Biotechnology for Biofuels

Decomposition of biomass for biogas production can be practiced under wet and dry fermentation conditions. In contrast to the dry fermentation technology, wet fermentation is characterized by a high liquid content and a relatively low total solid content. In this study, the composition and functional potential of a biogas-producing microbial community in an agricultural biogas reactor operating under wet fermentation conditions was analyzed by a metagenomic approach applying 454-pyrosequencing. The obtained metagenomic dataset and corresponding 16S rRNA gene amplicon sequences were compared to the previously sequenced comparable metagenome from a dry fermentation process, meeting explicitly identical boundary conditions regarding sample and community DNA preparation, sequencing technology, processing of sequence reads and data analyses by bioinformatics tools. High-throughput metagenome sequencing of community DNA from the wet fermentation process applying the pyrosequencing approach resulted in 1,532,780 reads, with an average read length of 397 bp, accounting for approximately 594 million bases of sequence information in total. Taxonomic comparison of the communities from wet and dry fermentation revealed similar microbial profiles with Bacteria being the predominant superkingdom, while the superkingdom Archaea was less abundant. In both biogas plants, the bacterial phyla Firmicutes, Bacteroidetes, Spirochaetes and Proteobacteria were identified with descending frequencies. Within the archaeal superkingdom, the phylum Euryarchaeota was most abundant with the dominant class Methanomicrobia. Functional profiles of the communities revealed that environmental gene tags representing methanogenesis enzymes were present in both biogas plants in comparable frequencies. 16S rRNA gene amplicon high-throughput sequencing disclosed differences in the sub-communities comprising methanogenic Archaea between both processes. Fragment recruitments of metagenomic reads to the reference genome of the archaeon Methanoculleus bourgensis MS2T revealed that dominant methanogens within the dry fermentation process were highly related to the reference. Although process parameters, substrates and technology differ between the wet and dry biogas fermentations analyzed in this study, community profiles are very similar at least at higher taxonomic ranks, illustrating that core community taxa perform key functions in biomass decomposition and methane synthesis. Regarding methanogenesis, Archaea highly related to the type strain M. bourgensis MS2T dominate the dry fermentation process, suggesting the adaptation of members belonging to this species to specific fermentation process parameters.

Lytic polysaccharide monooxygenases (LPMOs) are attracting large attention due their ability to degrade recalcitrant polysaccharides in biomass conversion and to perform powerful redox chemistry. We have established a universal Pichia pastoris platform for the expression of fungal LPMOs using state-of-the-art recombination cloning and modern molecular biological tools to achieve high yields from shake-flask cultivation and simple tag-less single-step purification. Yields are very favorable with up to 42 mg per liter medium for four different LPMOs spanning three different families. Moreover, we report for the first time of a yeast-originating signal peptide from the dolichyl-diphosphooligosaccharide-protein glycosyltransferase subunit 1 (OST1) form S. cerevisiae efficiently secreting and successfully processes the N-terminus of LPMOs yielding in fully functional enzymes. The work demonstrates that the industrially most relevant expression host P. pastoris can be used to express fungal LPMOs from different families in high yields and inherent purity. The presented protocols are standardized and require little equipment with an additional advantage with short cultivation periods.

Ethanol production from lignocellulosic feedstocks (also known as 2nd generation or 2G ethanol process) presents a great potential for reducing both ethanol production costs and climate change impacts since agricultural residues and dedicated energy crops are used as feedstock. This study aimed at the quantification of the economic and environmental impacts considering the current and future scenarios of sugarcane biorefineries taking into account not only the improvements of the industrial process but also of biomass production systems. Technology assumptions and scenarios setup were supported by main companies and stakeholders, involved in the lignocellulosic ethanol production chain from Brazil and abroad. For instance, scenarios considered higher efficiencies and lower residence times for pretreatment, enzymatic hydrolysis, and fermentation (including pentoses fermentation); higher sugarcane yields; and introduction of energy cane (a high fiber variety of cane). Ethanol production costs were estimated for different time horizons. In the short term, 2G ethanol presents higher costs compared to 1st generation (1G) ethanol. However, in the long term, 2G ethanol is more competitive, presenting remarkable lower production cost than 1G ethanol, even considering some uncertainties regarding technology and market aspects. In addition, environmental assessment showed that both 1G (in the medium and long term) and 2G ethanol can reduce climate change impacts by more than 80% when compared to gasoline. This work showed the great potential of 2G ethanol production in terms of economic and environmental aspects. These results can support new research programs and public policies designed to stimulate both production and consumption of 2G ethanol in Brazil, accelerating the path along the learning curve. Some examples of mechanisms include: incentives to the establishment of local equipment and enzyme suppliers; and specific funding programs for the development and use of energy cane.

Poly-γ-glutamic acid (γ-PGA) is a natural polymer with great potential applications in areas of agriculture, industry, and pharmaceutical. The biodiesel-derived glycerol can be used as an attractive feedstock for γ-PGA production due to its availability and low price; however, insufficient production of γ-PGA from glycerol is limitation. The metabolic pathway of Bacillus licheniformis WX-02 was rewired to improve the efficiency of glycerol assimilation and the supply of NADPH for γ-PGA synthesis. GlpK, GlpX, Zwf, and Tkt1 were found to be the key enzymes for γ-PGA synthesis using glycerol as a feedstock. Through combinational expression of these key enzymes, the γ-PGA titer increased to 19.20 ± 1.57 g/L, which was 1.50-fold of that of the wild-type strain. Then, we studied the flux distributions, gene expression, and intracellular metabolites in WX-02 and the recombinant strain BC4 (over-expression of the above quadruple enzymes). Our results indicated that over-expression of the quadruple enzymes redistributed metabolic flux to γ-PGA synthesis. Furthermore, using crude glycerol as carbon source, the BC4 strain showed a high productivity of 0.38 g/L/h, and produced 18.41 g/L γ-PGA, with a high yield of 0.46 g γ-PGA/g glycerol. The approach to rewiring of metabolic pathways enables B. licheniformis to efficiently synthesize γ-PGA from glycerol. The γ-PGA productivity reported in this work is the highest obtained in glutamate-free medium. The present study demonstrates that the recombinant B. licheniformis strain shows significant potential to produce valuable compounds from crude glycerol.

Arundo donax đã thu hút sự quan tâm trở lại như một ứng cử viên tiềm năng cho cây năng lượng sử dụng trong quá trình chuyển đổi biomass thành nhiên liệu lỏng và nhà máy sinh học. Điều này là do năng suất cao, khả năng thích ứng với điều kiện đất đai biên chế và phù hợp với sản xuất nhiên liệu sinh học và vật liệu sinh học. Mặc dù quan trọng, tài nguyên kính gene hiện có để hỗ trợ cải tiến loài này vẫn còn hạn chế. Chúng tôi đã sử dụng giải trình tự RNA (RNA-Seq) để lắp ráp và mô tả transcriptome lá của A. donax. Việc giải trình tự đã tạo ra 1249 triệu đoạn đọc sạch, được lắp ráp bằng cách sử dụng phương pháp single-k-mer và multi-k-mer thành 62,596 trình tự độc nhất (unitranscripts) với N50 là 1134 bp. Bộ phần mềm TransDecoder và Trinotate đã được sử dụng để thu được các trình tự mã hóa giả định và chú thích chúng thông qua việc ánh xạ tới các cơ sở dữ liệu UniProtKB/Swiss-Prot và UniRef90, tìm kiếm các transcript, protein, miền protein và peptid tín hiệu đã biết. Hơn nữa, các unitranscripts đã được chú thích bằng cách ánh xạ chúng tới các cơ sở dữ liệu không trùng lặp của NCBI, GO và đường dẫn KEGG thông qua Blast2GO. Transcriptome cũng được đặc trưng bởi các tìm kiếm BLAST để điều tra các transcript đồng hình của các gen chính liên quan đến các con đường chuyển hóa quan trọng như lignin, cellulose, purine và tổng hợp thiamine cũng như cố định carbon. Thêm vào đó, một tập hợp các transcript đồng hình của các gen chính liên quan đến sự phát triển khí khổng và các gen mã hóa cho protein liên quan đến căng thẳng (SAPs) đã được xác định. Ngoài ra, 8364 dấu hiệu lặp lại chuỗi đơn giản (SSR) đã được xác định và khảo sát. SSR dường như nhiều hơn ở các vùng không mã hóa (63.18%) so với vùng mã hóa (36.82%). Bộ dữ liệu SSR này đại diện cho danh mục dấu hiệu đầu tiên của A. donax. 53 SSRs (PolySSRs) sau đó được dự đoán là đa hình giữa các lắp ráp theo kiểu sinh thái đặc trưng, cho thấy sự biến đổi di truyền trong các kiểu sinh thái đã nghiên cứu. Nghiên cứu này cung cấp transcriptome lá đầu tiên có sẵn công khai cho cây năng lượng sinh học A. donax. Việc chú thích và mô tả chức năng của transcriptome sẽ rất hữu ích trong việc cung cấp cái nhìn sâu sắc về các cơ chế phân tử đặc trưng cho khả năng thích nghi cực kỳ của nó. Việc xác định các transcript đồng hình liên quan đến các con đường chuyển hóa chính mở ra một nền tảng cho việc chỉ đạo các nỗ lực cải tiến di truyền tương lai của loài này. Cuối cùng, các SSR đã được xác định sẽ tạo điều kiện cho việc khai thác đa dạng di truyền chưa được sử dụng. Transcriptome này sẽ có giá trị cho các nghiên cứu di truyền và gen học chức năng đang diễn ra trong cây trồng có giá trị kinh tế hàng đầu này.

A major obstacle, and perhaps the most important economic barrier to the effective use of plant biomass for the production of fuels, chemicals, and bioproducts, is our current lack of knowledge of how to efficiently and effectively deconstruct wall polymers for their subsequent use as feedstocks. Plants represent the most desired source of renewable energy and hydrocarbons because they fix CO2, making their use carbon neutral. Their biomass structure, however, is a barrier to deconstruction, and this is often referred to as recalcitrance. Members of the bacterial genus Caldicellulosiruptor have the ability to grow on unpretreated plant biomass and thus provide an assay for plant deconstruction and biomass recalcitrance. Using recently developed genetic tools for manipulation of these bacteria, a deletion of a gene cluster encoding enzymes for pectin degradation was constructed, and the resulting mutant was reduced in its ability to grow on both dicot and grass biomass, but not on soluble sugars. The plant biomass from three phylogenetically diverse plants, Arabidopsis (a herbaceous dicot), switchgrass (a monocot grass), and poplar (a woody dicot), was used in these analyses. These biomass types have cell walls that are significantly different from each other in both structure and composition. While pectin is a relatively minor component of the grass and woody dicot substrates, the reduced growth of the mutant on all three biomass types provides direct evidence that pectin plays an important role in biomass recalcitrance. Glycome profiling of the plant material remaining after growth of the mutant on Arabidopsis biomass compared to the wild-type revealed differences in the rhamnogalacturonan I, homogalacturonan, arabinogalactan, and xylan profiles. In contrast, only minor differences were observed in the glycome profiles of the switchgrass and poplar biomass. The combination of microbial digestion and plant biomass analysis provides a new and important platform to identify plant wall structures whose presence reduces the ability of microbes to deconstruct plant walls and to identify enzymes that specifically deconstruct those structures.

The large surplus of crude glycerol, as main low-value waste stream in biodiesel production, has led to the investigation of new possibilities for the production of value-added chemicals from this feedstock. New and efficient (bio-) catalysts are needed that are able to convert glycerol to versatile chemical building blocks. This would contribute to further develop away from a mainly petroleum based, to a sustainable, bio-based industry. One promising group of discussed building block chemicals are dicarbonic acids. Here, we report the efficient synthesis of malate from glycerol using Ustilago trichophora RK089, which was identified in a screening of 74 Ustilaginaceae. For economically feasible production that can compete with existing processes, a high productivity is required. By adaptive laboratory evolution, the growth and production rate were increased by 2.5- and 6.6-fold, respectively. Further medium optimization increased the final titer, yield, and overall production rate to 196 g L−1, 0.82 gmal g gly −1 , and 0.39 g L−1 h−1, respectively. This titer is the highest reported for microbial malate production, making U. trichophora TZ1 a promising microbial production host for malate from crude glycerol, especially since it is not genetically engineered. Since this production process starts from an industrial waste stream as substrate and yields an interesting platform chemical, which can be used to replace petro-chemicals, it greatly contributes to a sustainable bio-economy.