Research and Development

Microbial production of fuels via bioprocesses has emerged as an attractive alternative to the traditional production of fuels. Wiparat Siripong of the National Center for Genetic Engineering and Biotechnology in Thailand engineered the yeast Pichia pastoris to produce isobutanol from glucose and glycerol. The team diverted the amino acid intermediates to the 2-keto acid degradation pathway for higher alcohol production.

The engineered strain overexpressing the keto-acid degradation pathway produced 284 mg/L of isobutanol when fed with 2-ketoisovalerate. To improve the production of isobutanol, the team overexpressed a part of the amino acid l-valine biosynthetic pathway in the engineered strain. This led to a strain capable of producing 0.89 g/L of isobutanol. Fine-tuning the expression of bottleneck enzymes further improved the production titer of isobutanol by 43 times compared to the original strain.

This work will provide a route to establish P. pastoris as a versatile production platform for fuels and chemicals.

Synechocystis sp. PCC 6803 is an attractive organism for the production of alcohols. However, the produced alcohol is toxic to Synechocystis sp., hindering industrial applications. Hence, researchers want to engineer organisms with strong alcohol tolerance. Takuya Matsusako of Osaka University in Japan aimed to increase alcohol tolerance genes in Synechocystis sp. via adaptive laboratory evolution.

Isobutanol-tolerant strains of Synechocystis sp. PCC 6803 were obtained by long-term passage culture experiments using medium containing 2 g/L isobutanol. These evolved strains were capable of growing on medium with 5 g/L isobutanol, on which the parental strain could not grow. Analysis of the evolved strains revealed that they acquired resistance ability due to combinatorial malfunctions of several genes including slr1044 (mcpA) and slr0369 (envD), or slr0322 (hik43) and envD.

The tolerant strains demonstrated stress resistance against isobutanol as well as a wide variety of alcohols. And with the introduction of an ethanol-producing pathway into the evolved strain, its productivity successfully increased to 142% of the control strain.

Second-generation biofuels help decrease dependency on fossil fuels. To make biomass more suitable for biorefinery use, researchers need to better understand plant cell wall synthesis. Increasing the ratio of C6 to C5 sugars in the cell wall and decreasing the lignin content are two important targets in engineering of plants that are more suitable for biofuel production. Aude Aznar and Camille Chalvin of Lawrence Berkeley National Laboratory have identified genes involved in the synthesis of pectic galactan, including the GALS1 and URGT1.

Their team aimed to engineer plants with increased pectic galactan in stems. To do this, they used plants that were already engineered to have low xylan content, then further engineered them to overexpress GALS1, URGT1, and UGE2, a UDP-glucose epimerase. Finally, the high galactan and low xylan traits were again stacked with the low lignin trait, which was achieved by expressing the QsuB gene encoding dehydroshikimate dehydratase in lignifying cells.

These results show that increasing C6 sugar content, decreasing xylan, and reducing lignin content can be combined in an additive manner, leading to improved properties in terms of biofuel production, without any negative growth effects.

Energy Crops and Feedstocks for Biofuels Production

The function of Domain of Unknown Function 231-containing proteins (DUF231) is largely unknown. While previous studies have suggested that DUF231 proteins are related in O-acetyl substitution of hemicellulose and esterification of pectin, little is known about their function in woody plants. A study from Oak Ridge National Laboratory has revealed that one member of the DUF231 proteins in poplar (Populus deltoides), PdDUF231A, is involved in the acetylation of xylan and affects cellulose biosynthesis.

A total of 52 DUF231 proteins were identified in the poplar genome. In transgenic poplar lines overexpressing PdDUF231A, the research team from ORNL found that glucose and cellulose contents were increased, as well as the levels of cellulose synthesis-related genes. Further analysis revealed that total acetylated xylan was increased in the transgenic lines, leading to higher rate of glucose release. Plant biomass productivity was also increased in the transgenic lines.

PdDUF231A could be a promising target for genetic modification for biofuel production since biomass productivity and quality can both be improved through its overexpression.

The demand for higher yield Jatropha curcas L. is rapidly increasing due to its potential in biofuel production. However, genetic analysis of Jatropha and molecular breeding for higher yield have been hindered by the few molecular markers available.

Zhiqiang Xia of the Chinese Academy of Tropical Agriculture Sciences aimed to construct an ultrahigh-density linkage map for a Jatropha mapping population of 153 individuals. The genetic linkage map consisted of 3422 SNP and indel markers, which clustered into 11 linkage groups.

Using this map, 13 repeatable QTLs (reQTLs) for fruit yield traits were identified and mapped. Ten reQTLs, qNF-1, qNF-2a, qNF-2b, qNF-2c, qNF-3, qNF-4, qNF-6, qNF-7a, qNF-7b and qNF-8, control the number of fruits while the other three, qTWF-1, qTWF-2 and qTWF-3, control the total weight of fruits (TWF). There were also two candidate critical genes which may regulate Jatropha fruit yield.

This study is the first report of a Jatropha genetic linkage map construction where the markers used in this study showed great potential for QTL mapping. This map will be a useful tool for localization of other economically important QTLs and candidate genes for Jatropha.

Biofuels Processing

Production of milk products produces a huge amount of wastewater in the factory. This wastewater, called "acid whey", cannot be fed to animals in large quantities due to its acidity. It is rich in organic material and must be treated or transported to farms for use as fertilizer.

University of Tübingen scientists have recently developed a process that converts acid whey, a dairy byproduct, into biofuels without the use of additional chemicals. Led by Professor Lars Angenent, the research team used microbiome cultures similar to those in the human gut. The new bio-oil can be used in animal feed or as a fuel for airplanes when refined.

The team used tanks with several types of bacteria, called a reactor microbiome. They kept two microbiomes with different temperatures. The first hot microbiome (50°C) converts all the sugars into an intermediate acid. The second warm microbiome (30°C) then performs chain elongation until a product is formed with six to nine carbons. The team also investigated which bacteria had grown in the two different microbiomes.

More work is now needed to study if other types of wastewater can also be converted into these valuable chemicals.

Bioflocculation has been developed as an effective method to harvest microalgae. However, the high production cost of bioflocculants makes it difficult to scale up. Haipeng Guo of Lakehead University in China aimed to develop low-cost bioflocculants using untreated corn stover and a biomass-degrading bacterium, Pseudomonas sp. GO2.

Pseudomonas sp. GO2 showed excellent production of bioflocculants by directly hydrolyzing various biomasses. The untreated corn stover was selected as carbon source for bioflocculants' production due to its high flocculating efficiency. Biochemical analysis showed that bioflocculants contained 59.0% polysaccharides with uronic acid (34.2%), 32.1% protein, and 6.1% nucleic acid. In addition, the bioflocculants showed the highest flocculating efficiency and were stable over broad ranges of pH and temperature.

These results indicate that Pseudomonas sp. GO2 can directly use various untreated biomasses to produce low-cost bioflocculants. The produced bioflocculants showed high efficiency to harvest two green microalgae in a low GO2 fermentation broth/algal culture ratio.

Policy and Regulation

The Bangladesh legislation will now allow the use of ethanol with conventional fuels, however, it will not mandate production levels.

The government will be allowing a 5% blend of bioethanol with octane and gasoline. This move is said to be a response to the interest of several producers to set up biofuel plants. Plant owners will have to first get government permission before producing ethanol. Then all produce will be sent to the government-run Bangladesh Petroleum Corporation.

It is still unclear what feedstock will be used but will most likely be waste materials. However, government officials have questioned the necessity of the move since Bangladesh has negligible contributions to global greenhouse gas emissions.