yO_21DKrj1dafpQ50o7GhbV7X9yo/s1600/Glucose+fisher.jpg" /></a></span></div>
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<span style="font-family: Arial,Helvetica,sans-serif;">b) In solution glucose adopts a ring structure that can be shown as the Hawthorn projection. When it is in its ring structure it is also known as glucopyranose. Pyranose consists of any structure that has 5 carbons and 1 oxygen in the ring. Glucopyranose is therefore glucose as the 5carbon1oxygen ring structure.</span><br />
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<tr><td style="text-align: center;"><span style="font-family: Arial,Helvetica,sans-serif;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjd1WvHaYnCZPgBtbALuJ9JadwRwsucfLBksKs0x1jnI8gVoL1i0mmfKV-mxNSwLqNS-hrE_1HQADYhpfD4Z8Hk1HD6etaBN3-OTQ8AHXxAK8BNL82ZoC5eNnY3b7TYnNViZmRoKE03e2g/s1600/D-glucose+to+D-glucopyranose.gif" imageanchor="1" style="margin-left: auto; margin-right: auto;"><img border="0" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjd1WvHaYnCZPgBtbALuJ9JadwRwsucfLBksKs0x1jnI8gVoL1i0mmfKV-mxNSwLqNS-hrE_1HQADYhpfD4Z8Hk1HD6etaBN3-OTQ8AHXxAK8BNL82ZoC5eNnY3b7TYnNViZmRoKE03e2g/s1600/D-glucose+to+D-glucopyranose.gif" height="270" width="320" /></a></span></td></tr>
<tr><td class="tr-caption" style="text-align: center;"><span style="font-family: Arial,Helvetica,sans-serif;">Glucose in Fisher (top) and Hawthorn projections. Hawthorn projections show </span><span style="font-family: Arial,Helvetica,sans-serif;"><span style="font-family: "Arial","sans-serif";">α</span> (left) and </span><span style="font-family: Arial,Helvetica,sans-serif;"><!--[if gte mso 9]><xml>
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<![endif]--><span style="font-family: "Arial","sans-serif"; font-size: 11.0pt; line-height: 115%; mso-ansi-language: EN-US; mso-bidi-language: AR-SA; mso-fareast-font-family: Calibri; mso-fareast-language: EN-US; mso-fareast-theme-font: minor-latin;">β</span> (right) conformations (link). </span></span></td></tr>
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<br />
<span style="font-family: Arial,Helvetica,sans-serif;"><br /></span>
<span style="font-family: "Arial","sans-serif";">c)α</span><span style="font-family: Arial,Helvetica,sans-serif;">
vs <span style="font-family: "Arial","sans-serif"; font-size: 11.0pt; line-height: 115%; mso-ansi-language: EN-US; mso-bidi-language: AR-SA; mso-fareast-font-family: Calibri; mso-fareast-language: EN-US; mso-fareast-theme-font: minor-latin;">β</span>. You will notice that the glucose Fisher projection can form two types of Hawthorn projections. In the </span><span style="font-family: Arial,Helvetica,sans-serif;"><span style="font-family: "Arial","sans-serif";">α</span> projection, the OH at C1 points downwards (or is in trans or axial orientation) compared with the CH<span style="font-size: xx-small;">2</span>OH group whereas in the <span style="font-family: "Arial","sans-serif"; font-size: 11.0pt; line-height: 115%; mso-ansi-language: EN-US; mso-bidi-language: AR-SA; mso-fareast-font-family: Calibri; mso-fareast-language: EN-US; mso-fareast-theme-font: minor-latin;">β</span> projection, the OH at C1 points up (or is in cis or equitorial orientation) compared with the CH<span style="font-size: xx-small;">2</span>OH group. This is then written as </span><span style="font-family: Arial,Helvetica,sans-serif;"><span style="font-family: "Arial","sans-serif";">α</span>-D-glucose or </span><span style="font-family: Arial,Helvetica,sans-serif;"><span style="font-family: "Arial","sans-serif";">α</span>-D-glucopyranose for the trans conformation and <span style="font-family: "Arial","sans-serif"; font-size: 11.0pt; line-height: 115%; mso-ansi-language: EN-US; mso-bidi-language: AR-SA; mso-fareast-font-family: Calibri; mso-fareast-language: EN-US; mso-fareast-theme-font: minor-latin;">β</span>-D-glucose/glucopyranose for the cis conformation. Whether the glucose is </span><span style="font-family: Arial,Helvetica,sans-serif;"><span style="font-family: "Arial","sans-serif";">α </span>or <span style="font-family: "Arial","sans-serif"; font-size: 11.0pt; line-height: 115%; mso-ansi-language: EN-US; mso-bidi-language: AR-SA; mso-fareast-font-family: Calibri; mso-fareast-language: EN-US; mso-fareast-theme-font: minor-latin;">β</span> affects the binding to the next monomer and the type of enzymes that are able cut the bonds.</span><br />
<span style="font-family: Arial,Helvetica,sans-serif;"><br /></span>
<span style="font-family: Arial,Helvetica,sans-serif;">d) D vs L. You will also have noticed that I used the letter D. </span><br />
<br />
<span style="font-family: Arial,Helvetica,sans-serif;">The letters d and l (lower case) refer to the way in which plane polarised light would be rotated by a chiral center. A chiral center from our perspective is a carbon with for different groups attached.
Clockwise rotation means dextroroatorary (d) and anticlockwise rotation
is levorotatory (l). Dextro and Levo come from the latin for right and
left. </span><br />
<br />
<span style="font-family: Arial,Helvetica,sans-serif;">When sugars it gets a bit confusing because D and L (upper case) refers to the actual conformation of the chiral center at the carbon furthest from the carbonyl group in the Fisher projection (i.e. carbon 5) and in which direction the OH group on this carbon is pointing. D means that it is on the right side, L means that it is on the left side. You may also hear them called enantiomers. The D and L enatiomers are mirror images of each other. One particular carbon is referred to because there are often several chiral centers. Glucose has 4 for example. <b>Naturally, only the D form of glucose occurs.</b> </span><br />
<br />
<table align="center" cellpadding="0" cellspacing="0" class="tr-caption-container" style="margin-left: auto; margin-right: auto; text-align: center;"><tbody>
<tr><td style="text-align: center;"><span style="font-family: Arial,Helvetica,sans-serif;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEg2iuf7669X7gL8x-u2iYkwg3keDfD-D1UU7FIw5uPJk5SqdXMV-sKGGTeibZHn2MAVISnQZJCQErwfooDvkvIYrbVUrIMFhZ5vupGW3TEz8Pg7LyspqzoyRcGJy6rXE8iHwEhUni2QmiM/s1600/D+and+L+glucose.jpg" imageanchor="1" style="margin-left: auto; margin-right: auto;"><img border="0" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEg2iuf7669X7gL8x-u2iYkwg3keDfD-D1UU7FIw5uPJk5SqdXMV-sKGGTeibZHn2MAVISnQZJCQErwfooDvkvIYrbVUrIMFhZ5vupGW3TEz8Pg7LyspqzoyRcGJy6rXE8iHwEhUni2QmiM/s1600/D+and+L+glucose.jpg" height="278" width="400" /></a></span></td></tr>
<tr><td class="tr-caption" style="text-align: center;"><span style="font-family: Arial,Helvetica,sans-serif;">Fisher projections of L and D glucose from <a href="http://illuminolist.wordpress.com/tag/d-l-designation/">here</a>. * show chiral centers.</span></td></tr>
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<span style="font-family: Arial,Helvetica,sans-serif;"> </span><br />
<span style="font-family: Arial,Helvetica,sans-serif;">Because of the way in which chiral centers were discovered, (it was based on the conformation of glyceraldehyde), L or D does not always refer accurately to the rotation direction of plane polarised light. For example, amino acids naturally occur in the L-form but for historical reasons it does not follow that this molecule always rotate light anticlockwise. In fact chemists use the R/S system....but we won't go into that.....If you want to dig deeper, google it up and here is the <a href="http://en.wikipedia.org/wiki/Chirality_%28chemistry%29">wiki page</a> to get you started. Following any the links from which I have gather images will also give you more information.</span><br />
<br />
<span style="font-family: Arial,Helvetica,sans-serif;"> e) Joining <span style="font-family: "Arial","sans-serif"; font-size: 11.0pt; line-height: 115%; mso-ansi-language: EN-US; mso-bidi-language: AR-SA; mso-fareast-font-family: Calibri; mso-fareast-language: EN-US; mso-fareast-theme-font: minor-latin;">β</span>-D-glucopyranose together. So, celluose consist of D-anhydroglucopyranose joined together by <span style="font-family: "Arial","sans-serif"; font-size: 11.0pt; line-height: 115%; mso-ansi-language: EN-US; mso-bidi-language: AR-SA; mso-fareast-font-family: Calibri; mso-fareast-language: EN-US; mso-fareast-theme-font: minor-latin;">β</span>-1,4-glycosidic bonds to form an anhydrocellobiose unit. So, lets deconstruct. D means the D form, anhydro, means the loss of the hydroxyl group at the glycosidic bond (I think from carbon4) glucopyranose is the pyranose ring form of glucose. </span><span style="font-family: Arial,Helvetica,sans-serif;"><!--[if gte mso 9]><xml>
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<![endif]--><span style="font-family: "Arial","sans-serif"; font-size: 11.0pt; line-height: 115%; mso-ansi-language: EN-US; mso-bidi-language: AR-SA; mso-fareast-font-family: Calibri; mso-fareast-language: EN-US; mso-fareast-theme-font: minor-latin;">β</span> means that the OH group at C1 is in the cis conformation and 1,4 means that the glycosidic bond is between carbon 1 of the first glucose unit and carbon 4 of the second glucose unit. Each glucopyranose unit is rotated 180 degrees with respect to the previous one and together they form a repeating unit known has anhydrocellubiose. This is shown below. </span><br />
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<table align="center" cellpadding="0" cellspacing="0" class="tr-caption-container" style="margin-left: auto; margin-right: auto; text-align: center;"><tbody>
<tr><td style="text-align: center;"><span style="font-family: Arial,Helvetica,sans-serif;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEib1vpVoGuWFOZwxxp4zOCZu2pvmIRoTEKjwBegBkWSo2WmcjT6NYK-YoP639zYJQcDMZpY-xIpVbWMyVbwGzwxe3-9ZiJXErgog6_UIfx0J6kClcWj7vsI3eoM1Aq0S7LMU0kyD1qSZMQ/s1600/cellubiose.jpg" imageanchor="1" style="margin-left: auto; margin-right: auto;"><img border="0" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEib1vpVoGuWFOZwxxp4zOCZu2pvmIRoTEKjwBegBkWSo2WmcjT6NYK-YoP639zYJQcDMZpY-xIpVbWMyVbwGzwxe3-9ZiJXErgog6_UIfx0J6kClcWj7vsI3eoM1Aq0S7LMU0kyD1qSZMQ/s1600/cellubiose.jpg" height="308" width="640" /></a></span></td></tr>
<tr><td class="tr-caption" style="text-align: center;"><span style="font-family: Arial,Helvetica,sans-serif;">Structure of cellulose featuring anhydrocellobiose unit in the large square brackets (left) and cellulose I crystal structure (right) adapted from ref. 1.</span></td></tr>
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<span style="font-family: Arial,Helvetica,sans-serif;">You will notice that the structure on the left is not drawn in the Hawthorn projection but in what is known as the Chair conformation - very slightly looks like a chair, right? This is the most stable form for glucose. The other orientation is known as the boat conformation - for more on this look <a href="http://chemwiki.ucdavis.edu/Organic_Chemistry/Organic_Chemistry_With_a_Biological_Emphasis/Chapter__3%3A_Conformations_and_Stereochemistry/Section_3.2%3A_Conformations_of_cyclic_organic_molecules">here, point 3.2C. </a> Cellulose can exist in several crystalline forms but the native one is referred to as cellulose I. Sheets of cellulose I stack on top of each other and form a fibril. Since they all stack in the same way, different parts of the stack will have different properties. For example, one end will have all the reducing ends, the other all the non-reducing ends. The edge of the crystal will have the edges of the glucose units. All this presents elements for the cellulases to tackle. Surrounding the fibers is hemicellulose and surrounding this is lignin. Reminiscent of a previous post, the figure (adapted from reference 1) below shows a depiction of a microfibril. </span><br />
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<span style="font-family: Arial,Helvetica,sans-serif;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgo17tyDzcs1NrXzIptmS625E1cnHrNrmkZQsf5i599Xw5CqcwCqqRXNnYNHO-0nxErd0Lr_9MiUqqei2bFMfk6GLp27DHyWQKz57EvnGuoEVIW94TWWOJABdBx8qxEY6HPZjoSjRdden0/s1600/microfibril.jpg" imageanchor="1" style="margin-left: 1em; margin-right: 1em;"><img border="0" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgo17tyDzcs1NrXzIptmS625E1cnHrNrmkZQsf5i599Xw5CqcwCqqRXNnYNHO-0nxErd0Lr_9MiUqqei2bFMfk6GLp27DHyWQKz57EvnGuoEVIW94TWWOJABdBx8qxEY6HPZjoSjRdden0/s1600/microfibril.jpg" height="603" width="640" /></a></span></div>
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<span style="font-family: Arial,Helvetica,sans-serif;">OK! I think we are ready to tackle cellulases....</span>EmeraldBiologyhttp://www.blogger.com/profile/07468813270664433504noreply@blogger.com0tag:blogger.com,1999:blog-385372388482159249.post-84017660317263718292014-01-13T14:35:00.001-05:002014-01-26T21:44:14.195-05:00Biofuel life cycle: things to consider<span style="font-family: Arial,Helvetica,sans-serif;">Biofuels - gotta be good for everything right? No drilling, no using of reserves millions of years in the making; just using plants and bugs to get where you gotta go.....ok, so thats a teensy bit of an exaggeration, actually it could be quite a large exaggeration. Browsing my e-mail alert for the journal Trends in Biotechnology the other day, I came across this article, hot off the press: Sustainability considerations for integrated biorefineries by Adisa Azapagic at the University of Manchester (Trends in Biotechnolgy, Jan. 2014, Vol. 32, No. 1, page 1-4). Little did I know what lay in store. Environmentalists pride themselves (I like to think...) on considering the whole picture, which is what this article takes a stab at, rather competently I think. </span><br />
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<span style="font-family: Arial,Helvetica,sans-serif;">First off, when comparing biofuel vs petroleum fuel production and the overall impact this has, the life cycle from source to product must be taken into account.</span><br />
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<span style="font-family: Arial,Helvetica,sans-serif;"><b>A) green house gas emission </b>(GHG). Oh, I thought, what a doozy, biofuels win hands down. Not so fast my friends, in fact take a step back, if you will and take a gander at the lovely graph in figure 1, care of Dr. Azapagic. </span><br />
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<tr align="left"><td class="tr-caption">Figure 1. On a life-cycle basis, ethanol produced in an integrated biochemical refinery saves up to 104 g CO<sub>2</sub> eq./MJ compared to petrol (85 g CO<sub>2</sub> eq./MJ for petrol compared to −19 g CO<sub>2</sub>
eq./MJ for ethanol from UK poplar) owing to the credits for the
co-products, in this case electricity, lactic acid, and acetic acid.
Ethanol from sugar cane in Brazil saves 65 g CO<sub>2</sub> eq./MJ,
whereas ethanol from corn has much higher greenhouse gas (GHG) emissions
than petrol. Land-use change (LUC) can increase GHG emissions
significantly — in the case of biofuel from miscanthus to 310 g CO<sub>2</sub>
eq./MJ or 3.6 times higher than petrol. GHG emissions for all fuel
options are from ‘cradle to grave’, encompassing production of the
feedstocks and fuels as well as fuel combustion during use of vehicles.</td><td class="tr-caption"></td><td class="tr-caption"></td></tr>
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<span style="font-family: Arial,Helvetica,sans-serif;">You see the shock factor, "Ethanol from US corn"? - its GHG emissions are 1.5x HIGHER than from a fossil fuel refinery! This is because of nitrous oxide emissions from fertilisers applied to the corn fields. Well its not organically grown, is it?! UK wheat and Brazilian sugar cane do better but its not in the negative. Noooo, you don't get that until you start in on the 2nd generation biofuels (the lignocellulose feeds stocks) and then comes another shocker. Miscanthus, a second generation fibrous grass that has GHG emissons 3.6x higher than a fossil fuel.....arghhhhh! WHY?</span></div>
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<span style="font-family: Arial,Helvetica,sans-serif;"><b>B) LUC and Biodiversity</b> See those letters - LUC - land use change. This is the term for changing the use of the land from say forest to corn, or from forest to miscanthus or from farm use to forest - a biofuel feedstock forest. This can have two effects: GHG emissions change and biodiversity changes. Forest land in the UK replaced by miscanthus resulted in the 310 g CO<span style="font-size: xx-small;">2</span> eq./MJ (Figure 1). Biodiversity also decreased as a monoculture of miscanthus is obviously less diverse than a forest. However a feedstock forest could be more diverse than a field previously used for growing wheat, so the biodiversity issue works both ways. </span></div>
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<span style="font-family: Arial,Helvetica,sans-serif;"><b>C) Water use</b>. The source of biofuel feedstock varies a great deal in the amount of water required to grow it. Feedstock from agricultural waste or forestry waste requires little or no water, whereas energy crops such as miscanthus require more water than arable crops such as corn because of a longer growing season. Water has to be transported from somewhere and this will effect GHG and stress the place from which it was taken. Biorefineries themselves require relatively little water use. So, like biodivesity, water use can have positive or negative impact. Well, maybe not positive but at least, less negative. </span></div>
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<span style="font-family: Arial,Helvetica,sans-serif;"><b>D)</b> Other considerations in the bio(fuel) life cycle are environmental impacts such as soil pollution (acidification, human toxicity etc), emissions of sulfur dioxide, nitric oxide, nitrite etc. There are also economic considerations such as feedstock costs, capital cost (the commercial biorefineries using second generation feedstock have to be built - the US Department of Energy has a <a href="http://www1.eere.energy.gov/bioenergy/integrated_biorefineries.html">website</a> showing sites where they are being built) and the cost of the biofuel itself. Then there are social considerations such as jobs and regional development, health issues (e.g. pesticides cause cancer and death, particulate emission from biomass handling affects air quality), human labour rights, land availability and food prices (energy crops might displace food crops and drive up food prices) and affects on future generations.</span></div>
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<span style="font-family: Arial,Helvetica,sans-serif;">Curiously, the article does not mention the other hazards involved in
the petrolum industry such as the contamination of water ways by
fracking or the great environmental disasters caused during drilling for oil, many of which we never even hear about. The chance for that kind of disaster seems much less likely in biofuel production. </span><span style="font-family: Arial,Helvetica,sans-serif;"><br /></span></div>
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<span style="font-family: Arial,Helvetica,sans-serif;">Still, there is a GREAT DEAL to consider while we go about the nitty gritty of finding exactly how a cellulase chews ups cellulose. It really IS a<span style="font-size: x-large;"> BIG</span> picture but I believe the conclusion of the article; if managed correctly, biofuels really could be a very good thing.</span></div>
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<span style="font-family: Arial,Helvetica,sans-serif;">To read the article for yourself, go <a href="http://www.sciencedirect.com/science/article/pii/S0167779913002278">here</a>. </span></div>
EmeraldBiologyhttp://www.blogger.com/profile/07468813270664433504noreply@blogger.com0tag:blogger.com,1999:blog-385372388482159249.post-87105261900487463672014-01-05T22:30:00.000-05:002014-01-05T22:34:03.723-05:00Fuels for Biofuels part 4: lignocelluose as a biofuel: pretreatment.<span style="font-family: Arial,Helvetica,sans-serif;"><span style="font-size: small;">One of the problems with lignocellulosic feedstocks is the complexity of composition and the presence of lignin - the polymer that allows plants to fill their cells with a rigid mass and stand up straight - see my <a href="http://www.emeraldbiology.com/2013_07_01_archive.html">post</a> on plant structure. Wood and the fibrous stems of crops and other plants have an abundance of lignin and this needs to be broken down before the cellulose fibers that provide the edible stuff for micro-organisms becomes accessible. Simultaneously the hemicellulose and cellulose fibers need to be disrupted to make them more readily digestible. </span></span><br />
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<span style="font-family: Arial,Helvetica,sans-serif;"><span style="font-size: small;">Figure 1 shows the a schematic of conversion of lignocelluosic feedstock to biofuel. In this post I will discuss pretreatment processes (the green bit in Figure 1). </span></span><br />
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<tr><td class="tr-caption" style="text-align: center;"><span style="font-family: Arial,Helvetica,sans-serif;"><span style="font-size: small;">Figure 1. Converting lignocellulose to biofuel (4).</span></span></td></tr>
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<tr><td style="text-align: center;"><span style="font-family: Arial,Helvetica,sans-serif;"><span style="font-size: small;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhC20xu18dXFlOhD3QMiHT-6H1b6P7WzoVxUxaY_quprj76w2cCr1T1tpfdTmHah_fJUO4AAoPZ9BubG9xFJq_2FBi8sm_mmQY9ld2Pqf091PBBs99Tuo6JmVU-mphf0XW9VfWdyX-Ld6k/s1600/lignocellulose+to+biofuel.png" imageanchor="1" style="clear: left; margin-bottom: 1em; margin-left: auto; margin-right: auto;"><img border="0" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhC20xu18dXFlOhD3QMiHT-6H1b6P7WzoVxUxaY_quprj76w2cCr1T1tpfdTmHah_fJUO4AAoPZ9BubG9xFJq_2FBi8sm_mmQY9ld2Pqf091PBBs99Tuo6JmVU-mphf0XW9VfWdyX-Ld6k/s1600/lignocellulose+to+biofuel.png" height="356" width="640" /></a></span></span></td></tr>
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<span style="font-family: Arial,Helvetica,sans-serif;"><span style="font-size: small;">The basic process for biological conversion of lignocellulose to ethanol is a pretreatment to break down the wood polymers, followed by enzyme mediated hydrolysis of those polymers to produce monosaccharides that can then be fermented by micro-organism to produce ethanol. </span></span><br />
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<span style="font-family: Arial,Helvetica,sans-serif;"><span style="font-size: small;"><b>Pretreatment </b></span></span><br />
<span style="font-family: Arial,Helvetica,sans-serif;"><span style="font-size: small;">Even before pretreatment, the biomass must be converted to smaller pieces. For example, wood is shredded to chips and then further converted to fibers. Great energy savings can result if the chips are chemically treated <b>before</b> further milling to fibers (Zhu et al, 2010) i.e. the order of shredding and then pretreatment can be manipulated to give optimal results. There are different types of pretreatment and the type that is used depends a great deal on the biomass resource. For example one treatment that works well for wheat straw may be much less efficient for use with wood chips. Here are a few examples of pretreatments:</span></span><br />
<span style="font-family: Arial,Helvetica,sans-serif;"><span style="font-size: small;">1) Dilute acid pretreatment uses low levels of acid (e.g. 2% sulfuric acid) and heat (190dC). Compounds called furferals are produced which inhibit fermentation but are a value added co-product. The process works best with hardwoods rather than softwoods and also produces a condensed form of lignin that is of little value apart from as boiler fuel. </span></span><br />
<span style="font-family: Arial,Helvetica,sans-serif;"><span style="font-size: small;">2) Steam explosion (Figure 2) also uses dilute acid and combines chemical pretreatment with size reduction in one step increasing efficiency. However it is energy intensive, has relatively low sugar recovery, though this can be improved, and works better with hardwoods than with softwoods. It has not yet been commercially scaled (2010). </span></span><br />
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<tr><td style="text-align: center;"><span style="font-family: Arial,Helvetica,sans-serif;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjwRiBRk2Gz26KoIGMUUp-406nr8Q-P57QPHhb4qTVW_Q_PqKhoqEPs1e5PQLiqZ8W3NMjM0_s8HxB1wgBPbbnZfoftsmNju8gC7JUhNBi9QbtL1eBmSIbc1oD-eyDvJSN_kLH5zrRF_wk/s1600/Steam+exploaded+oil+palm+trunk+httpwww.intechopen.combookssustainable-degradation-of-lignocellulosic-biomass-techniques-applications-and-commercializationoptimization-of-delignification-and-enzyme-hydrolysis-of-steam-explo.jpg" imageanchor="1" style="clear: left; margin-bottom: 1em; margin-left: auto; margin-right: auto;"><img alt="" border="0" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjwRiBRk2Gz26KoIGMUUp-406nr8Q-P57QPHhb4qTVW_Q_PqKhoqEPs1e5PQLiqZ8W3NMjM0_s8HxB1wgBPbbnZfoftsmNju8gC7JUhNBi9QbtL1eBmSIbc1oD-eyDvJSN_kLH5zrRF_wk/s1600/Steam+exploaded+oil+palm+trunk+httpwww.intechopen.combookssustainable-degradation-of-lignocellulosic-biomass-techniques-applications-and-commercializationoptimization-of-delignification-and-enzyme-hydrolysis-of-steam-explo.jpg" height="150" title="" width="400" /></a></span></td></tr>
<tr><td class="tr-caption" style="text-align: center;"><span style="font-family: Arial,Helvetica,sans-serif;">Figure 2 <a href="http://www.intechopen.com/books/sustainable-degradation-of-lignocellulosic-biomass-techniques-applications-and-commercialization/optimization-of-delignification-and-enzyme-hydrolysis-of-steam-exploded-oil-palm-trunk-for-ethanol-p">Oil palm trunk chip (a) and steam exploed oil palm trunk pulp (b) </a></span></td></tr>
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<tr><td class="tr-caption" style="text-align: center;"><span style="font-family: Arial,Helvetica,sans-serif;"><span style="font-size: small;"><a href="http://www.intechopen.com/books/sustainable-degradation-of-lignocellulosic-biomass-techniques-applications-and-commercialization/optimization-of-delignification-and-enzyme-hydrolysis-of-steam-exploded-oil-palm-trunk-for-ethanol-p"> </a></span></span><br />
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<span style="font-family: Arial,Helvetica,sans-serif;"><span style="font-size: small;">3) Organosolv uses organic solvents such as ethanol, acetone, butanol or polyethylene glycol. To date ethanol has proven to be the most economical as it is cheap and easy to recover even if it is not the most efficient. Organosolv processes produce high quality lignin and celluose which has good digestibility. Large quantities of ethanol are required and the process is energy intense. As a result commercial viability lies in the recovery of the high quality value added products that are a by-product of this process. </span></span><br />
<span style="font-family: Arial,Helvetica,sans-serif;"><span style="font-size: small;">4) The most promising pretreatment however is the Sulfate Process (also known as the Kraft Process or Sulfite pretreatment to Overcome Recalcitrance of Lignocellulose (SORL). As above, dilute acid is used together with the addition of sulfite (for the chemical reaction, click<a href="http://en.wikipedia.org/wiki/Sulfite_process"> here)</a>. Conditions are relatively mild at 130-190dC and produce low amounts of fermentation inhibitors but also lignosulfonate which is a good value added coproduct. Additionally the process softens lignin by increasing its hydrophilicity which reduces energy consumption and aids downstream polymer digestion. Economy is also increased because the process can be carried out directly on wood chips without further size decrease. </span></span><br />
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<tr><td style="text-align: center;"><span style="font-family: Arial,Helvetica,sans-serif;"><span style="font-size: small;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhz1uVsz8J2vA9jsezOExXHTB707-HIWbEbqreuR0J0Mdi4r3UaMC1_aP_bIIg4Amk8sChni1nqtAmymlBt6pfYGbtJ593r6hPLU6vwQWSnblMSzMAfwxq-waJf6MTmAe2fGuf-SbxlBp0/s1600/ionic+liquids+wiki.JPG" imageanchor="1" style="clear: right; margin-bottom: 1em; margin-left: auto; margin-right: auto;"><img border="0" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhz1uVsz8J2vA9jsezOExXHTB707-HIWbEbqreuR0J0Mdi4r3UaMC1_aP_bIIg4Amk8sChni1nqtAmymlBt6pfYGbtJ593r6hPLU6vwQWSnblMSzMAfwxq-waJf6MTmAe2fGuf-SbxlBp0/s1600/ionic+liquids+wiki.JPG" height="304" width="320" /></a></span></span></td></tr>
<tr><td class="tr-caption" style="text-align: center;"><span style="font-family: Arial,Helvetica,sans-serif;"><span style="font-size: small;">Figure 3. <a href="http://en.wikipedia.org/wiki/Ionic_liquid">Sodium chloride compared with ionic liquid [bmim]NTf<sub>2</sub></a></span></span></td></tr>
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<span style="font-family: Arial,Helvetica,sans-serif;"><span style="font-size: small;">5) Ionic liquid treatment is a relatively recent approach using salts that are liquid at room temperature (figure 3) and that are able to dissolve cellulose and lignin. However, more research is needed into ionic salt recovery from the dissolved biomass before they can be commercially viable. </span><span style="font-size: small;"> </span></span><br />
<span style="font-family: Arial,Helvetica,sans-serif;"><span style="font-size: small;">6) Biological pretreatment. If you have had the good fortune of being able to walk through<table cellpadding="0" cellspacing="0" class="tr-caption-container" style="float: left; margin-right: 1em; text-align: left;"><tbody>
<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEh0cT6HKiDrn37L2Ykrck8PkiYbVStL5Zd8bo2fu1LL1OfQpWRHutCiuuBMArnVDNLaQL6TgCTQZYsQszK9uCZwnIOl_SEfn2m2kGCEfokTIi8PUpJ84Yn6xXMHOnCn15t_yBl7QKroGbc/s1600/8998353423_bed918bc42.jpg" imageanchor="1" style="clear: left; margin-bottom: 1em; margin-left: auto; margin-right: auto;"><img border="0" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEh0cT6HKiDrn37L2Ykrck8PkiYbVStL5Zd8bo2fu1LL1OfQpWRHutCiuuBMArnVDNLaQL6TgCTQZYsQszK9uCZwnIOl_SEfn2m2kGCEfokTIi8PUpJ84Yn6xXMHOnCn15t_yBl7QKroGbc/s1600/8998353423_bed918bc42.jpg" height="400" width="300" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;">Figure 4 -<a href="http://www.flickr.com/photos/dendroica/8998353423/in/photolist-eH9UYx-5pbtcg-5BK5Fs-cryT2W-ayt5MW-duNPNc-5F6cD6/lightbox/"> link</a></td></tr>
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<span style="font-family: Arial,Helvetica,sans-serif;"><span style="font-size: small;">
a quiet wood and step over or on soft rotting branches (Figure 4), you'll understand even better that there are biological processes capable of breaking down lignin. These involve enzymes and as we will discuss in the next post, enzymes are a major economic burden in biofuel production. Their advantage is that they require mild conditions and consolidation of the breakdown of lignin (pretreatment), cellulose hydrolysis and fermentation could potentially all be done in one step, substantially reducing cost. There'll be more on this in a subsequent post I think.</span></span><br />
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<span style="font-family: Arial,Helvetica,sans-serif;"><span style="font-size: small;">In each pretreatment scenario, the lignin and other non-digestible material must be separated from the degraded cellulose fibers. How this occurs depends on the pretreatment type and the details of the pretreatment. For example in organosolv pretreatment, temperature and solvent (e.g. ethanol) to water ratio effect the morphology of lignin. The many aspects of such fractionation are beyond the scope of this blog and at this moment, my interest! </span></span><br />
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<span style="font-family: Arial,Helvetica,sans-serif;"><span style="font-size: small;"><b>Engineering plants.</b></span></span><br />
<span style="font-family: Arial,Helvetica,sans-serif;"><span style="font-size: small;">As discussed in my <a href="http://www.emeraldbiology.com/2013_07_01_archive.html">April blog post</a>, wood consists of three polymers - cellulose, hemicellulose and lignin. Have a look back to remind yourself of their structures. Wood cellulose has a twist to the arrangement of the polymers and to be relatively amorphous, possible making it more amendable to breakdown. Soft and hardwood hemicellulose differs in the composition of the monosaccarhides that predominate and in chemical linkages between the monosaccharides. While cellulose and hemicellulose are polysaccharides, lignin is an unstructured polymer of <a href="http://www.emeraldbiology.com/p/bob-bug-and-friends.html">monolignols</a> and again the composition and structure of lignins varies between hardwoods and softwoods. As the chemical links joining the monomers are all different, so are the enzymes necessary to break them. In order to understand the best procedure for doing this, much research is performed into the structure and composition of the three polymers and to how changes in the genetic components of their synthesis pathways can ease the breakdown pathway during biofuel production without affecting plant vigor (3). Other avenues of genetically engineering plants have also been explored (5) and these include increasing the amount of cellulose, engineering plants to produce their own cellulase and reducing the amount of lignin in the plant. Of course, planting great swaths of genetically engineered trees or grasses is controversial. This topic will have its own post in the future.</span></span><br />
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<span style="font-family: Arial,Helvetica,sans-serif;"><span style="font-size: small;"><b>Thermal and biological conversion of wood feedstock</b></span></span><br />
<span style="font-family: Arial,Helvetica,sans-serif;"><span style="font-size: small;">As interesting aside, there are two ways of converting wood to biofuel:
biological and thermal. Above we discussed pretreatment for biological conversion. Thermal conversion (1) has two pathways: </span><span style="font-size: small;">gasification and
pyrolysis. Gasification uses high heat and pressure and yields carbon
monoxide and hydrogen from which a variety of fuels can be catalytically
obtained. Pyrolysis (breakdown by fire!) uses heat in the absence of
oxygen to generate bio-oils and char. Bio-oil can lead to a number of
different products but requires further treatment before it can be used.
Char is the solid carbon enriched material that remains. It can be used
as a soil fertilizer and perhaps as a method of carbon sequestration to
remove CO2 from the atmosphere in attempts to mitigate climate change.</span></span></div>
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<span style="font-family: Arial,Helvetica,sans-serif;"><span style="font-size: small;"><b>References</b></span></span><br />
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<span style="font-family: Arial,Helvetica,sans-serif;"><span style="font-size: small;">1) Pu, Y, Kosa M., Kalluri U, Tusakn GA, and Ragauskas AJ. 2011 Challenges of the utilization of wood polymers: how can they be overcome? Applied Microbiology and Biotechnology<span id="date" itemprop="datePublished"> September 2011</span>, <span id="volume-range">Volume 91</span>, Issue 6, <span id="page-range">pp 1525-1536</span></span></span><br />
<span style="font-family: Arial,Helvetica,sans-serif;"><span style="font-size: small;"><span id="page-range">2)</span><span id="page-range"><span id="page-range">Zhu, JY, Pan, X. and Zalesny Jr., RS. 2010 </span>Pretreatment of woody biomass for biofuel production:energy efficiency, technologies, and recalcitrance Appl Microbiol Biotechnol 87:847–857<br />3) </span><span role="menubar">Feltus FA and Vandenbrink JP.</span> 2012 Bioenergy
grass feedstock: current options and prospects for trait improvement
using emerging genetic, genomic, and systems biology toolkits. <span role="menubar">Biotechnol Biofuels </span>Nov 2;5(1):80</span></span><br />
<span style="font-family: Arial,Helvetica,sans-serif;"><span style="font-size: small;">4)Verardi, A, De Bari, I, Ricca, E, and Calabrò, V. Hydrolysis of Lignocellulosic Biomass:<br />Current Status of Processes and Technologies and Future Perspectives. 2013. www.intechopen.com </span></span><br />
<span style="font-family: Arial,Helvetica,sans-serif;"><span style="font-size: small;">5) Sticklen, M. 2006. Plant genetic engineering to improve biomass characteristics</span></span><br />
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<span style="font-family: Arial,Helvetica,sans-serif;"><span style="font-size: small;">for biofuels. Current Opinion Biotechnology. 17:315-319</span></span></div>
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EmeraldBiologyhttp://www.blogger.com/profile/07468813270664433504noreply@blogger.com0tag:blogger.com,1999:blog-385372388482159249.post-83406982499832546062013-08-24T13:21:00.000-04:002013-08-24T13:35:38.740-04:00Fuel for Biofuel 3: Cellulosic feedstocks<div class="separator" style="clear: both; text-align: center;">
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<a href="http://draft.blogger.com/blogger.g?blogID=385372388482159249" imageanchor="1" style="clear: right; float: right; margin-bottom: 1em; margin-left: 1em;"></a>In the last post, we established that cellulosic feedstock are somewhat more complicated in their molecular structure than a corn feedstock. What are the main feedstocks being considered for commercialisation? What are the fuels they are used to produce?<br />
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Corn and sugarcane are known as the feedstocks for first generation biofuels. Other feedstocks are called advanced biofuels but these still consist of ethanol and biodiesel. Biodiesel is produced from the triacylglycerides obtained from soy, oily seeds or the mesocarp of palm fruits (<a href="http://naturalnigerian.com/2012/02/pam-kernel-oil-vs-palm-oil/">click here for illustration</a>). More technically, a process known as trans-esterification converts the triacylglycerides to fatty acid methyl esters that can be directly used in engines. I hope to post on this too!<br />
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So what are the feedstocks current being exploited for advanced biofuels?<br />
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<b>C3 vs C4 vs CAM plants</b><br />
First, lets have little more plant education. One way in which plants can be classified is according to differences in their photosynthetic pathways. Photosynthesis is the use of the energy from sunlight to convert CO<span style="font-size: xx-small;">2</span> to an organic carbon molecule together with the release of oxygen. With this type of classification, 3 groups occur: C3, C4 and CAM. The first molecule produced by the use of CO<span style="font-size: xx-small;">2</span> in C3 plants is a 3 carbon molecule and a 4 carbon molecule in C4 plants. C3 plants operate more efficiently under cool wet conditions while C4 plants are more water efficient and well adapted to dryer and hotter conditions. CAM (or Crasseulean Acid Metabolism) are even more water efficient and survive well in arid dessert climates. One of the ways in which water loss is minimised is through regulation of the gas exchange pores on the underside of the leave; these are called stomata. In C3 plants they are open all the time, in C4 plants stomata open only during the day when photosynthesis can occur but in CAM plants, stomata open only during the cooler night. CO<span style="font-size: xx-small;">2</span> is gathered and stored as crasseulic acid during the night and then used during the day. As water loss can occur through the stomata, regulating when they are open, regulates the loss of water. Most plants are C3 (<span class="st"> rice, wheat, soybeans, potatoes, beans, fruit trees<i>)</i></span>. C4 plants include corn and many annual summer plants. CAM plants include catuses and agave plants.This is of course simply put. More details can be found in<a href="http://www.ehleringer.net/Jim/Publications/271.pdf"> this article</a>.<br />
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<b>Non-woody perennials.</b><br />
Popular contenders for cellulosic feedstock plants are the perennial grasses such as miscanthus, switch grass and elephant grass are all C4 plants and are more efficient at photosynthesis than C3 plants. <br />
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<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjCtrSjite5cvfzpm41t78NNQ02_TTbTPo8Y5KyjCEwRBKgVmuyGBNFu4thFxX3JiIM99FNQgJCXKiEWljiui4PyIQ2chOdjHsuobx6m65Rrp5g02bUPjEijfgMI1jYMw3P4QRY98vSvHE/s1600/Miscanthus_sinensis_ja01.jpg" imageanchor="1" style="clear: left; margin-bottom: 1em; margin-left: auto; margin-right: auto;"><img border="0" height="197" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjCtrSjite5cvfzpm41t78NNQ02_TTbTPo8Y5KyjCEwRBKgVmuyGBNFu4thFxX3JiIM99FNQgJCXKiEWljiui4PyIQ2chOdjHsuobx6m65Rrp5g02bUPjEijfgMI1jYMw3P4QRY98vSvHE/s320/Miscanthus_sinensis_ja01.jpg" width="320" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;">Miscanthus</td></tr>
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<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgZkqJAsEg5o1WKG3Ryjgqbf8cZuf6Ibf89PbGq4qnmKBhesdBbsHcF8TXUg0ppAu7aLH-d9-DT207ZllVuWYzk-NE-lcOQPSmRGgNuAOI9NR1aB3Fka32KXg1a_zwVKN983Q5NmuPejgI/s1600/Panicum_virgatum.jpg" imageanchor="1" style="clear: right; margin-bottom: 1em; margin-left: auto; margin-right: auto;"><img border="0" height="320" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgZkqJAsEg5o1WKG3Ryjgqbf8cZuf6Ibf89PbGq4qnmKBhesdBbsHcF8TXUg0ppAu7aLH-d9-DT207ZllVuWYzk-NE-lcOQPSmRGgNuAOI9NR1aB3Fka32KXg1a_zwVKN983Q5NmuPejgI/s320/Panicum_virgatum.jpg" width="256" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;">Switchgrass</td></tr>
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Additionally, their root system consist of a network of rhizomes that store energy and nutrients for the following season of growth allowing a fast regeneration time, drastically decreasing the amount of fertilizer needed and preventing soil erosion. Further the perennial grasses under consideration are native to Amercia (including switch grass, Praries grass and Big and Little Bluegrass, suggesting that the Northern Plains and Southeastern grasslands could be a source of advanced biofuel.<br />
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<b>Woody perennials. </b><br />
Woody perennials include fast growing trees such as Willow, SweetGum and Cottonwood.<br />
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<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjn7b1XMBS3W7LG1bEYJLuivUUZOKZTSVXbj9P7A7fp3Dic76K8vCSvfuoAQYzeXY1QyaWdda8E_liymCLlvjM36-S5hVtlmead6DhFDHe0VCIh2MBYGa3_flFHV_lNv3wjF_lgWbPgcGY/s1600/400px-Liquidambar_styraciflua_-_La_Hulpe_(1).JPG" imageanchor="1" style="clear: right; margin-bottom: 1em; margin-left: auto; margin-right: auto;"><img border="0" height="320" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjn7b1XMBS3W7LG1bEYJLuivUUZOKZTSVXbj9P7A7fp3Dic76K8vCSvfuoAQYzeXY1QyaWdda8E_liymCLlvjM36-S5hVtlmead6DhFDHe0VCIh2MBYGa3_flFHV_lNv3wjF_lgWbPgcGY/s320/400px-Liquidambar_styraciflua_-_La_Hulpe_(1).JPG" width="213" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;">Sweet Gum</td></tr>
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<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEiwt9WNCGQD7XVwuI7itu2zTH13YwC2Qz-6eJsLqloj-L31BkzE7Yvhaai11oZb-j4awFU-D3feg3C4LF488bpFzj21wkKIpvr4MeenhXsNPbhiiHqeXndhAiC8s0ZqQP4b3chcsxL0kYg/s1600/Starr_020803-0107_Hibiscus_tiliaceus.jpg" imageanchor="1" style="clear: left; margin-bottom: 1em; margin-left: auto; margin-right: auto;"><img border="0" height="240" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEiwt9WNCGQD7XVwuI7itu2zTH13YwC2Qz-6eJsLqloj-L31BkzE7Yvhaai11oZb-j4awFU-D3feg3C4LF488bpFzj21wkKIpvr4MeenhXsNPbhiiHqeXndhAiC8s0ZqQP4b3chcsxL0kYg/s320/Starr_020803-0107_Hibiscus_tiliaceus.jpg" width="320" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;">Cottonwood</td></tr>
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Like the perennial grasses they have several advantages over energy crops such as corn. First the amount of biomass produced for the same land area is much greater, they have better water use, don't require fertilization and deep root systems maintain soil structure and prevent erosion. Secondly, some species such as willow and eucalyptus are amendable to the technique of coppicing, where the plants are cut down to near ground level every 3-5 years. This allows for increased biomass and these species rapidly regenerate from the storage system contained in their roots.<br />
<br />
<b>New bioenergy crops.</b><br />
With modern agricultural practices and deforestation, large tracts of land have become semi arid. Plants such as the agave that uses CAM photosynthesis, is able to grow in such conditions and could be used to regenerate these semi-arid areas while providing a biofuel feedstock. Agave plants of different species have been under cultivation for many 100's of year for alcholic beverages (think Tequila!) and for sisal fibers to use in twine, paper, dartboards (!), handicrafts and mattresses for example. Sisal fibers could be a potential source for a biofuel feedstock.<br />
<br />
Another problem with modern agriculture is the salinization of irrigated land and it is estimated 1-2% of irrigated land is lost every year. Highly salt tolerant plants such as prairie cordgrass and Eucalyptus species are potential feedstocks and could also be used to desalinate lost arable land. <br />
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<b>Waste matter </b><br />
When I started considering biofuels from lignocellulosic feedstock, I was hopeful that a large proportion would be generated from the waste products of current industrial processes. To some extent this is true.<br />
Firstly there is corn stover. Stover are the leftover inedible fibrous stalks and leaves after the cobs have been harvested. It is estimated that 13.5 billions gallons of
ethanol could be generated from this stover. And while the bagasse produced after
sugarcane harvesting in Brazil is not used as a feedstock but is burnt and leads to production of 2giga
watts net electricity, this can also be classed as biofuel, just like wood burnt in your fireplace. However the costs of collection and
transportation to processing plants of corn stover and the loss of nutrients and risk of soil erosion could make this economically and environmentally untenable though this view depends on where you get the information. <br />
<br />
Many other crops such as wheat, rice and soybean leave a waste
product called residue after the harvest. This is also a potential
source of biofuel feedstock but is complicated by the same issues that
are involved in corn stover collection in that the decay of the residue
returns nutrients to the soil and prevents soil erosion by wind and
rain. The ins
and outs leading to an assessment of commerical viability are beyond the
scope of this post as they are many and complex. The <a href="http://www1.eere.energy.gov/bioenergy/pdfs/billion_ton_update.pdf">2011 US One Billion Update</a> provides an in depth analysis of all the different aspects that need to be considered.<br />
<br />
<b>Wood waste</b><br />
There are several sources of wood waste. The
first if from timberland (forests used for logging) and this includes
tree tops, branches, dead or rotten wood, small trees and non-commercial
trees. This waste is normal left in the forest to decay but could
provide a biofuel feedstock source. Careful management would be
necessary as some of this waste wood provides a return of nutrients to
the soil, habitats for insects and birds, fertiliser for new saplings
and prevention of soil erosion.<br />
<br />
Other wastewood comes
from forest thinning in timberland areas and non-logging forests where
biomass is removed to reduce the risk of forest fires becoming
catastrophic. Waste wood is also generated when timberland is converted
to non-forest land such as cropland and pasture roads. A last source of
waste wood is from urban wood which includes furniture, landscaping,
remodeling and construction. Much of this ends up in landfills. To me,
that seems like a wonderful source for generating biofuel.<br />
<br />
In the 2011 <a href="http://www1.eere.energy.gov/bioenergy/pdfs/billion_ton_update.pdf">US One Billion Update</a>, it is estimated that wood sources will produce up to 244 million tons of dry biomass.<br />
<br />
<b>Municipal waste (MSW)</b><br />
This is the matter that everyone discards on a daily basis. Food scraps, paper waste, plastic etc. From the point of view of biofuels, the most valuable items are paper and food. These need to be separated from the rest so despite the presence of a collection and transport system for MSW, the processing necessary in order to use it commercially as biofuel feedstock, is complicated. <b> </b><br />
<br />
<b>Animal Fat and Yellow grease.</b><br />
<div class="separator" style="clear: both; text-align: center;">
</div>
Animal
fats are generated from the slaughter of animals. However they are less
valuable as a feedstock as they have a tendency to crystallize at lower
temperatures. Yellow grease is the oil left over after cooking for example in
restaurants and is a mixture of plant and animal fats. It is in limited
supply but could be commercially viable for smaller biofuel operations. <br />
<br />
The table shows the total and projected tons of biomass available from different feedstocks for ethanol and biodiesel (from <a href="http://www1.eere.energy.gov/bioenergy/pdfs/billion_ton_update.pdf">2011 US One Billion Update</a>)<br />
<br />
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" /></a><br />
<br />
<br />
<br />
<br />
<b>Current commercial operations</b> <br />
According to <a href="http://www.environmentalleader.com/2013/03/12/cellulosic-ethanol-to-be-cost-competitive-by-2016/">Environmental Leader</a>, there are 11 competitive cellulosic ethanol plants currently operating and use of cellulosic feedstocks will be cost competitive with corn as a feedstock by 2016 and in fact many corn based plants are shutting down due to eroding profit margins. <a href="http://en.wikipedia.org/wiki/Cellulosic_ethanol_commercialization">Wikipedia</a> provides a table showing several companies in the US, their start dates and production capacities. A selection is shown in the table below and the feedstocks show that corn stover, wheat straw, wood waste and municiple waste are among the main ones in use. <br />
<br />
<br />
<table class="wikitable sortable jquery-tablesorter" style="height: 510px; width: 653px;"><thead>
<tr><th class="headerSort" role="columnheader button" tabindex="0" title="Sort ascending">Company</th>
<th class="headerSort" role="columnheader button" tabindex="0" title="Sort ascending">Location</th>
<th class="headerSort" role="columnheader button" tabindex="0" title="Sort ascending">Feedstock</th>
<th class="headerSort" role="columnheader button" tabindex="0" title="Sort ascending">Capacity (million gal/year)</th>
<th class="headerSort" role="columnheader button" tabindex="0" title="Sort ascending">Began Production</th>
<th class="headerSort" role="columnheader button" tabindex="0" title="Sort ascending">Type</th>
</tr>
</thead><tbody>
<tr>
<td><a href="http://en.wikipedia.org/wiki/Abengoa" title="Abengoa">Abengoa Bioenergy</a></td>
<td>Hugoton, KS</td>
<td>Wheat straw</td>
<td>25 - 30 <sup class="reference" id="cite_ref-Building_Cellulose_33-1"><a href="http://en.wikipedia.org/wiki/Cellulosic_ethanol_commercialization#cite_note-Building_Cellulose-33">[33]</a></sup><sup class="reference" id="cite_ref-34"><a href="http://en.wikipedia.org/wiki/Cellulosic_ethanol_commercialization#cite_note-34">[34]</a></sup></td>
<td>est. late 2013</td>
<td>Commercial</td>
</tr>
<tr>
<td><a href="http://en.wikipedia.org/wiki/DuPont" title="DuPont">DuPont</a></td>
<td>Nevada, IA</td>
<td>Corn stover</td>
<td>30<sup class="reference" id="cite_ref-41"><a href="http://en.wikipedia.org/wiki/Cellulosic_ethanol_commercialization#cite_note-41">[41]</a></sup></td>
<td>est. 2014</td>
<td>Commercial</td>
</tr>
<tr>
<td><a class="new" href="http://en.wikipedia.org/w/index.php?title=Fulcrum_BioEnergy&action=edit&redlink=1" title="Fulcrum BioEnergy (page does not exist)">Fulcrum BioEnergy</a></td>
<td>Reno, NV</td>
<td>Municipal solid waste</td>
<td>10</td>
<td>est. end of 2013</td>
<td>Commercial</td>
</tr>
<tr>
<td><a href="http://en.wikipedia.org/wiki/Gulf_Coast_Energy" title="Gulf Coast Energy">Gulf Coast Energy</a></td>
<td>Livingston, AL</td>
<td>Wood waste</td>
<td>0.3 <sup class="reference" id="cite_ref-42"><a href="http://en.wikipedia.org/wiki/Cellulosic_ethanol_commercialization#cite_note-42">[42]</a></sup></td>
<td>before 2008</td>
<td>Demonstration</td>
</tr>
<tr>
<td><a href="http://en.wikipedia.org/wiki/Mascoma_Corporation" title="Mascoma Corporation">Mascoma</a></td>
<td>Kinross, MI</td>
<td>Wood waste</td>
<td>20</td>
<td>est. 2014</td>
<td>Commercial</td>
</tr>
<tr>
<td><a class="mw-redirect" href="http://en.wikipedia.org/wiki/POET_LLC" title="POET LLC">POET LLC</a><sup class="reference" id="cite_ref-furth_43-0"><a href="http://en.wikipedia.org/wiki/Cellulosic_ethanol_commercialization#cite_note-furth-43">[43]</a></sup><sup class="reference" id="cite_ref-44"><a href="http://en.wikipedia.org/wiki/Cellulosic_ethanol_commercialization#cite_note-44">[44]</a></sup></td>
<td>Emmetsburg, IA</td>
<td>Corn stover</td>
<td>20 - 25</td>
<td>est. lat 2013<sup class="reference" id="cite_ref-45"><a href="http://en.wikipedia.org/wiki/Cellulosic_ethanol_commercialization#cite_note-45">[45]</a></sup></td>
<td>Commercial</td>
</tr>
<tr>
<td><a class="mw-redirect" href="http://en.wikipedia.org/wiki/POET_LLC" title="POET LLC">POET LLC</a><sup class="reference" id="cite_ref-46"><a href="http://en.wikipedia.org/wiki/Cellulosic_ethanol_commercialization#cite_note-46">[46]</a></sup></td>
<td>Scotland, SD</td>
<td>Corn stover</td>
<td>0.03</td>
<td>2008</td>
<td>Pilot</td></tr>
</tbody></table>
<br />
<b><br /></b>
<b>Concluding...this post</b><br />
Cellulosic feedstocks are on a roll and there are many sources. I hope to have a closer look at these in coming posts and to bring in the bugs! <br />
<b><br /></b>
<b>References and websites used:</b><br />
<a href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3342825/">Youngs and Somverville</a> 2012, Development of feedstocks for cellulosic biofuels. F1000 Reports Biology. Volume 4, Issue 10.<br />
<a href="http://www.environmentalleader.com/2013/03/12/cellulosic-ethanol-to-be-cost-competitive-by-2016/">Environmental Leader</a><br />
<a href="http://about.bnef.com/">Bloomberg New Energy Finance</a><br />
<a href="http://Biofuels Digest">Biofuels Digest</a><br />
<a href="http://www1.eere.energy.gov/bioenergy/pdfs/billion_ton_update.pdf">US One Billion Update </a><br />
Wikipedia; various<br />
<br />EmeraldBiologyhttp://www.blogger.com/profile/07468813270664433504noreply@blogger.com0tag:blogger.com,1999:blog-385372388482159249.post-56117747919482506452013-07-09T13:05:00.000-04:002014-01-26T21:48:15.683-05:00Fuel for Biofuels part 2: cellulosic feedstockIn Fuel for Biofuels part 1, I took a look at producing biofuel from corn. This has come to be known as first generation biofuel. The next generation biofuels can be produced from cellulosic feedstocks and by aquatic algae. In this part 2, I'm going to take a look a cellulosic feedstocks. In an attempt to make the posts more digestible, I'm going to make them shorter and hopefully more frequent.<br />
<br />
Previously we learnt that corn is a commercially popular feed stock because it is easily broken down having a relatively simple structure and requiring simpler chemical and enzymatic degradation processes. Cellulosic ethanol feedstocks include sawdust, forest thinnings, waste
paper, grasses, farm waste (e.g.,
corn stalks, wheat straw, and rice
straw), switchgrass and other perennial grasses. Where is the cellulosic material found in plants?<br />
<br />
<b>Plant cell wall macro structures</b><br />
There are many differences when plant cells are compared with animal cells, such as the presence of chloroplasts and a large central vacuole but the difference we are most interested in, is the cell wall which animal cells lack. The cell wall is one of the features of plants that gives a plant rigidity.<br />
<br />
The food generating cells of leaves have a thin cell well and these cells are known as parenchyma. The cells that make up young flexible stems are known as collenchyma. Their cell walls are thicker particularly at the corners where several cells intersect. The thicker the cell wall the less nutrients can get into the food production centers but collenchyma cells despite their thicker cell walls, are still alive. The cell wall consists of cellulose, hemicellulose and pectin. Collenchyma cells are able to stretch as the plant grows. Figure 1 illustrates the plant cell wall structure. You can see that the cellulose is arranged in microfibrils and a
magnified illustration of a microfibril is shown in figure 2. The highly
regular formation of the fibrils can easily be imagined (curiously it
reminds me of the highly structured formation of skeletal muscle).
Hemicellulose links the microfibrils together. In figure 1 cell wall illustration, you can also
see a molecule called pectin. Pectin is another polysaccharide and works to both give mechanical
strength to the cell wall and to improve impermeability. <br />
<table cellpadding="0" cellspacing="0" class="tr-caption-container" style="float: left; text-align: left;"><tbody>
<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjC-Ar4Ry-W4sA6gWtYBeg5XVrlSfYu_EJStYEK3Xdn4btL3UhSdBYZzmL6ZVCZ-58_S2eC9XEZTlV_uTJlGEg4e07ApBtoXypQGbzeh2HJMDWCG_uUGORbxt-88fKt033qBsLZFEGg8Ks/s1600/497px-Plant_cell_wall_diagram.svg.png" imageanchor="1" style="clear: left; margin-bottom: 1em; margin-left: auto; margin-right: auto;"><img border="0" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjC-Ar4Ry-W4sA6gWtYBeg5XVrlSfYu_EJStYEK3Xdn4btL3UhSdBYZzmL6ZVCZ-58_S2eC9XEZTlV_uTJlGEg4e07ApBtoXypQGbzeh2HJMDWCG_uUGORbxt-88fKt033qBsLZFEGg8Ks/s400/497px-Plant_cell_wall_diagram.svg.png" height="230" width="400" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;">Figure 1 Structure of plant cell wall <a href="http://en.wikipedia.org/wiki/Cell_wall">(Link)</a></td></tr>
</tbody></table>
<br />
<table cellpadding="0" cellspacing="0" class="tr-caption-container" style="float: right; margin-left: 1em; text-align: right;"><tbody>
<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEh5MdBbK6wtlLf9baSseHY15WISM_1R35zStWyC1sK8samGf4ySJk9U-BBd-GxGFmezoKhdX90EMWUHxguaYz0KZ1cAmLv1VgcENvvh_hEm-iJR7XIOZlag_SZAAWOkM_BQkOWOE36tR8E/s1600/fiber.gif" imageanchor="1" style="clear: right; margin-bottom: 1em; margin-left: auto; margin-right: auto;"><img border="0" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEh5MdBbK6wtlLf9baSseHY15WISM_1R35zStWyC1sK8samGf4ySJk9U-BBd-GxGFmezoKhdX90EMWUHxguaYz0KZ1cAmLv1VgcENvvh_hEm-iJR7XIOZlag_SZAAWOkM_BQkOWOE36tR8E/s400/fiber.gif" height="261" width="400" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;">Figure 2 Structure of cellulose microfibril (<a href="http://nutrition.jbpub.com/resources/chemistryreview9.cfm">link</a>).</td></tr>
</tbody></table>
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<a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjnR7-czQl36q5LMFcIR7ChrqmhAAPXWFBc9I-L5f07MjH1uoBWufMnarOwjLxaxrHcCNkV3zcKPT8UwmEy13UZoo4N0s-d1EKRLV93hj7acsz4TnbF_3v3VaD7f_SWlODof06bhq07OO4/s1600/F1.large.jpg" imageanchor="1" style="clear: left; float: left; margin-bottom: 1em; margin-right: 1em;"><br /></a></div>
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<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjJOwXChnDqwjeoQGeB4jmA4JK2GnM5y3MYunwCgEoKimJfxGgrIeUigGJW-2gOC7zw8b-Iz8_KlRx4PiCeT5ztDvV4B0FwZkWxDd0hGHxI2WmQNXPkAgSza3jDa9X7rR19ESWU5_bPUAo/s1600/primary_secondary_cell_wall_middle_lamella1330138249714.jpg" imageanchor="1" style="clear: right; margin-bottom: 1em; margin-left: auto; margin-right: auto;"><img border="0" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjJOwXChnDqwjeoQGeB4jmA4JK2GnM5y3MYunwCgEoKimJfxGgrIeUigGJW-2gOC7zw8b-Iz8_KlRx4PiCeT5ztDvV4B0FwZkWxDd0hGHxI2WmQNXPkAgSza3jDa9X7rR19ESWU5_bPUAo/s320/primary_secondary_cell_wall_middle_lamella1330138249714.jpg" height="320" width="313" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;">Figure 3 Electron micrograph of cell wall with secondary cell wall (<a href="http://www.studyblue.com/notes/note/n/topic-7-plant-form-/deck/2261434">Link</a>)</td></tr>
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To the inside of the primary cell wall in these cells, a secondary cell wall can be laid down and toughened
by the deposition of lignin. Lignin is water impermeable and is very
rigid, with great mechanical strength; without water the cell cannot
survive and dies. This layer of cells becomes known as sclerenchyma.<br />
<br />
Figure 3 shows an electron micrograph of the cell wall and you can really see how thick the secondary wall is <br />
<br />
<b>Molecular structures </b><br />
We are interested in the structure of these polymers because they will affect downstream processing of the cellulosic materials for biofuel.<br />
<br />
Cellulose consists of glucose monomers linked together by C1 and C4 as shown in Figure 4. <br />
<table cellpadding="0" cellspacing="0" class="tr-caption-container" style="float: left; margin-right: 1em; text-align: left;"><tbody>
<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgV5C6mZXhC7cQXKfDUskmVBCnJ2SzAEmYnGIFQEsv19SA9VjBE3gFCJ_cePRqv-ugmga6kWGao9PQ4bV2fNsNWcdN4Rs-YEtHRDwhpXT80RzHhgpqqI4QJEXC9635EUdNJB_8EDysVifY/s1600/385px-Cellulose_Sessel.svg.png" imageanchor="1" style="clear: left; margin-bottom: 1em; margin-left: auto; margin-right: auto;"><img border="0" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgV5C6mZXhC7cQXKfDUskmVBCnJ2SzAEmYnGIFQEsv19SA9VjBE3gFCJ_cePRqv-ugmga6kWGao9PQ4bV2fNsNWcdN4Rs-YEtHRDwhpXT80RzHhgpqqI4QJEXC9635EUdNJB_8EDysVifY/s200/385px-Cellulose_Sessel.svg.png" height="91" style="cursor: move;" width="200" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;">Figure 4 Monomer of cellulose</td></tr>
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The number of glucose units can be in the 10,000s and its regular structure allows it to pack tightly together giving strength.<br />
<br />
Hemicellulose on the other hand is amorphous and flexible with little structure strength. Hemicellulose consists mainly of 5 carbon sugar monomers such as xylose, mannose, galactose, rhammnose and arabinose. Figure 5 shows a picture of a common hemicellulose repeating unit with the different pentose sugars.<br />
<table cellpadding="0" cellspacing="0" class="tr-caption-container" style="float: left; margin-right: 1em; text-align: left;"><tbody>
<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhwv551mDN2z35NEOBRasbUvYto07R9Aa9dvdGka9uk8EU7SYV2Caacs3lDWF-vbhT6A3Fa5Px_qPVBHVkcFIgIctyNKUZzk_mZClzgXx8FQbJ-2a3a714b_yqxZZCDCsLWLcLGmWFwdbI/s1600/Hemicellulose.png" imageanchor="1" style="clear: left; margin-bottom: 1em; margin-left: auto; margin-right: auto;"><img border="0" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhwv551mDN2z35NEOBRasbUvYto07R9Aa9dvdGka9uk8EU7SYV2Caacs3lDWF-vbhT6A3Fa5Px_qPVBHVkcFIgIctyNKUZzk_mZClzgXx8FQbJ-2a3a714b_yqxZZCDCsLWLcLGmWFwdbI/s320/Hemicellulose.png" height="276" width="320" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;">Figure 5. Comon hemicellulose molecular motif</td></tr>
</tbody></table>
Pectin, like hemicellulose, also consists of several different monosaccharides and illustration of pectin is shwon in figure with the different regions shown in blue and the monosaccarhides illustrated as small differently coloured polygons, and the key to the polygons is given in the figure. <br />
<br />
<br />
Lignin is unusual in a structural polymer in being very irregular. It consist of three molecules called monolignol monomers: <i>p</i>-coumaryl alcohol, coniferyl alcohol, and sinapyl alcohol. Different plants have lignin with different proportions of each molecule. Figure 6 shows an example of a possible lignin structure. It makes me think of lace.<br />
<table cellpadding="0" cellspacing="0" class="tr-caption-container" style="float: left; margin-right: 1em; text-align: left;"><tbody>
<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjRu7neNVnOvbz454xZyr438-zeGfEgVBQEClB6rouwrm8d3eev1JIM9RGOIE__jjuN6z023-Kh4E6zZ1i06ihr8xCG9tmVFlMwslTnBKBHeInCJh_OPhjqqmOZEYBWsPkJCPqCxsir-9I/s1600/621px-Lignin_structure.svg.png" imageanchor="1" style="clear: left; margin-bottom: 1em; margin-left: auto; margin-right: auto;"><img border="0" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjRu7neNVnOvbz454xZyr438-zeGfEgVBQEClB6rouwrm8d3eev1JIM9RGOIE__jjuN6z023-Kh4E6zZ1i06ihr8xCG9tmVFlMwslTnBKBHeInCJh_OPhjqqmOZEYBWsPkJCPqCxsir-9I/s320/621px-Lignin_structure.svg.png" height="309" width="320" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;">Figure 6. Example of possible lignin structure.</td></tr>
</tbody></table>
So, wow, thats a bit more complex than the starch nolecule of corn. Interesting, the difference between starch and cellulose, both of which consist of chains of glucose molecules is the way in which the monomers are linked. In starch, animals have enzymes that can easily digest the link while they do not have the enzymes to easily digest the cellulose chemical links. How do we turn the complex structure of plant cell walls into biofuels? See the next post!<br />
<ul></ul>
<br />EmeraldBiologyhttp://www.blogger.com/profile/07468813270664433504noreply@blogger.com0tag:blogger.com,1999:blog-385372388482159249.post-51283430927485881832013-05-19T22:25:00.001-04:002013-05-19T22:33:57.545-04:00Fuel for Biofuel part 1<table cellpadding="0" cellspacing="0" class="tr-caption-container" style="float: right; height: 319px; margin-left: 1em; text-align: right; width: 245px;"><tbody>
<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEj5-0amy5rqNs5n1R3bk8UGGPFwcOggHmgRGkO-mXKuAzLegEJK_vXwN2zudKaGuRgDvQtiafJTuds9wI9aBIP_283JSLdhNsEPEFQ_IvuM-hq0UFxGEqPmU7EZI_xwr5LRVcdSPFss_b8/s1600/bug-blog-5.jpg" imageanchor="1" style="clear: right; margin-bottom: 1em; margin-left: auto; margin-right: auto;"><img border="0" height="320" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEj5-0amy5rqNs5n1R3bk8UGGPFwcOggHmgRGkO-mXKuAzLegEJK_vXwN2zudKaGuRgDvQtiafJTuds9wI9aBIP_283JSLdhNsEPEFQ_IvuM-hq0UFxGEqPmU7EZI_xwr5LRVcdSPFss_b8/s320/bug-blog-5.jpg" width="273" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;">Figure 1: Illustration of bacterial ethanol (EtOH) production from corn by<a href="http://naturallinestudio.com/"> <b>Natural Line Studio</b></a></td></tr>
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Last month we took a look at ocean plastic. It was pretty depressing and from the point of view of microbiology, a bit of a non-starter. So lets go back to land and biofuels, one of the next great hopes that makes use of various micro-organisms.<br />
<span style="color: #38761d;"><b> </b></span><br />
<span style="color: #38761d;"><b>Summarising the basics of the biofuel process</b></span><br />
In order for micro-organism to produce biofuel (Figure 1) they need a food source or feedstock. Feedstocks are generally made of starch (Figure 2) or lignocellulose
(fibrous parts of plants). These are polysaccharides of small subunits like monosaccharides
or disaccharides, just like plastics are polymers of
hydrocarbon monomers (see my January post). In Figure 2 each hexagon is a glucose (monosaccharide) molecule. A row of glucose molecule joined together, by glycosidic bonds, in this way is called amylose. When the glucose from the top row forms a bond with a glucose in the second row, it is called amylopectin. Therefore starch consists of amylose and amylopectin. <br />
<table cellpadding="0" cellspacing="0" class="tr-caption-container" style="float: left; height: 185px; text-align: left; width: 288px;"><tbody>
<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEiISTcPXin8SIdnC_12NQE61mR1FgqKhWK_IKYW_jwOsXVJRKbTKYeSQSukUskHMbZe-MUQD1xe2HIHR0fuACAog8CGVYTGhCOKv57l2HcV6VCEbVKR7S53phmEYOju5FO0xR2z6KO8KZs/s1600/1-6branch2.JPG" imageanchor="1" style="clear: left; margin-bottom: 1em; margin-left: auto; margin-right: auto;"><img border="0" height="122" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEiISTcPXin8SIdnC_12NQE61mR1FgqKhWK_IKYW_jwOsXVJRKbTKYeSQSukUskHMbZe-MUQD1xe2HIHR0fuACAog8CGVYTGhCOKv57l2HcV6VCEbVKR7S53phmEYOju5FO0xR2z6KO8KZs/s200/1-6branch2.JPG" width="200" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;">Figure 2.<b> <a href="http://academic.brooklyn.cuny.edu/biology/bio4fv/page/starch.html">Structure of starch</a></b></td></tr>
</tbody></table>
The first step is to break up the polymers so that the maximum number of chemical bonds are exposed and available to micro-organisms. Mechanical, heat and enzymatic action is used to achieved the breakdown of the polymers to edible chunks (monosaccharides and dissaccharides). These breakdown steps are followed by and often coincident with the fermentation step where micro-organisms use the released sugars as a carbon source to produce ethanol.You can imagine that the cost of this is not insignificant, so making it as efficient as possible is key. Biofuels include ethanol, butanol, biodiesel and a number of second generation fuels. Currently ethanol is the only commercially produced biofuel, a so-called first generation biofuel and we will start with this.<br />
<br />
<span style="color: #38761d;"><b>Ethanol</b></span><br />
Ethanol is the main biofuel produced globally (Fisher et al,. 2008). Ethanol is an
alcohol. Chemically, alcohols are denoted by a hydroxyl or -OH group (see structure in figure 3) <br />
<table cellpadding="0" cellspacing="0" class="tr-caption-container" style="float: left; margin-right: 1em; text-align: left;"><tbody>
<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEiM9v4JfR9OtjS9mlBqJepVe0yfUVSK-SpXLgnBxWQPRiYGf4xl4BwlwO4Sw0EdvmUxL9Njxn5MCNzJ0KsE0szSPqffR8UTG6wL39tcuXIpKVCIud2GVYfbEGG1DB_8t9XuEzMOplF13Ns/s1600/Ethanol.png" imageanchor="1" style="clear: left; margin-bottom: 1em; margin-left: auto; margin-right: auto;"><img border="0" height="114" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEiM9v4JfR9OtjS9mlBqJepVe0yfUVSK-SpXLgnBxWQPRiYGf4xl4BwlwO4Sw0EdvmUxL9Njxn5MCNzJ0KsE0szSPqffR8UTG6wL39tcuXIpKVCIud2GVYfbEGG1DB_8t9XuEzMOplF13Ns/s200/Ethanol.png" width="200" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;">Figure 3:<b><a href="http://www.google.com/imgres?imgurl=http://cornellbiochem.wikispaces.com/file/view/Ethanol/172801857/694x442/Ethanol&imgrefurl=http://cornellbiochem.wikispaces.com/Ethanol&h=303&w=529&sz=5&tbnid=uLKdeAYXJpUESM:&tbnh=72&tbnw=126&zoom=1&usg=__fzgreCuBi_bz2Tikgjc13rrVvao=&docid=uMebvvwCpcl7sM&sa=X&ei=rLGIUaqaB8b40gG6-IGwDA&ved=0CFMQ9QEwCQ&dur=2579"> Ethanol</a></b></td></tr>
</tbody></table>
and it is added to petrochemical
vehicle fuel to increase the octane number (see bottom of post for
octane number explanation) and to cut down on smog
inducing emissions. Flexible fuel vehicles have modified engines that
are able to use mixed gasoline/ethanol as fuel. A common flex fuel is
E85 (85% gasoline, 15% ethanol). Sugar cane in Brazil and corn in the
USA are the main feedstocks of bioethanol with very high conversion efficiencies
(i.e. % of feedstock input converted to ethanol) of between 90-95%
(Fisher et al, 2008). The problems with ethanol is that it is rather
different from gasoline. But hold on....just what IS gasoline or petrol?
It consists of a mix of hydrocarbons called alkanes, cycloalkanes and
olefins with 4-12 carbons in each molecule (Figure 4). Additives are
also present to enhance the use of the fuel in an engine such as MBTE in Figure 4. Structurally and chemically, these molecules are quite different from ethanol. <br />
<table cellpadding="0" cellspacing="0" class="tr-caption-container" style="float: right; margin-left: 1em; text-align: right;"><tbody>
<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgLQ3EUQ3L34wy-tPsZ8uxXx9qvkKbvgf8RJjpLrQ_OKzR6Q2EaMo1Ens9Z_1H6PR1gx-KtJBzgPCw6pSiBaMXO-3XkflOkx7mbze0dXh0QDQvk18YiUfHtxXlReE_kxWHOfU5kgk1XJGY/s1600/340px-GasolineComp.png" imageanchor="1" style="clear: right; margin-bottom: 1em; margin-left: auto; margin-right: auto;"><img border="0" height="92" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgLQ3EUQ3L34wy-tPsZ8uxXx9qvkKbvgf8RJjpLrQ_OKzR6Q2EaMo1Ens9Z_1H6PR1gx-KtJBzgPCw6pSiBaMXO-3XkflOkx7mbze0dXh0QDQvk18YiUfHtxXlReE_kxWHOfU5kgk1XJGY/s320/340px-GasolineComp.png" width="320" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;">Figure 4: Common components of gasoline</td></tr>
</tbody></table>
<br />
Since most cars can run on 10% ethanol mixed with gasoline, why does flexfuel only make up only 6% of fuel used (Solomon, 2010)?. The infrastructure present for piping gasoline around the country is extensive and obviously has taken considerable investment. Because ethanol can be corrosive to the materials of this infrastructure as well as to unadapted car engines (Fisher et al, 2008), a new infrastructure would be required specifically for flex fuel and adapting cars costs a couple of thousand dollars, something many people can't afford.<br />
<br />
The other big problem is the use of corn as a feedstock despite its good conversion efficiencies. This is because, when considering the impact of feedstocks for biofuel, there are numerous factors to consider apart from the hopeful reduction in climate change gases. These include water use, land use prior to corn growth, biodiversity decrease, pollution of water and air due to growth (pesticides, fertilisers), processing (needs electricity from oil) and socio-economic impact. There have been numerous studies on corn as an ethanol feedstock with varying outcomes. The overall message that comes through is that although corn as a feedstock is better than using petrochemcials, it is unsustainable and even at maximum capacity, could only support 20-30% of biofuel needs of the US (Solomon, 2010). Though sugar cane as a feedstock fares better than corn, there are other feedstocks known as cellulosic (sometimes called lignocellulosic) material (includes paper, wood, cardboard, fibrous plant material) that have a much lower impact as they can come from waste products of current industries, not from the new growth of a dedicated crop. Interestingly, one of the main public concerns of corn as an ethanol feedstock is the increase in food prices as more land is devoted to corn for ethanol rather than corn for food. However the situation is more complex and in fact also depends on oil prices (Solomon, 2010). The % of corn that is used for food is the smallest of the corn uses at 10% and animal feed is the largest at 48%. Fuel takes up about 23% (Solomon, 2010).<br />
<br />
So if cellulosic material is so much better, why aren't we already using that? The commercial popularity of corn is because cornstarch is readily available, a relatively simple polysaccharide (figure 2) and therefore more easily converted to ethanol. Cornstarch also makes up 70-72% of the dry weight of a corn kernal. Lignocellulosic materials have a more complex structure and requires more enzymes and different types of micro-organisms to digest it; because of this, they are not yet commercially viable. I will discuss this in my next post but for now, lets get back to ethanol and the types of micro-organisms that are used to convert it to ethanol. <br />
<span style="color: #38761d;"><b><br /></b></span>
<span style="color: #38761d;"><b>Ethanol producing micro-organisms</b></span><br />
<i>Saccharomyces cerevisiae </i>also known as budding yeast or bakers yeast is the micro-organism of choice in commercial bioethanol production (Figure 5). <i>Zymomonas mobilis </i> has also been heavily studied but <i>S. cerevisiae</i> remains top bug, mainly because <i>Zymomonas mobilis</i> need a specific subset of monosaccharides in order to produce ethanol where as <i>S. cerevisiae </i>is less of a picky eater (Bai et al, 2008). Conversion of corn to ethanol can be done through dry milling (67% of ethanol is produced this way) or by wet milling (33% ethanol is produced this way). Bothast and Schlicher, 2005 give an excellent summary as follows<br />
<table cellpadding="0" cellspacing="0" class="tr-caption-container" style="float: right; margin-left: 1em; text-align: right;"><tbody>
<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhvep8QVoqGty8qIHDIoaqMkRJU1on_frsk7t3bhArw_h32xNTQwAwMS3uNBrEdsM2f10wYCb0N7NnQWyPaLBlAcSAiReQ2BSm6_jpmpV0irtt2UCzaGoFWfjWYWq-uNjxc-IwHt0PdviA/s1600/3.1.4_fungi_2.png" imageanchor="1" style="clear: right; margin-bottom: 1em; margin-left: auto; margin-right: auto;"><img border="0" height="131" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhvep8QVoqGty8qIHDIoaqMkRJU1on_frsk7t3bhArw_h32xNTQwAwMS3uNBrEdsM2f10wYCb0N7NnQWyPaLBlAcSAiReQ2BSm6_jpmpV0irtt2UCzaGoFWfjWYWq-uNjxc-IwHt0PdviA/s200/3.1.4_fungi_2.png" width="200" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;">Figure 5:<a href="http://www.microbiologyonline.org.uk/about-microbiology/introducing-microbes/fungi"> Electron micrograph of <i>S. cerevisiae</i></a></td></tr>
</tbody></table>
<br />
"The <b>wet milling</b> process is more capital- and energy intensive, as the grain must first be separated into its components, including starch, fiber, gluten, and germ. The germ is removed from the kernel and corn oil is extracted from the germ. The remaining germ meal is added to fiber and the hull to form corn gluten feed. Gluten is also separated to become corn gluten meal, a high-protein animal feed. In the wet milling process, a starch solution is separated from the solids and fermentable sugars are produced from the starch. These sugars are fermented to ethanol. Wet mill facilities are true “biorefineries”, producing a number of high-value products. In the <b>dry grind</b> process, the clean corn is ground and mixed with water to form a mash. The mash is cooked and enzymes are added to convert starch to sugar. Then yeast is added to ferment the sugars, producing a mixture containing ethanol and solids. This mixture is then distilled and dehydrated to create fuel-grade ethanol. The solids remaining after distillation are dried to produce distillers’ dried grains with protein and are sold as animal feed supplement."<br />
<br />
It is in the fermentation process that <i>S. cerevisiae</i> uses the released glucose in the respiratory process of glycolysis. Remember back to your school biology? Glucose is broken down to pyruvate with the coincident production of the energy rich molecule ATP. Pyruvate can have many fates but one of them that occurs under anaerobic conditions is the production of ethanol. In animals cells lactic acid is produced instead of ethanol. But we have a problem. In both wet mill and dry grind conditions, harsh conditions are used - low pH and accumulating ethanol. Glycoamylase, one of the enzymes that is used to converts starch into glucose works well in a pH of 4.5 and is continuously added during the fermentation process to ensure that all startch is converted to glucose. Now <i>S. cerevisiae</i> is one of the bugs I have personal experience with. Generally a neutral to slightly acidic pH (between pH 5-7) is best for growth and optimum temperatures were 30-37 degrees Celsius depending on the experiment. According to Bohast and Schlicher, 2005, fermentation occurs at 32 degress Celsius (no problem there) but the pH can sink to below 4. Ethanol concentration reach between 10-12% which is toxic and osmotic pressure is also a problem. How do the industrial yeast manage? <br />
<br />
<span style="color: #38761d;"><b>Engineering yeast for biofuel production</b></span><br />
<span style="color: #38761d;"><span style="color: black;">To enable the yeast to be at maximum efficiency for converting glucose to ethanol, many changes can be made to its genome. <i>S. cerevisiae</i> is a model organism, meaning that it is widely used as a model for studying genetic and protein interactions from which inferences can be made about similar genes and proteins in higher eukaryotic cells such as our own. The genetic manipulation of <i>S. cerevisae</i> is well studied and has a wide repertory of molecular techniques which are much easier to perform than those necessary for mammalian cells. So, what has been done to allow <i>S. cerevisiae </i>to cope with high ethanol production (here's where it gets a bit more technical....)</span></span><br />
<span style="color: #38761d;"><span style="color: black;"></span></span><br />
<span style="color: #38761d;"><span style="color: black;">Increased tolerance to stress is a complex metabolic process and it is not usually the case that you can alter one gene and not affect anything else. Having said this, single gene manipulations have had some success. For example, increased expression of single amino acid transport systems have been shown to have protective effects and overexpression of the amino acid tryptophane permease genes (<i>TRP1-5)</i> have resulted in improved ethanol tolerance with little negative impact to growth. Other single manipulations of genes involved in uracil and galactose synthesis have also improved ethanol tolerance.</span></span><br />
<span style="color: #38761d;"><span style="color: black;"></span></span><br />
<span style="color: #38761d;"><span style="color: black;">Examples of other approaches that affect many genes are directed evolution and global transcription machinery engineering (gTME). In directed evolution, yeast is exposed to fermentation conditions, cells that survive are grown up and rexposed to fermentation conditions. Different batches are made testing for high ethanol and heat resistance for example. In this way, advantageous mutations accumulate. Increases in ethanol tolerance of 62 fold and heat tolerance of 89 fold have been reported (Zhao and Bai, 2009). gTME is the introduction of a randomly mutated copy of a transcription factor that controls transcription of a large set of genes. The transcription factor will then alter the transcriptional controls depending on where in the genome it is introduced. Subjecting the engineered yeast to fermentation conditions will result in the survival of those yeast cells with improved ethanol tolerance. For example - 69% more ethanol production has been reported on a laboratory scale using this method (Zhao and Bai, 2009). </span></span><br />
<br />
<span style="color: #38761d;"><span style="color: black;">Another rather impressive aspect is the control of flocculation. Flocculation is the tendency of yeast to clump together. Self flocuclation is controlled by expression of certain genes and, by varying the stirring speed in the fermentor, the size of the floc can be also be controlled. Zhao and Bai, 2009 report on a study where the optimal floc size was found to be 300 um (micrometers) producing the highest cell viability when subjected to ethanol shock with concomitant improved ethanol yield. </span></span><br />
<span style="color: #38761d;"><span style="color: black;"></span></span><br />
<span style="color: #38761d;"><span style="color: black;">These are just some examples of how the yeast genetics can be manipulated to improve ethanol yield and you can see how industrial yeast ends up being very different from the yeast used to brew beer or bake bread and also demonstrates the flexibility of this micro-organism. </span></span><br />
<br />
<span style="color: #38761d;"><b>Conclusion</b></span><br />
<span style="color: #38761d;"><span style="color: black;">Here, I have talked about the use of corn as a feedstock for bioethanol and we have seen that it is likely not a long term solution to our energy woes. Several second generation biofuels are now being made from other feedstocks and with other micro-organisms. The use of corn is only the tip of the iceberg in terms of what is possible for the environmentally friendly production of fuel. Wait up for my next post to read about this!</span></span><br />
<br />
<a href="http://chemistry.about.com/od/chemistryglossary/a/octanenumberdef.htm"><b><span style="color: #38761d;">Definition of octane number</span></b></a>: Octane number (or rating) is a value used to indicate the resistance of a motor fuel to knock. Octane numbers are based on a scale on which isooctane is 100 (minimal knock) and heptane is 0 (bad knock).
<b> </b><br />
<b>Examples:</b> A gasoline with an octane number of 92 has the same knock as a mixture of 92% isooctane and 8% heptane.<br />
<a href="http://chemistry.about.com/cs/howthingswork/a/aa070401a.htm"><b>Knock</b></a>: compressed gasoline-air mixtures have a tendency to ignite prematurely rather than burning smoothly. This creates engine <i>knock</i>, a characteristic rattling or pinging sound in one or more cylinders <br />
<br />
<b><span style="color: #38761d;">References</span></b>
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<a href="http://draft.blogger.com/null" name="_ENREF_1"><b style="mso-bidi-font-weight: normal;"><span style="mso-ascii-font-family: Calibri; mso-hansi-font-family: Calibri; mso-no-proof: yes;">Bai, F. W., W. A. Anderson and M. Moo-Young</span></b></a><span style="mso-bookmark: _ENREF_1;"><span style="mso-ascii-font-family: Calibri; mso-hansi-font-family: Calibri; mso-no-proof: yes;"> (2008). "Ethanol
fermentation technologies from sugar and starch feedstocks." <u>Biotechnol
Adv</u> <b style="mso-bidi-font-weight: normal;">26</b>(1): 89-105</span></span><span style="mso-ascii-font-family: Calibri; mso-hansi-font-family: Calibri; mso-no-proof: yes;">.</span></div>
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<a href="http://draft.blogger.com/null" name="_ENREF_2"><b style="mso-bidi-font-weight: normal;"><span style="mso-ascii-font-family: Calibri; mso-hansi-font-family: Calibri; mso-no-proof: yes;">Bothast, R. J. and M. A. Schlicher</span></b></a><span style="mso-bookmark: _ENREF_2;"><span style="mso-ascii-font-family: Calibri; mso-hansi-font-family: Calibri; mso-no-proof: yes;"> (2005). "Biotechnological processes for
conversion of corn into ethanol." <u>Appl Microbiol Biotechnol</u> <b style="mso-bidi-font-weight: normal;">67</b>(1): 19-25.</span></span><span style="mso-ascii-font-family: Calibri; mso-hansi-font-family: Calibri; mso-no-proof: yes;"></span></div>
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<a href="http://draft.blogger.com/null" name="_ENREF_3"><b style="mso-bidi-font-weight: normal;"><span style="mso-ascii-font-family: Calibri; mso-hansi-font-family: Calibri; mso-no-proof: yes;">Solomon, B. D.</span></b></a><span style="mso-bookmark: _ENREF_3;"><span style="mso-ascii-font-family: Calibri; mso-hansi-font-family: Calibri; mso-no-proof: yes;"> (2010). "Biofuels and sustainability." <u>Ann N Y Acad Sci</u> <b style="mso-bidi-font-weight: normal;">1185</b>: 119-134.</span></span><span style="mso-ascii-font-family: Calibri; mso-hansi-font-family: Calibri; mso-no-proof: yes;"></span></div>
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<a href="http://draft.blogger.com/null" name="_ENREF_4"><b style="mso-bidi-font-weight: normal;"><span style="mso-ascii-font-family: Calibri; mso-hansi-font-family: Calibri; mso-no-proof: yes;">Zhao, X. Q. and F. W. Bai</span></b></a><span style="mso-bookmark: _ENREF_4;"><span style="mso-ascii-font-family: Calibri; mso-hansi-font-family: Calibri; mso-no-proof: yes;"> (2009). "Mechanisms of yeast stress tolerance and its manipulation
for efficient fuel ethanol production." <u>J Biotechnol</u> <b style="mso-bidi-font-weight: normal;">144</b>(1): 23-30.</span></span></div>
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<br />EmeraldBiologyhttp://www.blogger.com/profile/07468813270664433504noreply@blogger.com0tag:blogger.com,1999:blog-385372388482159249.post-91530987812534086722013-05-01T23:15:00.004-04:002013-05-09T22:21:53.847-04:00Farmed fish - pros and cons<br />
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<a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEiTfPuQthjA0KLs9tITMdwEcjmfrWYX_W6zFizVpccnt5DHx2Bbsd-dQYC_XRIxwq3FxPgcMi460azPyTSWCFsn81H_3NLAhm9JaFWG867SDglWuXV1FANAt3YKgq_n7rp6xngTt8TuM40/s1600/Jumping+fish.jpeg" imageanchor="1" style="clear: left; float: left; margin-bottom: 1em; margin-right: 1em;"><img border="0" height="142" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEiTfPuQthjA0KLs9tITMdwEcjmfrWYX_W6zFizVpccnt5DHx2Bbsd-dQYC_XRIxwq3FxPgcMi460azPyTSWCFsn81H_3NLAhm9JaFWG867SDglWuXV1FANAt3YKgq_n7rp6xngTt8TuM40/s320/Jumping+fish.jpeg" width="320" /></a></div>
<b><span style="font-size: small;"><span style="font-family: Arial,Helvetica,sans-serif;">A piece of information I really wanted to add to the last post (Plastic Oceans, April 2013) where I started off by talking about declining fish stocks are the pros and cons of eating farmed fish in order to preserve ocean fish stocks. An excellent review of this topic is given<span style="font-size: large;"> <a href="http://www.pbs.org/kqed/oceanadventures/episodes/amazon/indepth-fishfarming.html">here</a></span>. In summary, fish farming can be beneficial but needs to be carefully managed and regulated because feeding farmed fish, managing their waste and keeping them contained can all become risks to the environment and consumer. As with anything you buy at the supermarket, if you can afford the time and energy to research where and how it arrives in the store, you can at least make more informed decisions. </span></span></b><br />
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<b><span style="font-size: small;"><span style="font-family: Arial,Helvetica,sans-serif;">Next post coming next week: Fuel for biofuel.....</span></span></b><br />
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<br />EmeraldBiologyhttp://www.blogger.com/profile/07468813270664433504noreply@blogger.com0tag:blogger.com,1999:blog-385372388482159249.post-58667384380494900392013-04-07T11:37:00.000-04:002013-09-20T22:25:07.112-04:00Plastic oceans<table cellpadding="0" cellspacing="0" class="tr-caption-container" style="float: right; margin-left: 1em; text-align: right;"><tbody>
<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEiBlthF1R64MWkJq3ZOdRd4VE7Ql_WOQB1F3KxR2YHV4wIxpwXN3DmhUIjajznOg4mHpTFrof6iPe3jO0tUnlbljbACif4ZRjNHRWXKX8WCpNZUogX6Ihh_OlzKGvIUC9IoXOR4MPNqYsU/s1600/Blog-%234-artwork.jpg" imageanchor="1" style="clear: right; margin-bottom: 1em; margin-left: auto; margin-right: auto;"><img border="0" height="273" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEiBlthF1R64MWkJq3ZOdRd4VE7Ql_WOQB1F3KxR2YHV4wIxpwXN3DmhUIjajznOg4mHpTFrof6iPe3jO0tUnlbljbACif4ZRjNHRWXKX8WCpNZUogX6Ihh_OlzKGvIUC9IoXOR4MPNqYsU/s320/Blog-%234-artwork.jpg" width="320" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;">Figure 3. Bacteria and ocean plastic. Artwork: <a href="http://www.behance.net/MarkWSlater" target="_blank">Mark W. Slater</a></td></tr>
</tbody></table>
You may have heard, lately, that fish stocks are rapidly declining. I thought this was something new, as in general knowledge in the last couple of years. Well, its not. In 2003, the National geographic published an article stating that fish stocks had declined by <b>90%</b>. 90%. An article in 2011 by Juan-Jordá et al, (1) looked at tuna and its relatives and show a 60% decline in the last 50 years. Clearly, dear Readers, there is a problem and its pretty much slamming us in the face. Apart from overfishing, our old frenemy plastics has a large part to play. <br />
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What happens to plastic that enters the ocean? That plastic beach ball I let go of on holiday when I was 10 and that bobbed out to sea before we could get a hold on its slippery surface. Plastic used by the fishing industry, plastic accidentally or purposely dumped in the ocean, plastic from land use that finds it way from rivers to the ocean, dumping from commercial liners (to name a few) all make up the debris. The plastic that floats is swept around by the oceans currents and collects in gigantic slow moving pools including the 5 major oceanic gyres. The orgnanisation "5 gyres" has a great <a href="http://5gyres.org/" target="_blank">website</a> showing the location of the gyres and how the plastic gets there. The debris is not visible from space as it is dispersed and often below the surface of the water but the concentration of plastic, chemical sludge and other debris in the open sea (also called the pelagic zone) is much higher than in non-gyre areas.<br />
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<table align="center" cellpadding="0" cellspacing="0" class="tr-caption-container" style="margin-left: auto; margin-right: auto; text-align: center;"><tbody>
<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgJIP6AUNaUuP1kzTbUUthzyscyABRBU7xpaUEHTHe0GRrkwL7-Kxn5Mo98MmB5pe09cjslAymXO33NfBngzBXZiLPHrBCXtuDzsT7Q5r9pu-jeXP1CMajEKXuXoNbCI23dNFxrU_xV7GM/s1600/blog4.jpg" imageanchor="1" style="margin-left: auto; margin-right: auto;"><img border="0" height="406" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgJIP6AUNaUuP1kzTbUUthzyscyABRBU7xpaUEHTHe0GRrkwL7-Kxn5Mo98MmB5pe09cjslAymXO33NfBngzBXZiLPHrBCXtuDzsT7Q5r9pu-jeXP1CMajEKXuXoNbCI23dNFxrU_xV7GM/s640/blog4.jpg" width="640" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;">Figure 1. Potential pathwys of transportation of microplastics and its biological interactions (6)</td></tr>
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Once in the water, numerous evils result (Figure 1). First plastic is weakened and broken down by several types of degradation: biodegradation, photodegradation (action from sunlight), thermooxidative degradation (slow oxidative breakdown at moderate temperatures) and hydroysis through reaction with water<span style="font-size: small;"> (<span style="font-size: small;">2)</span></span>. It ends up breaking down further and further until it reaches sandsized and then microscopic particles. At this point it is in the neuston layer of the ocean which is the region at the surface and just below the surface of the water. Neuston can also be used to describe organisms that live in this region. For example, a <a href="http://en.wikipedia.org/wiki/Water_strider" target="_blank">water strider (UK: skater)</a> is a neustonic organism. Neustonic plastic in the garbage patches reaches densities up to 7 fold higher than zooplankton - the small organims that drift in the ocean and are found at the start of the food chain. <br />
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You can easily imagine the danger to wild life of entangling fishing line or an ingested bottle cap. Study of birds, fish and other aquatic organisms find that they are loaded with plastic (Figure 2)(3,4,5). If this is the consequence of larger organisms eating plastic we can see by eye, what happens to the broken down plastic that we need a magnifying glass or microscope to see? Microplastics are defined as plastic particles less than 5mm in size (6) and of course even small organisms can eat these. The authors of the article in (7) have conducted a study on the size, mass and type of plastic they found in testing 748 samples from the western North Atlantic Ocean and 88% was less then 10mm long. Smaller organisms can eat these particles and they in turn can be eaten by organisms further up the food chain. What happens to the eaten plastic? For the larger organisms such as fish, birds and even whales it can be lethal [Figure 2, (1-6)]. But what about microplastics and - wouldn't they just pass straight through? As we've seen in
the previous posts, during plastic manufacture, chemicals are added to
change the properties of the the plastic to suit their end products or
to make them more biodegradeable. Additionally, monomers that were not
properly incorporated during polymer production, chemicals that result from breakdown and
chemicals unrelated to the manufacture of the plastic but included as
part of the product (e.g. detergents in household cleaners, engine oil etc) will also
end up in the water - they are known as persistent organic pollutants
(POPs) and can by highly toxic (2). Some of these chemicals have been
found to adsorb to microplastics and therefore will also end up in
organisms and probably become part of their cellular make up and work
their way up the food chain [<a href="http://5gyres.org/" target="_blank">5 gyres website</a>; (2)] and eventually, one would assume getting passed on to us.<br />
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<table cellpadding="0" cellspacing="0" class="tr-caption-container" style="float: left; text-align: left;"><tbody>
<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgcbEIE9_Vzggb89NYHG17YiKsGg3BD992H4MLidauTKvp7Fz1P0WFbWl6voVosw26LDcgTpeYUCStMNtLHItDDp-xUaZiuBSeh3LD3_zy3jrZwRtdFxCgS-_5h-f_j05Vc_Gb_OFCfMIw/s1600/fetchObject.png" imageanchor="1" style="clear: left; margin-bottom: 1em; margin-left: auto; margin-right: auto;"><img border="0" height="320" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgcbEIE9_Vzggb89NYHG17YiKsGg3BD992H4MLidauTKvp7Fz1P0WFbWl6voVosw26LDcgTpeYUCStMNtLHItDDp-xUaZiuBSeh3LD3_zy3jrZwRtdFxCgS-_5h-f_j05Vc_Gb_OFCfMIw/s320/fetchObject.png" width="224" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;">Figure 2. Dead Laysan albatross chick with plastic in its stomach (1).</td></tr>
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I'm only scratching the surface of this topic and since my topic in this blog has so far been micro-organsims but lets get back on task: do micro-organisms have anything to do with microplastics? The straight answer is yes but not much. Of the 4 types of degradation mentioned above, biodegradation is the least contributory. It is certain that microplastics are colonized by micro-organisms (Figure 3) but they have little impact on their generation and how much they have to do with their further breakdown has not been studied - as far as I can see. They could of course feed off POPs that adsorb to the plastics and contribute to the chemical status of these as they exist in the oceans or are eaten by other organisms. There is some suggestion that the bacteria that colonize ocean plastic come from the <i>Vibrio</i> Genus (<a href="http://www.microbemagazine.org/index.php?option=com_content&view=article&id=3715:marine-microbes-not-much-help-degrading-ocean-floating-plastics&catid=860&Itemid=1162" target="_blank">see this link for a summary and reference</a>), which is also the Genus that causes cholera. Whether or not the species of Vibrio is pathogenic remains unknown. <br />
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Considering how much plastic we use for the amount of time we have used it and our careless manners with regard to our planet, there must be a quite a bit of plastic in the ocean. I haven't been able to get a substantiated figure but according to <a href="http://sanfrancisco.cbslocal.com/2011/09/12/good-question-how-much-plastic-garbage-is-in-the-ocean/" target="_blank">CBS</a>, Jim Oswald of the Marine Mammal Center in San Francisco, California, USA says 300 billion pounds or 150 million tons (or 43 million elephants!).<br />
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Is it all doom and gloom and no fish and chips? Is there anything we can do about it? YES - try to use less plastic! Much easier said then done but think about it. The best form of being green is to reduce consumption. Recycling is good but if there's no demand for your recycle material, it will still end up in a landfill or incinerator. Reusing is good too but reducing is best. I am starting to consider how I can reduce my plastic consumption. For example instead of buying a plastic tub of hummus to take to a gathering, I could make it myself with cans (tins) of garbanzo beans. I don't need to put my fruit and veggies in little plastic bags at the supermarket I shop at. I can search out farmers markets and bring my own reusable bag. I was searching the internet and came across Beth Terry who blogs about living without or very little plastic on her <a href="http://myplasticfreelife.com/" target="_blank">my plastic free life</a>. Would you take a look and adopt some strategies for reducing the plastic in your life and maybe, just maybe reducing your chances of being part of marine life destruction. All life is linked - if they suffer, we suffer, even if you only feel it as no tuna in the supermarket. <br />
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<span style="color: #274e13;"><b>References. </b></span><br />
1. Juan-Jordá MJ, Mosqueira I, Cooper AB, Freire J, Dulvy NK. 2011 Global
population trajectories of tunas and their relatives. Proc Natl Acad Sci
USA. Dec 20; 108(51): 20650-5 <br />
2.<i> </i>Andrady L. Microplastics in the marine environment, 2011, Marine Pollution Bulletin, 62, 1596-1605.<br />
3. Young LC, Vanderlip C, Duffy DC, Afanasyev V, Shaffer SA (2009) Bringing
Home the Trash: Do Colony-Based Differences in Foraging Distribution
Lead to Increased Plastic Ingestion in Laysan Albatrosses. PLoS ONE
4(10)<br />
4. Mrosovsky, N, Ryan GD, James MC, Leatherback turtles: the menace of plastic. 2011<span role="menubar"> Proc Natl Acad Sci U S A.</span> Dec 20;108(51):20650-5.<br />
<div class="cit">
5. Boerger CM, Lattin GL, Moore SL, Moore CJ. 2010<span role="menubar"> Mar Pollut Bull.</span> <span class="highlight">Plastic</span> ingestion by planktivorous <span class="highlight">fishes</span> in the North Pacific Central Gyre. Dec; 60(12):2275-8<br />
<div class="cit">
6. Wright S.L, Richard C. Thompson RC, Galloway TS. 2013 The physical impacts of microplastics on marine organisms: A review. Environmental Pollution. In press.<br />
7. M<span style="font-size: small;">orét-Ferguson S, Law KL, Proskurowski G, Murphy EK, Peacock EE, Reddy CM</span>. 2010. The size, mass, and composition of plastic debris in the western North Atlantic Ocean. <span role="menubar">Mar Pollut Bull.</span> 2010 Oct;60(10):1873-8.<br />
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<a href="http://www.ncbi.nlm.nih.gov/pubmed?term=Mor%C3%A9t-Ferguson%20S%5BAuthor%5D&cauthor=true&cauthor_uid=20709339"></a><br />
<span style="color: #38761d;">__________________________________________________________________________________</span><br />
<span style="color: #38761d;"><b>Lifestyle magic: Reduce your plastic use.</b></span><br />
<span style="color: #38761d;"><span style="color: black;">Use reusable shopping bags (stash extra's in your car/backpack/briefcase, refill water bottles - metal, refill coffee cup (metal), buy from bulk bins for rice, grains etc, don't bag your fruit, go to farmer's markets if possible and bring your own bag. Write to your supermarket to reduce packaging. </span></span><br />
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<span style="color: #38761d;"><span style="color: black;"><br /></span> </span></div>
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EmeraldBiologyhttp://www.blogger.com/profile/07468813270664433504noreply@blogger.com0tag:blogger.com,1999:blog-385372388482159249.post-40795051276417860472013-02-28T15:15:00.001-05:002013-02-28T15:15:05.030-05:00This month has been extremely busy and more entertaining activities have had to be curtailed. I hope to post by the end of March. I'm sorry for the postponement. Keep being green!EmeraldBiologyhttp://www.blogger.com/profile/07468813270664433504noreply@blogger.com0tag:blogger.com,1999:blog-385372388482159249.post-66845705071771826422013-01-31T21:08:00.000-05:002014-01-26T21:46:52.556-05:00Plastic bugs<br />
<span style="color: #38761d;"><span style="font-family: Times,"Times New Roman",serif; font-size: small;"><b>PLASTIC DEGRADATION - NOT THE NITTY GRITTY</b></span></span><br />
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<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEi7iiwEBDBA64r2BeKEmD9hLK_MqW7ZfI3rrAGvqNOX0X3ZNMMhAsewzh9TEeEcxe8RKnh600rIqL1mDHo7cj9LcAwRfCL04su-LQKDBEbTbUEaUQX4I9W9x32WVo4joUiaV4oL5iP92AQ/s1600/blog-3.jpg" imageanchor="1" style="clear: right; margin-bottom: 1em; margin-left: auto; margin-right: auto;"><img border="0" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEi7iiwEBDBA64r2BeKEmD9hLK_MqW7ZfI3rrAGvqNOX0X3ZNMMhAsewzh9TEeEcxe8RKnh600rIqL1mDHo7cj9LcAwRfCL04su-LQKDBEbTbUEaUQX4I9W9x32WVo4joUiaV4oL5iP92AQ/s400/blog-3.jpg" height="400" width="285" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;">Figure 1. Bacteria producing PHA. Art by <a href="http://www.behance.net/MarkWSlater" target="_blank">Mark W. Slater</a></td></tr>
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<span style="font-family: Times,"Times New Roman",serif; font-size: small;">Here's where I thought we would get into some real biochemistry to explore the pathways involved in breaking down plastics. Turns out, the material gets a little heavy f<span style="font-size: small;">or the scope of this blog</span> (lots of experimental co<span style="font-size: small;">nditions and little consensus) </span>so there<span style="font-size: small;"> wi</span>ll be a little biochemistry and then we are going to turn to properly biodegradable plastics produced by micro-organisms. </span><span style="font-size: small;">Lets first return to my <span style="font-size: small;">favorite</span> plastic, polyurethane and specifically the polyester type of polyurethane (PU) that is more amendable to bio-degradation (see the previous blog for help with understanding the why!). </span><br />
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<span style="font-family: Times,"Times New Roman",serif; font-size: small;">It was evident from the beginning of polyurethane production and use, that the material was susceptible to fungal degradation. Tests were done<span style="font-size: small;"> with pur<span style="font-size: small;">ified enzymes and </span></span>in soil <span style="font-size: small;">where<span style="font-size: small;"> </span></span>fungal communities were discovered to have a number of PU degrading species. However the best results were obtained when a biostimulant was added such as Impranil DNT, a readily <span style="font-size: small;">digestible</span> form of PU<span style="font-size: small;"> - sort of like baby formula for fungi</span>. The addition of cultures of known PU biodegrading fungi <span style="font-size: small;">wa<span style="font-size: small;">s</span> </span>also helpful in increasing biodegradation of PU by native soil fungi. </span><span style="font-family: Times,"Times New Roman",serif; font-size: small;"><span style="font-size: small;">The enzyme<span style="font-size: small;">s were <span style="font-size: small;">thought</span> to be extracellu<span style="font-size: small;">lar (i.e. pumped out of the fun<span style="font-size: small;">gus) and to be</span></span> a com<span style="font-size: small;">bination of <span style="font-size: small;">ones that could cut bonds in the mid<span style="font-size: small;">dle of the polymer at rand<span style="font-size: small;">om pos<span style="font-size: small;">itions <span style="font-size: small;">(</span></span></span></span></span></span></span></span></span>endo<span style="font-size: small;">enzymes</span>) and those <span style="font-size: small;">that chopped of<span style="font-size: small;">f <span style="font-size: small;">monomers from the end of the polymer chain <span style="font-size: small;">(</span></span></span></span>exoenzy<span style="font-size: small;">mes</span>)<span style="font-size: small;">.<span style="font-size: small;"> </span></span>In general, an additive like yeast extract also needed to be added as a carbon source to creat<span style="font-size: small;">e more extensive degradation,<span style="font-size: small;"> though fungi that could <span style="font-size: small;">utilize PU as the sole carbon source were <span style="font-size: small;">identified</span></span></span></span> <span style="font-family: Times,"Times New Roman",serif; font-size: small;"><span style="font-family: Times,"Times New Roman",serif;">(Howard et al., 2012)</span></span><span style="font-size: small;">. <span style="font-size: small;"><span style="font-size: small;"><span style="font-size: small;"> </span></span></span>Recently, an endo<span style="font-size: small;">phytic fungus</span> from the Amazon rainforest has been identified that is able to efficiently use PU alone as a carbon source without additives </span><span style="font-size: small;"><span style="font-size: small;"><span style="font-size: small;"><span style="font-size: small;"><span style="font-size: small;"> </span></span></span>(Russell et al., 2011)</span>. Although this is promising, it has as only been tested in small scale laboratory experiments (w<span style="font-size: small;">ith I<span style="font-size: small;">mpranil DN<span style="font-size: small;">T)</span></span></span> and <span style="font-size: small;">I'm not sure how it compares with th<span style="font-size: small;">e fungi that were identi<span style="font-size: small;">fied earlier (Howard, 2012)</span></span></span>. </span><br />
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<span style="font-family: Times,"Times New Roman",serif; font-size: small;">On the bacterial side, several species have been identified as degrading PU. Enzymes from the <i>Pse</i><i>udom</i><i>onas</i> genus have been characterized and fall into a class that resemble lipolases<span style="font-size: small;"> </span>which are enzymes that degrade lipids (fats). Interestingly, when the sequences of the genes are compared to each other and to other lipolases, it was shown that they come from genetically diverse backgrounds and do not have a common parent. This suggests that several branches of the evolutionary tree thought up the same idea at different time points<span style="font-size: small;"> (Howard, 2012). </span> </span><br />
<span style="font-size: small;"><br /></span>
<span style="font-family: Times,"Times New Roman",serif; font-size: small;">However, all these test have been done under laboratory like conditions on a small scale and the general impression from the literature is that most plastic that is currently produced cannot be degraded sufficiently efficiently to make industrial efforts economical. Efforts are therefore turning towards more ecofriendly biodegradable plastics and also to plastics produced from renewable sources (biopolymers) rather than from petrochemicals. So lets leave the degradation of plastics and turn to plastic biosynthesis. </span><br />
<span style="font-size: small;"><br /></span>
<span style="color: #38761d;"><span style="font-size: small;"><b>PLASTIC FACTORIES</b></span></span><br />
<table cellpadding="0" cellspacing="0" class="tr-caption-container" style="float: left; margin-right: 1em; text-align: left;"><tbody>
<tr><td style="text-align: center;"><span style="font-size: small;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgVb_O4Z1AnmookDZk3fSOQgkY_q-Fp5WKZ5ER5EDgol3lYlTKS070_NG8RZ5V20xpCcH4lzN2hTY8SWG3rSQrZ4z2aAAzA449RO58vj-BDppCnVZf6VVvjxKqA1hXuAx6ZHQpok9mmaIs/s1600/PHA+in+bacteria.jpg" imageanchor="1" style="clear: left; margin-bottom: 1em; margin-left: auto; margin-right: auto;"><img border="0" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgVb_O4Z1AnmookDZk3fSOQgkY_q-Fp5WKZ5ER5EDgol3lYlTKS070_NG8RZ5V20xpCcH4lzN2hTY8SWG3rSQrZ4z2aAAzA449RO58vj-BDppCnVZf6VVvjxKqA1hXuAx6ZHQpok9mmaIs/s320/PHA+in+bacteria.jpg" height="268" width="320" /></a></span></td></tr>
<tr><td class="tr-caption" style="text-align: center;"><span style="font-size: small;">Figure 2. PHA granules in bacterium (Kunasundari and Sudesh, 2011)</span></td></tr>
</tbody></table>
<span style="font-size: small;">Since we are in the business of investigating what micro-organisms can do for us, is it really true that bacteria or fungi can make plastic? YES IT IS! Many <b>bio</b>polymers use micro-organisms in a fermentation step to breakdown a feedstock such as corn or mollases to produce a monomer. Examples of these plastic monomers include lactic acid used in polylactic acid (PLA) plastic and succinic acid used in Poly(butylene succinate adipate)( PBSA) (Wolf 2005). However, only one type of plastic [polyhydroxy alkanoates (PHA)] is produced directly by bacteria (Figure 1 and 2). Just like there any many types of PU or polyethylene, there are many types of PHA. Monomer units of different types of PHA are shown in figure 3. Polyhydroxybutyrate (PH<span style="font-size: small;">B/PH3B</span>) which is a PHA, was first discovered as far back as 1926 (Castilho et al, 2009), but because of the expense of production and the lack of technology and<span style="font-size: small;"> </span>the bliss of ignorance of the consequences of using oil for everything, the discovery languished. Recently with making our world into a rubbish pit by filling it with landfills, the finite supply of oil and the worsening consequences of churning out CO<span style="font-size: xx-small;">2</span>, much more effort has been devoted to research into bringing down the costs of bioplastic production. </span><br />
<span style="font-size: small;"> </span><br />
<table align="center" cellpadding="0" cellspacing="0" class="tr-caption-container" style="float: right; margin-left: 1em; text-align: right;"><tbody>
<tr><td style="text-align: center;"><span style="font-size: small;"><a href="http://upload.wikimedia.org/wikipedia/commons/thumb/2/24/Polyhydroxyalkanoates.png/800px-Polyhydroxyalkanoates.png" imageanchor="1" style="clear: left; margin-bottom: 1em; margin-left: auto; margin-right: auto;"><img alt="File:Polyhydroxyalkanoates.png" border="0" src="http://upload.wikimedia.org/wikipedia/commons/thumb/2/24/Polyhydroxyalkanoates.png/800px-Polyhydroxyalkanoates.png" height="197" width="400" /></a></span></td></tr>
<tr><td class="tr-caption" style="text-align: center;"><span style="font-size: small;">Figure 3. Monomer units of different PHAs</span></td></tr>
</tbody></table>
<span style="font-size: small;">As we are environmentally focused (reduce, reuse, recycle) lets first look at the use of inexpensive feedstocks as one of the aspects for bringing down production costs of PHA. Feedstock can either be liquid and used in submerged fermenation processes (SMF) or from more solid material used in solid state fermentation (SSF) processes. SMF is where the liquid feedstock is mixed in with the bacteria in one large agitating container. A few SMF feedstocks include molasses and sugar cane liquor (by-products of sugar production), sugar (which while not as cheap as molasses is less expensive than glucose), starch based substances such as wheat pearlings (the bran husk from the wheat kernel that is removed by rollers in a process known as pearling), cellulosic materials (e.g. sugar cane bagasse hydrolysate; the leftovers from sugar cane stalks after the sugar has been removed), hemicellulosic substances (e.g. xylose; a monosaccharide in hemicellulose found for example in corn husks and wood pulping byproducts) and whey based waste material (a large and mostly wasted resource from the dairy industry). SMF feedstocks also include organic waste matter such as swine waste liquor, malt waste from spent barley, millet refuse from brewing brewing and waste from olive oil mills. There is therefore great potential to couple some of these industries with PHA production. SSF feestocks on the other hand, used moist solid particles in substrate beds where the feedstock is the support surface (i.e. the surface on which the bacteria grow). SSF is a potential alternative for disposal of agricultural waste such as rice bran, cassava bagasse and cakes from vegetable oil extraction. Instead of needing to dispose of these via incineration or landfill, they could become an extra source of revenue. </span><br />
<span style="font-size: small;"><br /></span>
<span style="font-size: small;">PHA productivity in grams of PHA per kilogram feedstock per hour can range from 0.02 to 2.57 g/kg/h (Castilho et al, 2009). The top producer <span style="font-size: small;">in the article by Castilho et al., 2<span style="font-size: small;">009, </span></span>used recombinant <i>E. coli</i> containing PH<span style="font-size: small;">B</span> genes from the bacterium <i>A. latus</i> in a whey + salts + citric acid + trace metals SMF fed-batch reactor (read on for what this is!). The experiments were done by Ahn et al, 2000. <span style="font-size: small;">A r</span>ecombinant <span style="font-size: small;">micro-organism </span>means that <span style="font-size: small;">it</span> has an engineered genetic composition compared with its original state. In this case, a plasmid with <span class="st">PHB synthesis genes from the bacterium </span><i><span class="st">Alcaligenes</span> </i><i>latus </i>(now known as <i>Azahydromonas lata</i>) were added to the <i>E. coli</i> bacteria on a plasmid. A<a href="http://en.wikipedia.org/wiki/Plasmid" target="_blank"> plasmid</a> is a (relatively) small piece of circular DNA that contains all the necessary components for autonomous replication within the bacteria. Genes of choice can be inserted into the DNA of the plasmid and the plasmid can then be inserted into a new bacterium so that the effect of the genes can be studied. Certain strains of <i>E. co</i><i>li </i>are <span style="font-size: small;">laboratory</span> work horses, allowing the genes of more obscure, more finicky bacteria to be studied more easily. The autonomous replication property of a plasmid means that several copies of the plasmid exist within the bacteria and when the bacteria divides, some of them will remain in each daughter cell, ensuring that the bacteria retain the ability to <span style="font-size: small;">synthesize</span> PH<span style="font-size: small;">B</span>. </span><br />
<span style="font-size: small;"><br /></span>
<span style="font-size: small;">So now we have the <i>E. coli</i> PH<span style="font-size: small;">B</span> factory, what happens next<span style="font-size: small;">?</span> Ahn et al, 2000 use a fed batch reactor. This is a container with a stirring mechanism and probes by which the oxygen and pH can be monitored. At the beginning of fermentation, less than half the maximum volume of culture <span style="font-size: small;">wa</span>s added. As the lactose concentration f<span style="font-size: small;">e</span>ll (lactose is the sugar carbon source utilized from the whey by the bacteria) the pH r<span style="font-size: small;">ose</span> and more whey feedstock <span style="font-size: small;">was</span> added to bring the pH back down, thus increasing the volume in the reactor. The authors found that the oxygen concentration had a significant role in PHB productivity. The growth of the bacteria could be divided into 2 phases; a fast growth phase where high (40%) oxygen was optimal to achieve high bacterial density and a slow growth phase with lower oxygen levels where PHB production increased. This makes sense as PHB is stored as an energy reserve during lea<span style="font-size: small;">n times like <span style="font-size: small;">low oxygen levels</span>. </span>They also found that PHB production was highest during oxygen lowering so that with a step wise decrease in oxygen concentration from 40% to 30% to 15%, the highest PHB productivity of 2.57g/kg/L was achieved. The only <span style="font-size: small;">disappointment</span> for me with this study, was that the whey feedstock was not obtained from a dairy but from bought whey powder<span style="font-size: small;"> so</span> the experiments <span style="font-size: small;">was</span> not as "real" world as <span style="font-size: small;">the</span> could have been. </span><br />
<span style="font-size: small;"><br /></span>
<span style="font-size: small;">After expression in bacteria, the PHA must be extracted and this is estimated to be up to 50% of the cost of PHA production (Fiorese et al, 2009). In comparison with biosynthesis, the extraction process has been much less researched. Kunasundari and Sudesh, 2011 provide a review. The extraction process consists of pretreatment of the collected bacteria to weaken the cell wall and cell membrane (for an understanding of bacterial cell structure, take a look <a href="http://micro.digitalproteus.com/morphology2.php" target="_blank">here</a>). Following pretreatment, an extraction process is used to isolate the PHA from the other cell components. Extraction has commonly been done with organic solvents such as chloroform and this clearly presents enviromental consequences as well as being uneconomical unless the solvents can be recycled. Fiorese et al, 2009 use 1,2-propylene carbonate in the extraction process which is a much less toxic solvent than something like chloroform and can also be recycled, thus reducing costs. Major factors of any extraction process are purity, the affect on the homogeneity and breakdown of the PHA (i.e. shortening of the plastic polymer molecules). The standards for these qualities will depend on the end product for which the PHA is to be used. </span><br />
<span style="font-size: small;"><br /></span>
<span style="font-size: small;">Other extraction procedures being investigated include chemical or enzymatic disruption and mechanical disruption. Each method has advantages and drawbacks. For example, enzymatic digestion involves mild conditions and therefore provide an environment for <span style="font-size: small;">a </span>high molecular weight, high homogeneity and pure end product. On the downside, enzymes are expensive to produce and have finite life times. Mechanical disruption is advantageous because no chemicals are involved, therefore no contaminants are added that ne<span style="font-size: small;">ed to be removed later </span>and there are no <span style="font-size: small;">added</span> polluting solvents to deal with. However, there is a high capital investment cost of the mechanical disruption machinery, processing times are long to ensure maximum disruption and scale up is difficult. As of 2009, PHA plastic <span style="font-size: small;">is betwe<span style="font-size: small;">en 3-1<span style="font-size: small;"><span style="font-size: small;">7 <span style="font-size: small;">times more e<span style="font-size: small;">xp<span style="font-size: small;">ensive to <span style="font-size: small;">produce than petro<span style="font-size: small;">chemical based plastics (Castilho et<span style="font-size: small;"> al<span style="font-size: small;">., 2009).</span></span></span></span></span></span></span></span></span></span></span></span><br />
<span style="font-size: small;"><br /></span>
<span style="font-size: small;"> Having said that<span style="font-size: small;">,</span> there are a couple of companies producing PHA industrially. Metabolix in Cambridge, MA, USA, produced 50,000 tons of its PHB product Mirel in alliance with ADM between 2004 and 2012 (it has since closed). <span style="font-family: Times,"Times New Roman",serif;"><span style="color: black;">"Meredian, Inc </span></span><span style="color: black;"><span style="font-family: Arial;"><span style="font-family: Times,"Times New Roman",serif;">has a pilot
plant in Georgia, USA that produces 13,600 metric tons per year of PHA.
Construction is beginning at the site for a plant that will produce
91,000 metric tons per year of PHA</span>.</span></span><span style="font-family: Times,"Times New Roman",serif;"><span style="color: black;">
Tianjin
Green Bioscience and DSM operate a 10,000 metric ton per year PHA plant
in China. Tianan Biologic Material Co. is boosting its capacity in China
to 10,000 metric tons per year. About a dozen other companies operate
lab or pilot scale PHA
facilities"(Smock, 2012).</span></span></span><br />
<span style="font-size: small;"><br /></span>
<span style="font-size: small;">Can PHA plastics be used in the same way as petroleum based plastics? Currently<span style="font-size: small;">,</span> the main application of PHA is used for making thin plastic films such as plastic bags and food packaging which is an application that otherwises uses polyethylene or polyurethane plastics. With further research, PHA plastics could also be used for other products such as tissue engineering materials, bioimplant
materials and<a href="http://en.wikipedia.org/wiki/Smart_materials" target="_blank"> smart materials</a> (Chen, 2010)</span>. <span style="font-size: small;">The major advantage of PHAs produced from biowaste, <span style="font-size: small;">especially</span> when coupled with a dairy or sugar production plant for example, is that they are easily biodegradable. When (yes I do say <i>when</i>) the cost of their production <span style="font-size: small;">is</span> brought down sufficiently and their properties altered<span style="font-size: small;">,</span> they could be a significant source of plastic. </span><br />
<span style="font-size: small;"><br /></span>
<span style="font-size: small;">While this post has been about PHA (mainly), biopolymers produced from waste products initially broken down by micro-<span style="font-size: small;">organisms</span> and then made into polymers through other processes, is also an alternative<span style="font-size: small;"> </span>technology. Some of these biopolymers are not <span style="font-size: small;">biodegradable</span>. In 2005, the European Commission provided an interesting (and long) report on the "Techno-economic Feasibility of Large scale Production of Bio-based Polymersin Europe" (Wolf, 2005). It provides exactly what the title says and generally comes out on the side of biobased plastics as necessary for the environment, company reputations (being green is good marketing) and for scientific innovation, highlighting that green technology is about much more the reducing our environmental impact and the safety of future generations - as if this wasn't enough! </span><br />
<br />
<span style="font-size: small;"><span style="font-size: small;">Next post: Feb 28th. Right now the subject is a toss up - more on PHA or heading to the ocean to look at our pl<span style="font-size: small;">astic pollution th<span style="font-size: small;">ere.</span></span></span> </span> <br />
<span style="font-size: small;"><br /></span>
<span style="font-size: small;"><u><b>References:</b></u></span><br />
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recombinant Escherichia coli with a highly concentrated wheysolution." <u>Appl.
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3624-3627.</span></span></div>
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and solid-state fermentation." <u>Bioresour Technol</u> <b>100</b>(23): 5996-6009.</span></span></div>
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<span style="font-size: small;"><span style="color: #38761d;"><a href="http://draft.blogger.com/blogger.g?blogID=385372388482159249" name="_ENREF_3"><b>Chen, G.-Q.</b></a>, Ed. (2010). <u>Plastics from Bacteria:Natural Functions and Applications
(Google eBook)</u>. Microbiology Monographs.</span></span></div>
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<span style="font-size: small;"><span style="color: #38761d;"><a href="http://draft.blogger.com/blogger.g?blogID=385372388482159249" name="_ENREF_5"><b>Howard, G</b>. (2012). Polyurethane
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Dantzler, D. Hickman, J. Jee, F. M. Kimovec, D. Koppstein, D. H. Marks, P. A. Mittermiller,
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<span style="font-size: small;"><span style="color: #38761d;">___________________________________________________________________________________</span></span><br />
<span style="color: #38761d; font-size: small;"><b>Lifestyle Magic:<span style="color: black;"> <span style="color: #38761d;">Unblocking your sink</span></span></b></span><br />
<span style="color: #38761d; font-size: small;"><span style="color: black;">The other day, I was confronted by a non-draining bathroom sink. I googled it up and read that spooning down a bit of baking soda followed by apple cider vinegar, does wonders. And it does. You don't even need a spoon. I poured baking soda down, followed by the vinegar and enjoyed the frothing reaction</span></span><span style="font-size: small;"><b>. </b><span style="color: black;">After several pours, the sink drained beautifully, not an ugly chemical in sight.</span></span><br />
<span style="font-size: small;"><br /></span>
<span style="font-size: small;">Tip about baking soda shampoo from last post. I found that my hair does best on 1.5 tablespoons of backing soda to 2 cups of water. Less or more gives a less glossy result. Your hair could be different! </span>
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</script>EmeraldBiologyhttp://www.blogger.com/profile/07468813270664433504noreply@blogger.com0tag:blogger.com,1999:blog-385372388482159249.post-58401283198483299842013-01-06T16:05:00.000-05:002014-01-26T21:51:03.720-05:00Microbial degradation of plastics<table cellpadding="0" cellspacing="0" class="tr-caption-container" style="float: right; margin-left: 1em; text-align: right;"><tbody>
<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEiuQZC-2MOSexuSMMizl1MumcEENp6QKhqEXlThY_P7k7pOGLnzSco5T8ZBKVbl5yYSeZgHULXn-zqjJ-raarQGs_4wckZWdMur2REvSVJQPq_H5fFtt8el50cS7RCE8mHFGfRIYsAm18o/s1600/Emerald+blog+%232.jpg" imageanchor="1" style="clear: right; margin-bottom: 1em; margin-left: auto; margin-right: auto;"><img border="0" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEiuQZC-2MOSexuSMMizl1MumcEENp6QKhqEXlThY_P7k7pOGLnzSco5T8ZBKVbl5yYSeZgHULXn-zqjJ-raarQGs_4wckZWdMur2REvSVJQPq_H5fFtt8el50cS7RCE8mHFGfRIYsAm18o/s320/Emerald+blog+%232.jpg" height="320" width="320" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;">Title figure: Mark W. Slater (<a href="https://www.facebook.com/NaturalLineStudio?ref=ts&fref=ts" target="_blank">Natural Line Studio</a>)</td><td class="tr-caption" style="text-align: center;"><br /></td><td class="tr-caption" style="text-align: center;"><br /></td><td class="tr-caption" style="text-align: center;"><br /></td></tr>
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<![endif]--><span style="font-size: small;">My first post covered general topics of the capabilities of micro-organisms with regard to the manufacture and destruction of plastics. Now for the beginning of the slightly more nitty gritty. I'll start off with what plastics are, followed by what makes them amendable (or not) to bio-degradation. Lastly we'll see if the technology is at a stage where it can be used on an industrial scale. </span><br />
<span style="font-size: small;">
</span><br />
<span style="font-size: small;"><b>What are plastics: </b></span><br />
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<table cellpadding="0" cellspacing="0" class="tr-caption-container" style="clear: left; float: left; margin-bottom: 1em; text-align: left;"><tbody>
<tr><td style="text-align: center;"><span style="font-size: small;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEiZV6aoN-7mLhZ3P1LcbZWuadLLeyLthgnJF-PbdJ7G4RXgKrhR_Ywd5eQqqNfnEpV5QBvOp0TsoVv-BBAXuzwvsB4G34kUjsM2kYZLovOndy7tY17NuZdL4o-vwXm9NES-KTVPshDpa7s/s1600/PE+monomer.jpg" imageanchor="1" style="clear: left; margin-bottom: 1em; margin-left: auto; margin-right: auto;"><img border="0" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEiZV6aoN-7mLhZ3P1LcbZWuadLLeyLthgnJF-PbdJ7G4RXgKrhR_Ywd5eQqqNfnEpV5QBvOp0TsoVv-BBAXuzwvsB4G34kUjsM2kYZLovOndy7tY17NuZdL4o-vwXm9NES-KTVPshDpa7s/s1600/PE+monomer.jpg" height="162" width="200" /></a></span></td></tr>
<tr><td class="tr-caption" style="text-align: center;"><span style="font-size: small;"><u>Figure 1A. Ethylene </u></span><br />
<span style="font-size: small;"><u>large ball - carbon atoms</u></span><br />
<span style="font-size: small;"><u>small balls - hydrogen atoms </u></span></td></tr>
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<table cellpadding="0" cellspacing="0" class="tr-caption-container" style="clear: left; float: left; margin-bottom: 1em; text-align: left;"><tbody>
<tr><td style="text-align: center;"><span style="font-size: small;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEiJFn04gH3tSwhNIdodv5jWHPLElk-rqLoXwgJ_jaOqG9lXYHhCBYTUsC9hxnxpLpWs3GOWdo2j6kOqOEOSYNwsVA5GM0rfbL_JMqKrakMOA1tBhR6xvSETc47aYLuMCKGtGtnNimd_-eg/s1600/polyethylene.jpg" imageanchor="1" style="clear: left; margin-bottom: 1em; margin-left: auto; margin-right: auto;"><img border="0" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEiJFn04gH3tSwhNIdodv5jWHPLElk-rqLoXwgJ_jaOqG9lXYHhCBYTUsC9hxnxpLpWs3GOWdo2j6kOqOEOSYNwsVA5GM0rfbL_JMqKrakMOA1tBhR6xvSETc47aYLuMCKGtGtnNimd_-eg/s1600/polyethylene.jpg" height="162" width="200" /></a></span></td></tr>
<tr><td class="tr-caption" style="text-align: center;"><span style="font-size: small;"><u>Figure 1B: Polyethylene</u></span><br />
<span style="font-size: small;"><u>hydrogen atoms not shown </u></span></td></tr>
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<tr><td style="text-align: center;"><span style="font-size: small;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjb23Rw8baB_E7FYtHtgxSkvCmFYoU03d_Vw2e7P61lk5vPAEQoPBx71K40UkK_wKV_nrPmsV2gQLJP2YeaDs5pzMxMOfSlrqfy_YfdAyaCj8rNLq3mLWMXcpU2zK7wbJ0HPLD6zF6aOZE/s1600/LDPE+ball+and+stick.jpg" imageanchor="1" style="clear: left; margin-bottom: 1em; margin-left: auto; margin-right: auto;"><img border="0" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjb23Rw8baB_E7FYtHtgxSkvCmFYoU03d_Vw2e7P61lk5vPAEQoPBx71K40UkK_wKV_nrPmsV2gQLJP2YeaDs5pzMxMOfSlrqfy_YfdAyaCj8rNLq3mLWMXcpU2zK7wbJ0HPLD6zF6aOZE/s1600/LDPE+ball+and+stick.jpg" height="161" width="200" /></a></span></td></tr>
<tr><td class="tr-caption" style="text-align: center;"><span style="font-size: small;"><u>Figure 2: low density polyethylene (LDPE)</u></span><br />
<span style="font-size: small;"><u>hydrogen atoms not shown </u></span></td></tr>
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<span style="font-size: small;">Plastic forms such a daily part of our lives – packaging, toys, electronics, furniture, clothing (fleece, waterproof clothing), shoes, building materials; the list is endless. And when you think of the different forms
plastic takes, the diversity is mind boggling<span style="font-size: small;">; t</span>hin and foldable, thick and rigid, filled with
air, thread like, crinkly, fun to stamp on (i.e. bubble wrap) etc. </span><br />
<br />
<span style="font-size: small;">Plastics can be divided into two types. 1. Thermoplastics and 2. Thermoset plastics.</span><br />
<br />
<span style="font-size: small;"><b>Thermoplastics</b> are formed from alkenes. The simplest alkene is ethylene (CH2=CH2) (also called ethene) and is shown in figure 1A. During polymerization the double bond between the two carbons is broken, resulting in a single bond between the two carbons and the use of the two halves of the second single bond to form two new single bonds with two new ethylene molecules on either side of the original ethylene. Thus a chain of ethylene molecules -(CH-CH)n- as in figure 1B is generated. This is called polyethylene or PE. Other thermoplastics are polyvinyl chloride and polystyrene. Modification of the monomer or mixing two o<span style="font-size: small;">r</span> more different monomers together in the chain, changes the properties of the end plasic such as its temperature tolerance or flexibility. Modi<span style="font-size: small;">fication of the chain <span style="font-size: small;">also changes the end plastic.<span style="font-size: small;"> F</span></span><span style="font-size: small;"></span></span>or example, by adding side chains to the central strain of PE, causes the chains to pack less tightly, which decrease density<span style="font-size: small;"> creating low density polyethylene (LDEP - figure 2, r<span style="font-size: small;"><span style="font-size: small;">representative</span> <span style="font-size: small;">illustration</span></span>) </span>and allows it to be stretched into thin sheets such as in your supermarket plastic bag. </span><br />
<br />
<span style="font-size: small;">The single carbon bond in thermoplastics is stable, resistant to degradation and hydrolytic cleavage which is the cleavage of chemical bonds by the use of water. Thermoplastics have been generally regarded as non-biodegradable. Unfortunately, they make up the majority of our plastic use. In 2004 thermoplastics made up 92% of the distributed plastic resins in the USA (ref 1). </span><br />
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<tr><td style="text-align: center;"><span style="font-size: small;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgSJyQfAmrkKF2cgzkPhMOBAGjNkdLumDijTivtBCyMON-k_C-xgRl8UvJ0r06CIk0uHjBxnLS-5LkXkZ2LF9pqqI-fEVuRnVpnRCphFvxv2Y4So2fJRbrtX8ZV6EAm3Hc3lZFvkBbhr4I/s1600/Amide+and+Ester+reactions.jpg" imageanchor="1" style="clear: left; margin-bottom: 1em; margin-left: auto; margin-right: auto;"><img border="0" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgSJyQfAmrkKF2cgzkPhMOBAGjNkdLumDijTivtBCyMON-k_C-xgRl8UvJ0r06CIk0uHjBxnLS-5LkXkZ2LF9pqqI-fEVuRnVpnRCphFvxv2Y4So2fJRbrtX8ZV6EAm3Hc3lZFvkBbhr4I/s400/Amide+and+Ester+reactions.jpg" height="295" width="400" /></a></span></td></tr>
<tr><td class="tr-caption" style="text-align: center;"><span style="font-size: small;">Figure 3 Ester and Amide reactions</span></td></tr>
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<br />
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<tr><td style="text-align: center;"><span style="font-size: small;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEg0uGuWKpq03BzXEuht02PaA79q-967w1FFtycDBNXDx5mh7cObjpQcDuLo_nRNL_0Pf08HDN8pCPFrRLB-Yz4zFDv9L9pYf7NO1f3QEgI7cEE3lisIXPCop0PcWTM_uN1FKYAbdr9Ipgs/s1600/urethane+reactin+simple.jpg" imageanchor="1" style="clear: right; margin-bottom: 1em; margin-left: auto; margin-right: auto;"><img border="0" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEg0uGuWKpq03BzXEuht02PaA79q-967w1FFtycDBNXDx5mh7cObjpQcDuLo_nRNL_0Pf08HDN8pCPFrRLB-Yz4zFDv9L9pYf7NO1f3QEgI7cEE3lisIXPCop0PcWTM_uN1FKYAbdr9Ipgs/s400/urethane+reactin+simple.jpg" height="325" width="400" /></a></span></td></tr>
<tr><td class="tr-caption" style="text-align: center;"><span style="font-size: small;">Figure 4 Urethane reaction</span></td></tr>
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<span style="font-size: small;">The second type of plastic are<b> thermoset plastics.</b> These plastics are made by joining an alcohol (R-OH) or amine (R-NH<span style="font-size: xx-small;">2</span>) with a carbonate group (R-C(O)OH) in a process known as condensation as a water molecule is released and an ester (-C(O)OC(H<span style="font-size: xx-small;">2</span>)-.or amide (-C(O)NC(H<span style="font-size: xx-small;">2</span>)-) is formed (figure 3). Examples of thermoset plastics are polyesters and polyurethanes. Urethane is little more complex as it involves the reaction of an isocynate group with an alcohol group to generate a urethane group (figure 4). You will notice that no water is produced and this is not a condensation reaction but the result is an amide<span style="font-size: small;">-</span>ester bond or urethane bond. <span style="font-size: small;">The small black dots in the structures <span style="font-size: small;"><span style="font-size: small;">represent</span> carbons and the R groups, <span style="font-size: small;">the rest of the molecule. R1 a<span style="font-size: small;">nd R2<span style="font-size: small;"><span style="font-size: small;"><span style="font-size: small;"> in <i>poly</i>urethane </span>would then need co<span style="font-size: small;">ntain </span>another isocy<span style="font-size: small;">anate and alc<span style="font-size: small;">o<span style="font-size: small;">hol reactive group attached <span style="font-size: small;">to <span style="font-size: small;">whatever structure that was inbet<span style="font-size: small;">ween such as an alky or <span style="font-size: small;">aryl group (figure 5). In th<span style="font-size: small;">is way<span style="font-size: small;">,</span></span> both ends <span style="font-size: small;">of <span style="font-size: small;">the molecu<span style="font-size: small;">le could react to form a </span></span></span></span></span></span></span></span></span></span></span></span></span></span></span></span><i>poly</i><span style="font-size: small;">urethane chain.<span style="font-size: small;"> From the differences in t<span style="font-size: small;">he R groups, <span style="font-size: small;">its apparent that the pol<span style="font-size: small;">yurethanes can encompass great diversity (ref 2). </span></span></span></span></span>The ester, amide or urethane link is susceptible to enzymes produced by bacteria and fungi and is thus biodegradable or at least more biodegradable than the thermoplastics. </span><br />
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<tr><td style="text-align: center;"><span style="font-size: small;"><img alt="File:Polyurethane.png" src="http://upload.wikimedia.org/wikipedia/commons/thumb/c/ca/Polyurethane.png/800px-Polyurethane.png" height="160" style="margin-left: auto; margin-right: auto;" width="400" /></span></td></tr>
<tr><td class="tr-caption" style="text-align: center;"><span style="font-size: small;">Figure 5. Polyurethane synthesis reaction with a di-isocyante (2 isocyanate groups) and a diol (2 -OH groups) </span></td></tr>
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<span style="font-size: small;"><b>Biodegradation</b></span><br />
<span style="font-size: small;">Now that we have some basic ideas about plastic, lets see how biodegradation works. We know that thermoset plastics, because of their chemical composition are biodegradeable. Does that mean that addition of a few choice micro-organisms will cause automatic meltdown? Not quite. Polyesters for example consist of two types: aliphatic and aromatic polyesters.<b> </b>Though there is some controversy, aliphatic polyesters are thought to be biodegradable but aromatic polyesters are not (ref 1). <span style="font-size: small;">Aromatic polyesters</span> contain six carbon rings and form plastics used to make carpet, clothing and soda bottles. Al<span style="font-size: small;">iphatic polyesters</span> are rather weak plastics and are used to make medical and scientific items such as tissue scaffolds and dissolving drug delivery systems. Polyurethane is a different type of plastic to polyester (though sometime refered to as the polyester <span style="font-size: small;">polyuret<span style="font-size: small;">hane) </span></span>and is used in many product<span style="font-size: small;">s</span> including furniture, paints and construction materials and its biodegradation has been shown to be due to the microbial destruction of the ester bonds (ref 2). Additional<span style="font-size: small;">ly, <span style="font-size: small;">when plastics are made, add<span style="font-size: small;">itives are blen<span style="font-size: small;">ded that improve their <span style="font-size: small;">envi<span style="font-size: small;">romental stability <span style="font-size: small;">such as <span style="font-size: small;">their re<span style="font-size: small;">si<span style="font-size: small;">stance to heat and light<span style="font-size: small;"> and these generally hinder biod<span style="font-size: small;">egr<span style="font-size: small;">adation. <span style="font-size: small;">In biodegr<span style="font-size: small;">adation research, ways in which plastic stab<span style="font-size: small;">ility can be maintained w<span style="font-size: small;">hile imp<span style="font-size: small;">roving susceptibility to mic<span style="font-size: small;">ro<span style="font-size: small;">-organisms are investigated. </span></span></span></span></span></span></span></span></span></span></span></span></span></span></span></span></span></span></span></span>Various ways have also been devised to improve the poor biodegradation of thermoplastics. Before micro-organisms can attack the long polymer chains, they need to be transformed into bite size pieces of 500 Da or less. As many plast<span style="font-size: small;">ics</span> are resistant to conditions that allow the chains to be broken up, <span style="font-size: small;">additives</span> called pro-oxidants are mixed in with the polymers that make the plastics hydrophilic and <span style="font-size: small;">catalyze</span> the breakdown of the hydrocarbon chains during photolysis or thermolysis (see t<span style="font-size: small;">itle figure above)</span>. </span><span style="font-size: small;"><span style="font-size: small;">Pro-oxidants consist of transient metal ions (e.g <span style="font-size: small;">cob<span style="font-size: small;">alt or mangan<span style="font-size: small;">ese) </span></span></span>added in the fo<span style="font-size: small;">rm of <span style="font-size: small;">an organic ligand complex<span style="font-size: small;">. <span style="font-size: small;">The chem<span style="font-size: small;">istry is beyo<span style="font-size: small;">nd the scope of this blog (for now!) but see ref 3 if you want to d<span style="font-size: small;">ig in<span style="font-size: small;">.</span></span></span></span></span></span></span></span> </span> In addition to pro-oxidants, other truly biodegradable material such as starch are added to the polymers and this enhances the biodegradation process. </span><br />
<br />
<span style="font-size: small;">What are the bacteria and fungi that are used to chew up
plastics once it is in a compatible form? Of course its variable! Wi<span style="font-size: small;">thout getting too specific about genus and species, <span style="font-size: small;">l</span></span>ets
look at two examples - the thermoplastic polyethylene and the thermoset
plastic polyurethane.</span><br />
<span style="font-size: small;"><b><br /></b></span>
<span style="font-size: small;"><b>Biodegradation of thermoplastic<span style="font-size: small;"> polyethylene</span></b></span><br />
<span style="font-size: small;">PE takes up to 1000 years to degrade in the environment and makes up 64% of the plastic mass produce each year globally (ref<span style="font-size: small;">. 4</span>). To put that in perspective 500,000,000 (500 billion) to 1,000,000,000,000 (1 trillion) plastic bags are used annually, world wide. And thats not counting the PE thats used to make all the other products. It threatens wildlife, domestic life, marine life and gets into the food chain as its broken down by the action of heat and sunlight. Only a fraction is recycled <span style="font-size: small;">(ref. 4)</span>. In<span style="font-size: small;"> fact<span style="font-size: small;">, <span style="font-size: small;">the EPA says that only 8% of all plastic is recycled! </span></span></span>As mentioned above, because of the lack of atoms that are subject to electrophilic or nucleophilic attack such as oxgen and nitrogen, PE is regarded as essentially resistant to microbial attack. Pro-oxidants incorporated into the PE during manufacture and resident mainly in pockets of the PE that are less crystalline and more amorphous, are then used post-consumer to break up the chains of PE using heat or light. The broken up chains are susceptible to<span style="font-size: small;"> microbial</span> digestion (<span style="font-size: small;"><span style="font-size: small;">see <span style="font-size: small;">t</span>itle figure <span style="font-size: small;">at top of blog</span></span>)</span>.<span style="font-size: small;"> </span>For e<span style="font-size: small;">xample, the bacteria </span><i>Rhodococcous rhodocrous<span style="font-size: small;">, </span></i><span style="font-size: small;">is</span><i> </i>part of a genus able to metabolise a diverse array of substances due to their robust physiology and large genome that incorporates an array of catabolic genes<span style="font-size: small;">. </span> <span style="font-size: small;">These are</span> genes that make products able to break down (catabolise) many different substances an<span style="font-size: small;">d this genus is used for<span style="font-size: small;"> other <span style="font-size: small;">environmental p<span style="font-size: small;">urposes as well as PE degre<span style="font-size: small;">dation (ref 5)</span></span></span></span></span>. A comprehensive table of the different bacterium tried is given in reference <span style="font-size: small;">3 and 4</span> and the authors admit that while consumption occurs in the pockets of PE broken up by pro-oxidant action, further, more complete degradation does not occur. Some success was met mixing pro-oxidant<span style="font-size: small;"> h</span>eat treated PE into compost where up to 70% conversion of PE to carbon dioxide was achieved, perhaps because diverse populations of bacteria are present able to produce a more diverse <span style="font-size: small;">set <span style="font-size: small;">of</span></span> enzymes and <span style="font-size: small;">thus </span>support each other<span style="font-size: small;">.</span> However from the review arti<span style="font-size: small;">cle<span style="font-size: small;"> (ref 4) </span></span>published this year, <span style="font-size: small;">it can be seen that while </span>some bio-degradation is possible<span style="font-size: small;">, we are a long way </span>from having a commercially viable process <span style="font-size: small;">f<span style="font-size: small;">or bio<span style="font-size: small;">-<span style="font-size: small;">degradation <span style="font-size: small;">of PE</span></span></span></span></span>. </span><span style="font-size: small;"><span style="font-size: small;"><span style="font-size: small;">F</span>or the small dent it can make in improving our current and future envi<span style="font-size: small;">ronment - <span style="font-size: small;">k</span></span></span>eep reaching for your canvas bags and avoid extra packaging wherever possible<span style="font-size: small;">.</span></span><br />
<span style="font-size: small;"><br /></span>
<span style="font-size: small;"><span style="font-size: small;"><b><span style="font-size: small;">Biode<span style="font-size: small;">g<span style="font-size: small;">r<span style="font-size: small;">adation of Thermoset plastic, polyuret<span style="font-size: small;">hane.</span></span></span></span></span></b></span> </span><br />
<span style="font-size: small;">So much for PE! What about polyurethane? In 2004, 2,722,000 tons of polyurethane was produced in the United States which encompasses 25% of the market. To put that more visually, the average African male elephant weighs 12,000lbs. There are 2000lbs in a US ton. So, in 2004 about 227 African Elephants could have been <span style="font-size: small;">modeled</span> from of polyurethane. And thats only a quarter...in 2004. Production has assuredly increased since then. The mind boggles.</span><br />
<span style="font-size: small;"><br /></span>
<span style="font-size: small;"><span style="font-size: small;">As descr<span style="font-size: small;">ibed a<span style="font-size: small;">bove, <span style="font-size: small;">p</span></span></span></span>olyurethane is made from isocynate precursors interacting with polyol (precursors containing at least one -OH group). Because of the diversity of the precursors available, polyurethane (PU) can make many different types of products. The bond between the two precursors is called an amide ester or <span style="font-size: small;">ure<span style="font-size: small;">thane (fig<span style="font-size: small;">ure 4).</span></span> When the first poly<span style="font-size: small;">uret<span style="font-size: small;">hanes were made it was notice<span style="font-size: small;">d</span> that they were degr<span style="font-size: small;">aded by fungi<span style="font-size: small;"> and efforts were made to make them less susceptible by mixing in add<span style="font-size: small;">itives (ref 2)<span style="font-size: small;">. <span style="font-size: small;">It was <span style="font-size: small;">also noted <span style="font-size: small;">the <span style="font-size: small;">degr<span style="font-size: small;">edation <span style="font-size: small;">by enzymes was <span style="font-size: small;">dependent</span> on the<span style="font-size: small;"> chemical structure of the PU and that more am<span style="font-size: small;">o<span style="font-size: small;">rphous regions were <span style="font-size: small;">degraded</span> more<span style="font-size: small;"> ra<span style="font-size: small;">pidly. </span></span></span></span></span></span></span></span></span></span></span></span></span></span></span></span></span></span>As the amount of plastics <span style="font-size: small;">in landfills beca<span style="font-size: small;">me a problem, the biodegr<span style="font-size: small;">ad<span style="font-size: small;">ab<span style="font-size: small;">ility was looked on as an<span style="font-size: small;"> advantage, as long as it did not comp<span style="font-size: small;">romise the function of the plas<span style="font-size: small;">tic. </span></span></span></span></span></span></span></span>There are a number of fungi and bacteria that are efficient at degrading various PUs.<span style="font-size: small;"> <span style="font-size: small;">H</span>owever<span style="font-size: small;">, until very recently, </span>biostimulation (<span style="font-size: small;">micro-org<span style="font-size: small;">anism growth stimulus by addition of growth promoting <span style="font-size: small;">material such as yeast extract) and bio<span style="font-size: small;">augmentation (<span style="font-size: small;">enrichment of p<span style="font-size: small;">articular micro-organ<span style="font-size: small;">ism in a<span style="font-size: small;">n environment by <span style="font-size: small;">external addition of those micro<span style="font-size: small;">-organisms) were necessary for optimal degr<span style="font-size: small;">adation. The most re<span style="font-size: small;">cent r<span style="font-size: small;">esearch <span style="font-size: small;">(</span>ref. 6) has i<span style="font-size: small;">dentified a species<span style="font-size: small;"> of fungus <span style="font-size: small;">isolated </span> from the Amazon<span style="font-size: small;">ian rainf<span style="font-size: small;">or<span style="font-size: small;">est<span style="font-size: small;"><span style="font-size: small;"> called <i>Pestalotiopsis<br />microspora</i>, which, remarkabl<span style="font-size: small;">y</span></span></span></span></span></span></span></span></span></span></span></span></span></span></span></span></span></span></span></span></span></span> can live on PU alone. This suggests that th<span style="font-size: small;">ese t<span style="font-size: small;">ype of fungi<span style="font-size: small;"> are a source of effi<span style="font-size: small;">cient PU biodegr<span style="font-size: small;">aders.</span></span></span></span></span></span><br />
<br />
<span style="font-size: small;"><b>So, A<span style="font-size: small;">bbracadabra?</span></b> </span><br />
<span style="font-size: small;"><span style="font-size: small;">Fro<span style="font-size: small;">m these <span style="font-size: small;">two examples we <span style="font-size: small;">can <span style="font-size: small;">summarize</span> tha<span style="font-size: small;">t</span></span></span> PE is so<span style="font-size: small;">me way from being</span> biodegr<span style="font-size: small;">ad<span style="font-size: small;">able by current techniques and though PU is much further <span style="font-size: small;">along<span style="font-size: small;">, the<span style="font-size: small;"> technology is <span style="font-size: small;">s<span style="font-size: small;">till <span style="font-size: small;">at the research s<span style="font-size: small;">tage. </span></span></span></span></span></span></span></span></span></span></span>Is bioremediation of soils of plastics of any sort a commercial venture yet? It tu<span style="font-size: small;">rns out the my two ex<span style="font-size: small;">amples gi<span style="font-size: small;">ve a rough idea of the state of affair - <span style="font-size: small;">lots of encouraging research but no large sca<span style="font-size: small;">le <span style="font-size: small;">applications yet. <span style="font-size: small;">We live in hope.</span></span></span></span></span></span></span></span><br />
<span style="font-size: small;"><br /></span>
<span style="font-size: small;"><b>When plastic goes, what do you get instead?</b></span><br />
<span style="font-size: small;">O<span style="font-size: small;">k, <span style="font-size: small;">popping the plasti<span style="font-size: small;">c landfill b<span style="font-size: small;">allo<span style="font-size: small;">on needs a sharper needle, but say we <span style="font-size: small;">did have a large s<span style="font-size: small;">c<span style="font-size: small;">ale technology availa<span style="font-size: small;">ble - </span></span></span></span></span></span></span></span></span>what would happen to the plastic once the microbes get their proverbial teeth into it? Well, boys and girls, thats the story for next time! Await the delights, January 30th!<b> </b>or there abouts<b>. </b></span><br />
<span style="font-size: small;"><br />NB: I think there's been some difficulty posting comments which I have addressed. If you would like to post a comment, use the little arrow to select a pull down tab. From the resulting pull down menu, you can select an ID or you can comment as annonymous, which is the last option. You can also e-mail me at emeraldbiology@gmail.com</span><br />
<br />
<span style="font-size: small;"><b>References </b></span><br />
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<a href="http://draft.blogger.com/blogger.g?blogID=385372388482159249" name="_ENREF_1"><span style="mso-ascii-font-family: Calibri; mso-hansi-font-family: Calibri; mso-no-proof: yes;">1.<span style="mso-tab-count: 1;"> </span>Zheng, Y., E.K. Yanful,
and A.S. Bassi, <i style="mso-bidi-font-style: normal;">A review of plastic waste
biodegradation.</i> Crit Rev Biotechnol, 2005. <b style="mso-bidi-font-weight: normal;">25</b>(4): p. 243-50.</span></a><span style="mso-ascii-font-family: Calibri; mso-hansi-font-family: Calibri; mso-no-proof: yes;"></span></div>
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<a href="http://draft.blogger.com/blogger.g?blogID=385372388482159249" name="_ENREF_2"><span style="mso-ascii-font-family: Calibri; mso-hansi-font-family: Calibri; mso-no-proof: yes;">2.<span style="mso-tab-count: 1;"> </span>Howard, G., <i style="mso-bidi-font-style: normal;">Polyurethane Biodegradation</i>, in <i style="mso-bidi-font-style: normal;">Microbial Degradation of Xenobiotics</i>, S.
Singh, Editor. 2012, Springer-Verlag Berlin, Heidelberg. p. 371-392.</span></a><span style="mso-ascii-font-family: Calibri; mso-hansi-font-family: Calibri; mso-no-proof: yes;"></span></div>
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<a href="http://draft.blogger.com/blogger.g?blogID=385372388482159249" name="_ENREF_3"><span style="mso-ascii-font-family: Calibri; mso-hansi-font-family: Calibri; mso-no-proof: yes;">3.<span style="mso-tab-count: 1;"> </span>Koutny, M., J. Lemaire,
and A.M. Delort, <i style="mso-bidi-font-style: normal;">Biodegradation of polyethylene
films with prooxidant additives.</i> Chemosphere, 2006. <b style="mso-bidi-font-weight: normal;">64</b>(8): p. 1243-52.</span></a><span style="mso-ascii-font-family: Calibri; mso-hansi-font-family: Calibri; mso-no-proof: yes;"></span></div>
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<a href="http://draft.blogger.com/blogger.g?blogID=385372388482159249" name="_ENREF_4"><span style="mso-ascii-font-family: Calibri; mso-hansi-font-family: Calibri; mso-no-proof: yes;">4.<span style="mso-tab-count: 1;"> </span>Sangale, M., M.
Shahnawaz, and A. Ade, <i style="mso-bidi-font-style: normal;">A Review on
Biodegradation of Polythene: The Microbial Approach.</i> Bioremediation &
Biodegradation, 2012. <b style="mso-bidi-font-weight: normal;">3</b>(10).</span></a><span style="mso-ascii-font-family: Calibri; mso-hansi-font-family: Calibri; mso-no-proof: yes;"></span></div>
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<a href="http://draft.blogger.com/blogger.g?blogID=385372388482159249" name="_ENREF_5"><span style="mso-ascii-font-family: Calibri; mso-hansi-font-family: Calibri; mso-no-proof: yes;">5.<span style="mso-tab-count: 1;"> </span>Larkin, M.J., L.A. Kulakov,
and C.C. Allen, <i style="mso-bidi-font-style: normal;">Biodegradation and
Rhodococcus--masters of catabolic versatility.</i> Curr Opin Biotechnol, 2005. <b style="mso-bidi-font-weight: normal;">16</b>(3): p. 282-90.</span></a><span style="mso-ascii-font-family: Calibri; mso-hansi-font-family: Calibri; mso-no-proof: yes;"></span></div>
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<a href="http://draft.blogger.com/blogger.g?blogID=385372388482159249" name="_ENREF_6"><span style="mso-ascii-font-family: Calibri; mso-hansi-font-family: Calibri; mso-no-proof: yes;">6.<span style="mso-tab-count: 1;"> </span>Russell, J.R., et al., <i style="mso-bidi-font-style: normal;">Biodegradation of Polyester Polyurethane by
Endophytic Fungi.</i> Applied and Environmental Microbiology, 2011. <b style="mso-bidi-font-weight: normal;">77</b>(17): p. 6076-6084.</span></a><span style="mso-ascii-font-family: Calibri; mso-hansi-font-family: Calibri; mso-no-proof: yes;"></span></div>
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</span><span style="font-size: small;"><b><br /></b></span>
<span style="font-size: small;"><b>Lifestyle magic - the soda wash.</b></span><br />
<span style="font-size: small;">Wash your hair with baking soda. 2 tablespoons of soda to 2-3 cups of water. Use as much or little as you need per wash. I've been doing this for the last 6 weeks and I'm really pleased. I have fine, fly away hair (alas, NOT a crowning glory) that needs frequent washing. <span style="font-size: small;">My hair is as clean an<span style="font-size: small;">d fluff<span style="font-size: small;">y as<span style="font-size: small;"> with any shampoo BUT - less comes out during brushing and my scalp do<span style="font-size: small;">es not itch as it often <span style="font-size: small;">did with sha<span style="font-size: small;">mp<span style="font-size: small;">oos. Neither do I need conditioner. If you do, an <span style="font-size: small;">alt<span style="font-size: small;">n<span style="font-size: small;">ative is <span style="font-size: small;">dilute apple cider vinegar with honey added optionally<span style="font-size: small;">. </span></span></span></span></span></span></span></span></span></span></span></span></span></span><span style="font-size: small;"><br /></span></div>
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</script>EmeraldBiologyhttp://www.blogger.com/profile/07468813270664433504noreply@blogger.com0tag:blogger.com,1999:blog-385372388482159249.post-2975287728400504912012-12-01T08:53:00.000-05:002014-01-26T21:52:55.534-05:00Bacteria for the EnvironmentI am a biochemist and microbiologist. As the latter, I have mainly been involved with bacteria and fungi that cause disease. For some years, I have been increasingly interested in how micro-organisms can be used for the environment. My enthusiastic self exclaims to anyone who will listen "There are bugs out there that can metabolise anything!". Really? Anything? This sounds just a little wooly. What can these organisms really do for the environment and how do they do it for themselves and for us? What are the pros and cons? Are they really a solution to our oil addiction?<br />
<br />
This blog is an exploratory journey for me, to satisfy my own curiosity and anyone else who cares to read it; this first post is introductory, to see what the very general fields are in terms of bacteria. And I do mean bacteria, but I hope to delve into fungi and algae along the way. In the following installments, there will be more references and research. I aim to present facts as accurately as possible while not getting too serious. For now I'll just raise some questions - come back to find out the answers!<br />
<br />
<b>Bacteria eat our waste. </b><br />
<table cellpadding="0" cellspacing="0" class="tr-caption-container" style="float: right; margin-left: 1em; text-align: right;"><tbody>
<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEi54HMXqP5Rp2YKbuuJVKqYFjphtKr8wivF3q6JFKPxDae5VcgMInvBIBzenhlHo6dUPS6yuxx8b6Nt4R3L86NsmJPiFUKqGvmRFYq2CjNONEnJfx19zaLAq678LNFaHK7sP7olbyB-n1o/s1600/ida+oct+blog.jpg" imageanchor="1" style="clear: right; margin-bottom: 1em; margin-left: auto; margin-right: auto;"><img border="0" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEi54HMXqP5Rp2YKbuuJVKqYFjphtKr8wivF3q6JFKPxDae5VcgMInvBIBzenhlHo6dUPS6yuxx8b6Nt4R3L86NsmJPiFUKqGvmRFYq2CjNONEnJfx19zaLAq678LNFaHK7sP7olbyB-n1o/s1600/ida+oct+blog.jpg" height="200" width="200" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;">Artwork: Mark W. Slate (<a href="http://www.naturallinestudio.com/" target="_blank">www.naturallinestudio.com</a>)</td></tr>
</tbody></table>
We all know that bacteria and fungi are the engines of composting. If you make your compost rich and warm then the degradation goes that much faster. Make your compost pile out in the open and the microorganism will work their magic, but perhaps they'll be a little slower. They will take care of all the organic waste - paper, food scraps, fecal matter, plant waste, man-made biodegradeable items etc. There seems to be more awareness of the need for composting to reduce what we put in our landfills and to save resources, with communities turning to industrial composting but how wide spread is this?<br />
<br />
Its not only organic matter that bacteria can degrade. They can also use “unnatural” materials like plastics, organic solvents and heavy metal contaminants as a resource though sometimes we have to help them along, either by engineering their genomes or by providing an optimal environment.<br />
<br />
As bacteria live everywhere, degradation of waste doesn't only happen in soil, it goes on in water too. One of most disturbing aspects of our modern life are the great swirls
of plastic debris pooling in the Sargasso Sea in the North Atlantic Ocean, driven there by currents. The plastic is ground down as stones are
ground down to sand and floats as a layer of microscopic debris beneath the
surface of the ocean. Bacteria are able to breakdown these microscopic
particles, slowing down the increase in size of the pool of plastic.Without human intervention, are bacteria helping to clean up the oceans? <br />
<br />
So could this be a magic bullet? All we need to do is add a load of bacteria on to some contaminated land or a mound of empty water bottles and Abracadabra! a nice pile of nutrient rich sludge is made to use on the field for next year’s crop? Unfortunately and predictably, it is not so easy. Many bacteria are rather finicky in their growth conditions and while you can produce great results in the optimal conditions of the lab, once you put your industrious devourers into a more natural environment where there are lower levels of nutrients and competing, better adapted bacteria, sucking up those nutrients and releasing antibiotics to fight off any newcomers, life becomes much more of a challenge. And then there is the issue of bacterial waste. Once the molecules of plastic are broken down in the sea or in the soil or even in the lab, what do they become; something nice and safe or something more toxic than the initial product?
For example do the bacteria eating plastic in the Sargasso Sea produce toxins that end up in fish and then migrate up the food chain to us? <br />
<br />
<b>Bacteria produce plastics, fuel</b><br />
I’ve just devoted a couple of paragraphs to how bacteria degrade plastic but actually, they can also be used as tiny manufacturing plants. It seems a great deal to ask that they make what we are hoping they will break down. However, bacteria can and are being engineered to produce plastic and fuels. Some bacteria even secrete organic solvents so that all you need to do is to centrifuge away the bacteria and suck off the secreted products layered on top. There is even research into getting bacteria to produce electricity!<br />
<br />
BUT we humans need alot of fuel and alot of plastic - a gigantic amount if you compare it to the size of a bacterium or even a few million bacteria. Can we have really have our cake and eat it too? How close are we to producing plastic or fuel from bacteria on a scale that would supply our demand? And yes, there are some small and not so small bumps in the road to circumnavigate. What do you feed the bacteria? Plastics??? What do you do with the bacterial waste products? Bacteria are living organisms with finite life spans. How do you remove the dead cells? To keep costs down and production up, optimal conditions must be established and maintained and this requires a good deal of experimentation and optimization, first on a laboratory scale and then on an industrial scale.<br />
<br />
I hope that wets your appetite for the next installment which should be published on November 30th - tentatively titled "Bacterial degradation of plastic - part 1".<br />
<br />
<br />
<a name='more'></a><span style="background-color: #d9ead3;"><span style="color: #38761d;"><b>LifeStyle Magic (adding little green improvements to your homelife)</b></span></span><br />
<br />
We've recently started making our own muesli/granola. Its super simple and much cheaper than what you buy at the supermarket. Google it up or ask me for my recipe!
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