Wednesday, May 11, 2011


The journal Applied and Environmental Microbiology (one of my favorite reads) has some interesting articles on the use of Genetically Modified Organisms (GMOs) for the production of fuel and plastics precursors. While still in the research stage there is some hope that mixing and matching novel genes from different species coupled with appropriate control of gene expression can induce bacterial cultures to produce useful quantities of either fuel or bioplastics (or there precursors). The first article (below) is explained in some detail in a press release from the Oak Ridge National Laboratory in the US.

Metabolic Engineering of Clostridium cellulolyticum for Production of Isobutanol from Cellulose

Wendy Higashide, Yongchao Li, Yunfeng Yang, and James C. Liao

Producing biofuels directly from cellulose, known as consolidated bioprocessing, is believed to reduce costs substantially compared to a process in which cellulose degradation and fermentation to fuel are accomplished in separate steps. Here we present a metabolic engineering example for the development of a Clostridium cellulolyticum strain for isobutanol synthesis directly from cellulose. This strategy exploits the host's natural cellulolytic activity and the amino acid biosynthesis pathway and diverts its 2-keto acid intermediates toward alcohol synthesis. Specifically, we have demonstrated the first production of isobutanol to approximately 660 mg/liter from crystalline cellulose by using this microorganism.

From the Oak Ridge (edited) press release:

BESC scores a first with isobutanol directly from cellulose (March 2011)

In the quest for inexpensive biofuels, cellulose proved no match for a bioprocessing strategy and genetically engineered microbe developed by researchers at the Department of Energy's BioEnergy Science Center.

The team's work, published online in Applied and Environmental Microbiology, represents across-the-board savings in processing costs and time, plus isobutanol is a higher grade of alcohol than ethanol.

"Unlike ethanol, isobutanol can be blended at any ratio with gasoline and should eliminate the need for dedicated infrastructure in tanks or vehicles," said Liao. "Plus, it may be possible to use isobutanol directly in current engines without modification."

“Compared to ethanol, higher alcohols such as isobutanol are better candidates for gasoline replacement because they have an energy density, octane value and Reid vapor pressure - a measurement of volatility - that is much closer to gasoline”, Liao said.

While cellulosic biomass like corn stover and switchgrass is abundant and cheap, it is much more difficult to utilize than corn and sugar cane. This is due in large part because of recalcitrance, or a plant's natural defenses to being chemically dismantled.

While some Clostridium species produce butanol, these organisms typically do not digest cellulose directly. Other Clostridium species digest cellulose but do not produce butanol. None produce isobutanol, an isomer of butanol.

"In nature, no microorganisms have been identified that possess all of the characteristics necessary for the ideal consolidated bioprocessing strain, so we knew we had to genetically engineer a strain for this purpose," Li said.

Also at Science Daily - Inexpensive Biofuels: Isobutanol Made Directly from Cellulose

In a later edition of AEM, US and Japanese researchers (including some of the same team above) engineered E. coli to produce 1-Butanol in even higher quantities. This paper is open access.

Driving Forces Enable High-Titer Anaerobic 1-Butanol Synthesis in Escherichia coli

Claire R. Shen, Ethan I. Lan, Yasumasa Dekishima, Antonino Baez, Kwang Myung Cho and James C. Liao

1-Butanol, an important chemical feedstock and advanced biofuel, is produced by Clostridium species. Various efforts have been made to transfer the clostridial 1-butanol pathway into other microorganisms. However, in contrast to similar compounds, only limited titers of 1-butanol were attained. In this work, we constructed a modified clostridial 1-butanol pathway in Escherichia coli to provide an irreversible reaction catalyzed by trans-enoyl-coenzyme A (CoA) reductase (Ter) and created NADH and acetyl-CoA driving forces to direct the flux. We achieved high-titer (30 g/liter) and high-yield (70 to 88% of the theoretical) production of 1-butanol anaerobically, comparable to or exceeding the levels demonstrated by native producers. Without the NADH and acetyl-CoA driving forces, the Ter reaction alone only achieved about 1/10 the level of production. The engineered host platform also enables the selection of essential enzymes with better catalytic efficiency or expression by anaerobic growth rescue. These results demonstrate the importance of driving forces in the efficient production of nonnative products.

The introduction can be read to get the general idea but what it means in simpler language is…

The researchers genetically modified E. coli so that it could produce enzymes that made 1-butanol, the same enzymes used by Clostridia. But increased yield only occurs under special conditions (‘high driving force’). To force the organism to produce higher amounts of butanol several anaerobic (without oxygen) metabolic pathways (energy producing) were deleted. By deleting these pathways the organism accumulated increased levels of a chemical (NADH) which is required to produce the butanol. To make this work another enzyme system had to be modified. Thus genes from another species were introduced. Finally a gene from Clostridia that may limit the reaction was deleted. If you did high school chemistry this is essentially  Le Chatelier's Principle. Increasing the concentration of NADH and lowering the activation energy increases the production of butanol. The produced butanol was continually removed by gas sparging – thus also favouring production of more butanol.

Two important points to note: in this paper the team produced 30g/L of butanol compared to ~0.6g/L in the first paper, and in this second paper the source was not Cellulose but glucose. Once these two concepts are linked than we can expect the first small scale trials to quickly follow.

In the same edition, another team has some of the latest research in the production of bio-plastic using Palm Oil as feed stock.

Production of Poly(3-Hydroxybutyrate-co-3-Hydroxyhexanoate) from Plant Oil by Engineered Ralstonia eutropha Strains

Charles F. Budde, Sebastian L. Riedel, Laura B. Willis, ChoKyun Rha, and Anthony J. Sinskey

The polyhydroxyalkanoate (PHA) copolymer poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) [P(HB-co-HHx)] has been shown to have potential to serve as a commercial bioplastic. [deleted]

Our group has engineered two R. eutropha strains that accumulate high levels of P(HB-co-HHx) with significant HHx content directly from palm oil, one of the world's most abundant plant oils. The strains express a newly characterized PHA synthase gene from the bacterium Rhodococcus aetherivorans I24. [deleted]

This study resulted in two engineered strains for production of P(HB-co-HHx) from palm oil. In palm oil fermentations, one strain accumulated 71% of its cell dry weight as PHA with 17 mol% HHx, while the other strain accumulated 66% of its cell dry weight as PHA with 30 mol% HHx.

Whether or not using Palm Oil as the feed stock  is a good thing… I’m not sure.

And finally, my personal observation is that somewhere between 20 – 30% (maybe more) of men do not wash there hands (at all or well) after going to the toilet (including #1s and #2s). In any case, if the toilet uses a bulk refill type detergent dispenser then washing hands may actually increase bacterial contamination!

Bacterial Hand Contamination and Transfer after Use of Contaminated Bulk-Soap-Refillable Dispensers

Carrie A. Zapka, Esther J. Campbell, Sheri L. Maxwell, Charles P. Gerba, Michael J. Dolan, James W. Arbogast, and David R. Macinga

Bulk-soap-refillable dispensers are prone to extrinsic bacterial contamination, and recent studies demonstrated that approximately one in four dispensers in public restrooms are contaminated. The purpose of this study was to quantify bacterial hand contamination and transfer after use of contaminated soap under controlled laboratory and in-use conditions in a community setting. Under laboratory conditions using liquid soap experimentally contaminated with 7.51 log10 CFU/ml of Serratia marcescens, an average of 5.28 log10 CFU remained on each hand after washing, and 2.23 log10 CFU was transferred to an agar surface. In an elementary-school-based field study, Gram-negative bacteria on the hands of students and staff increased by 1.42 log10 CFU per hand (26-fold) after washing with soap from contaminated bulk-soap-refillable dispensers. In contrast, washing with soap from dispensers with sealed refills significantly reduced bacteria on hands by 0.30 log10 CFU per hand (2-fold). [deleted]

These results demonstrate that washing with contaminated soap from bulk-soap-refillable dispensers can increase the number of opportunistic pathogens on the hands and may play a role in the transmission of bacteria in public settings.

Possibly conducted to support sales of sealed soap refills.

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