Synergistic degradation of lignocellulose by fungi and bacteria in boreal forest soil

Thesis (M.S.) University of Alaska Fairbanks, 2015Boreal forests contain an estimated 28% of the world's soil carbon, and currently act as a significant global carbon sink. Plant-derived lignocellulose is a major component of soil carbon, and its decomposition is dependent on soil bacteria and fungi. In order to predict the fate of this soil carbon and its potential feedbacks to climate change, the identities, activity, and interactions of soil microbial decomposer communities must be better understood. This study used stable isotope probing (SIP) with ¹³C-labeled lignocellulose and two of its constituents, cellulose and vanillin, to identify microbes responsible for the processing of lignocellulose-derived carbon and examine the specific roles that they perform. Results indicate that multiple taxa are involved in lignocellulose processing, and that certain taxa target specific portions of the lignocellulose macromolecule; specifically, fungi dominate the degradation of lignocellulose and cellulose macromolecules, while bacteria scavenge aromatic lignocellulose monomers. Major fungal taxa involved in lignocellulose degradation include Ceratobasidium, Geomyces, and Sebacina, among others. Bacterial taxa processing lignocellulose and cellulose included Cellvibrio and Mesorhizobium in high abundance relative to other taxa, although Burkholderia were the primary vanillin consumers. These results elucidate some of the major players in lignocellulose decomposition and their specific roles in boreal forest soil. This information provides knowledge of small-scale microbial processes that dictate ecosystem-level carbon cycling, and can assist in predictions of the fate of boreal forest carbon stocks

125th anniversary review: fuel alcohol: current production and future challenges

Global research and industrial development of liquid transportation biofuels are moving at a rapid pace. This is mainly due to the significant roles played by biofuels in decarbonising our future energy needs, since they act to mitigate the deleterious impacts of greenhouse gas emissions to the atmosphere that are contributors of climate change. Governmental obligations and international directives that mandate the blending of biofuels in petrol and diesel are also acting as great stimuli to this expanding industrial sector. Currently, the predominant liquid biofuel is bioethanol (fuel alcohol) and its worldwide production is dominated by maize-based and sugar cane-based processes in North and South America, respectively. In Europe, fuel alcohol production employs primarily wheat and sugar beet. Potable distilled spirit production and fuel alcohol processes share many similarities in terms of starch bioconversion, fermentation, distillation and co-product utilisation, but there are some key differences. For example, in certain bioethanol fermentations, it is now possible to yield consistently high ethanol concentrations of ~20% (v/v). Emerging fuel alcohol processes exploit lignocellulosic feedstocks and scientific and technological constraints involved in depolymerising these materials and efficiently fermenting the hydrolysate sugars are being overcome. These so-called secondgeneration fuel alcohol processes are much more environmentally and ethically acceptable compared with exploitation of starch and sugar resources, especially when considering utilisation of residual agricultural biomass and biowastes. This review covers both first and second-generation bioethanol processes with a focus on current challenges and future opportunities of lignocellulose-to-ethanol as this technology moves from demonstration pilot-plants to full-scale industrial facilities

Succession of physiological stages hallmarks the transcriptomic response of the fungus Aspergillus niger to lignocellulose.

BackgroundUnderstanding how fungi degrade lignocellulose is a cornerstone of improving renewables-based biotechnology, in particular for the production of hydrolytic enzymes. Considerable progress has been made in investigating fungal degradation during time-points where CAZyme expression peaks. However, a robust understanding of the fungal survival strategies over its life time on lignocellulose is thereby missed. Here we aimed to uncover the physiological responses of the biotechnological workhorse and enzyme producer Aspergillus niger over its life time to six substrates important for biofuel production.ResultsWe analysed the response of A. niger to the feedstock Miscanthus and compared it with our previous study on wheat straw, alone or in combination with hydrothermal or ionic liquid feedstock pretreatments. Conserved (substrate-independent) metabolic responses as well as those affected by pretreatment and feedstock were identified via multivariate analysis of genome-wide transcriptomics combined with targeted transcript and protein analyses and mapping to a metabolic model. Initial exposure to all substrates increased fatty acid beta-oxidation and lipid metabolism transcripts. In a strain carrying a deletion of the ortholog of the Aspergillus nidulans fatty acid beta-oxidation transcriptional regulator farA, there was a reduction in expression of selected lignocellulose degradative CAZyme-encoding genes suggesting that beta-oxidation contributes to adaptation to lignocellulose. Mannan degradation expression was wheat straw feedstock-dependent and pectin degradation was higher on the untreated substrates. In the later life stages, known and novel secondary metabolite gene clusters were activated, which are of high interest due to their potential to synthesize bioactive compounds.ConclusionIn this study, which includes the first transcriptional response of Aspergilli to Miscanthus, we highlighted that life time as well as substrate composition and structure (via variations in pretreatment and feedstock) influence the fungal responses to lignocellulose. We also demonstrated that the fungal response contains physiological stages that are conserved across substrates and are typically found outside of the conditions with high CAZyme expression, as exemplified by the stages that are dominated by lipid and secondary metabolism

RNAseq reveals hydrophobins that are involved in the adaptation of aspergillus nidulans to lignocellulose

Background Sugarcane is one of the world’s most profitable crops. Waste steam-exploded sugarcane bagasse (SEB) is a cheap, abundant, and renewable lignocellulosic feedstock for the next-generation biofuels. In nature, fungi seldom exist as planktonic cells, similar to those found in the nutrient-rich environment created within an industrial fermenter. Instead, fungi predominantly form biofilms that allow them to thrive in hostile environments. Results In turn, we adopted an RNA-sequencing approach to interrogate how the model fungus, Aspergillus nidulans, adapts to SEB, revealing the induction of carbon starvation responses and the lignocellulolytic machinery, in addition to morphological adaptations. Genetic analyses showed the importance of hydrophobins for growth on SEB. The major hydrophobin, RodA, was retained within the fungal biofilm on SEB fibres. The StuA transcription factor that regulates fungal morphology was up-regulated during growth on SEB and controlled hydrophobin gene induction. The absence of the RodA or DewC hydrophobins reduced biofilm formation. The loss of a RodA or a functional StuA reduced the retention of the hydrolytic enzymes within the vicinity of the fungus. Hence, hydrophobins promote biofilm formation on SEB, and may enhance lignocellulose utilisation via promoting a compact substrate-enzyme-fungus structure. Conclusion This novel study highlights the importance of hydrophobins to the formation of biofilms and the efficient deconstruction of lignocellulose

Prospection and Evaluation of (Hemi) Cellulolytic Enzymes Using Untreated and Pretreated Biomasses in Two Argentinean Native Termites

Saccharum officinarum bagasse (common name: sugarcane bagasse) and Pennisetum purpureum (also known as Napier grass) are among the most promising feedstocks for bioethanol production in Argentina and Brazil. In this study, both biomasses were assessed before and after acid pretreatment and following hydrolysis with Nasutitermes aquilinus andCortaritermes fulviceps termite gut digestome. The chemical composition analysis of the biomasses after diluted acid pretreatment showed that the hemicellulose fraction was partially removed. The (hemi) cellulolytic activities were evaluated in bacterial culture supernatantsof termite gut homogenates grown in treated and untreated biomasses. In all cases, we detected significantly higher endoglucanase and xylanase activities using pretreated biomasses compared to untreated biomasses, carboxymethylcellulose and xylan. Several protein bands with (hemi) cellulolytic activity were detected in zymograms and two-dimensionalgel electrophoresis. Some proteins of these bands or spots were identified as xylanolytic peptides by mass spectrometry. Finally, the diversity of cultured cellulolytic bacterial endosymbionts associated to both Argentinean native termite species was analyzed. This study describes, for the first time, bacterial endosymbionts and endogenous (hemi) cellulases of two Argentinean native termites as well as their potential application in degradation of lignocellulosic biomass for bioethanol production.Fil: Ben Guerrero, Emiliano. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; ArgentinaFil: Arneodo Larochette, Joel Demián. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Microbiología y Zoología Agrícola; ArgentinaFil: Bombarda Campanha, Raquel. Ministerio da Agricultura Pecuaria e Abastecimento de Brasil. Empresa Brasileira de Pesquisa Agropecuaria; BrasilFil: Oliveira, Patrícia Abrão de. Ministerio da Agricultura Pecuaria e Abastecimento de Brasil. Empresa Brasileira de Pesquisa Agropecuaria; BrasilFil: Labate, Mônica T. Veneziano. Universidade de Sao Paulo; BrasilFil: Cataldi, Thaís Regiani. Universidade de Sao Paulo; BrasilFil: Campos, Eleonora. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Cataldi, Ángel Adrián. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Labate, Carlos A.. Universidade de Sao Paulo; BrasilFil: Rodrigues, Clenilson Martins. Ministerio da Agricultura Pecuaria e Abastecimento de Brasil. Empresa Brasileira de Pesquisa Agropecuaria; BrasilFil: Talia, Paola Monica. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Development of a D-xylose fermenting and inhibitor tolerant industrial Saccharomyces cerevisiae strain with high performance in lignocellulose hydrolysates using metabolic and evolutionary engineering

Background: The production of bioethanol from lignocellulose hydrolysates requires a robust, D-xylose-fermenting and inhibitor-tolerant microorganism as catalyst. The purpose of the present work was to develop such a strain from a prime industrial yeast strain, Ethanol Red, used for bioethanol production. Results: An expression cassette containing 13 genes including Clostridium phytofermentans XylA, encoding D-xylose isomerase (XI), and enzymes of the pentose phosphate pathway was inserted in two copies in the genome of Ethanol Red. Subsequent EMS mutagenesis, genome shuffling and selection in D-xylose-enriched lignocellulose hydrolysate, followed by multiple rounds of evolutionary engineering in complex medium with D-xylose, gradually established efficient D-xylose fermentation. The best-performing strain, GS1.11-26, showed a maximum specific D-xylose consumption rate of 1.1 g/g DW/h in synthetic medium, with complete attenuation of 35 g/L D-xylose in about 17 h. In separate hydrolysis and fermentation of lignocellulose hydrolysates of Arundo donax (giant reed), spruce and a wheat straw/hay mixture, the maximum specific D-xylose consumption rate was 0.36, 0.23 and 1.1 g/g DW inoculum/h, and the final ethanol titer was 4.2, 3.9 and 5.8% (v/v), respectively. In simultaneous saccharification and fermentation of Arundo hydrolysate, GS1.11-26 produced 32% more ethanol than the parent strain Ethanol Red, due to efficient D-xylose utilization. The high D-xylose fermentation capacity was stable after extended growth in glucose. Cell extracts of strain GS1.11-26 displayed 17-fold higher XI activity compared to the parent strain, but overexpression of XI alone was not enough to establish D-xylose fermentation. The high D-xylose consumption rate was due to synergistic interaction between the high XI activity and one or more mutations in the genome. The GS1.11-26 had a partial respiratory defect causing a reduced aerobic growth rate. Conclusions: An industrial yeast strain for bioethanol production with lignocellulose hydrolysates has been developed in the genetic background of a strain widely used for commercial bioethanol production. The strain uses glucose and D-xylose with high consumption rates and partial cofermentation in various lignocellulose hydrolysates with very high ethanol yield. The GS1.11-26 strain shows highly promising potential for further development of an all-round robust yeast strain for efficient fermentation of various lignocellulose hydrolysates

Connecting Carbon Capture with Oceanic Biomass Production

The climate change believed by anthropogenic emission is not isolated but tightly coupled with other issues including biodiversity loss and ocean acidification etc., and in order to prevent the potential serious impacts, both political and technological methods are being tried for greenhouse mitigation. Dimming the income sunlight by some “geoengineering” approaches currently seem ruinously expensive and technically difficult, and would not prevent the increase of greenhouse gases (GHGs) in atmosphere and ocean acidification, so capturing carbon to reduce the environmental concentration of carbon dioxide (CO2) and promoting renewable energy development for the reduction of using fossil fuels are very necessary. Biofuels derived from natural and agricultural biomass could be deployed for power production and existing transportation needs. The current economics are more favorable for conversion of edible biomass into biofuels, which could spend plenty of freshwater and farmlands, compete with food supply, and create a “carbon debt” with local ecosystem destruction by deforestation to expand biofuel-crop production. So it is vital to develop processes for converting non-edible feedstock such as lignocellulose and microalgae into biofuels.
 Compared with lignocellulose, microalgae have higher growth rates, don’t need plenteous freshwater for irrigating, and can grow in the conditions that are not favorable for terrestrial biomass growth. The current limitation of microalgal biofuels is the microalgae cultivation cost, and to compensate the high cost of microalgal biofuels, three suggestions are propounded here. (i) Using ships as the platforms of cultivating microalgae, producing biofuels, and transporting feedstock and products on a large scale on subtropical oligotrophic oceans, where the ocean’s least productive waters are formed with compared peaceful surface condition and poor marine communities. (ii) Operating different kinds of oceanic biomass productions for high-value products to compensate the cost of microalgal biofuels. Different kinds of microalgae and macroalgae (seaweeds) could be cultivated for biofuels, chemicals, healthy food, and feed for breeding economic marine species to satisfy the accelerating demands for seafood supply and simultaneously mitigate the fast decline of wild stocks. (iii) Constituting financial subsidies to make CO2 as the feedstock of microalgae cultivation for free, and exact quantifying the carbon captured in biomass products and the CO2 reduction that these products would provide by displacing natural and nonrenewable carbon resources, to take part in the international carbon-credit trading markets and sell the offsets. In a word, this article mainly talks about trying to find a way that connect CO2 capture with renewable energy development, and partially combat against deforestation, loss of biodiversity, shortage of food, and decline of marine lives etc., if possible

Assembly in vitro of rhodococcus jostii RHA1 encapsulin and peroxidase DypB to form a nano-compartment

Rhodococcus jostii RHA1 peroxidase DypB has been recently identified as a bacterial lignin peroxidase. The dypB gene is co-transcribed with a gene encoding an encapsulin protein, shown in Thermotoga maritima to assemble to form a 60-subunit nano-compartment, and DypB contains a C-terminal sequence motif thought to target the protein to the encapsulin nanocompartment. R. jostii RHA1 encapsulin protein has been overexpressed in R. jostii RHA1, and purified as a high molecular weight assembly (Mr >106). The purified nanocompartment can be denatured to form a low molecular weight species by treatment at pH 3.0, and can be re-assembled to form the nanocompartment at pH 7.0. Recombinant DypB can be assembled in vitro with monomeric encapsulin to form an assembly of similar size and shape to the encapsulin-only nanocompartment, assessed by dynamic light scattering. The assembled complex shows enhanced lignin degradation activity per mg DypB present, compared with native DypB, using a nitrated lignin UV-vis assay method. The measured stoichiometry of 8.6 µmoles encapsulin/µmol DypB in the complex is comparable to the value of 10 predicted from the crystal structure