Phenotypic and genomic characterization of Methanothermobacter wolfeii strain BSEL, a CO2-capturing archaeon with minimal nutrient requirements.

A new variant of Methanothermobacter wolfeii was isolated from an anaerobic digester using enrichment cultivation in anaerobic conditions. The new isolate was taxonomically identified via 16S rRNA gene sequencing and tagged as M. wolfeii BSEL. The whole genome of the new variant was sequenced and de novo assembled. Genomic variations between the BSEL strain and the type strain were discovered, suggesting evolutionary adaptations of the BSEL strain that conferred advantages while growing under a low concentration of nutrients. M. wolfeii BSEL displayed the highest specific growth rate ever reported for the wolfeii species (0.27 ± 0.03 h-1) using carbon dioxide (CO2) as unique carbon source and hydrogen (H2) as electron donor. M. wolfeii BSEL grew at this rate in an environment with ammonium (NH4+) as sole nitrogen source. The minerals content required to cultivate the BSEL strain was relatively low and resembled the ionic background of tap water without mineral supplements. Optimum growth rate for the new isolate was observed at 64°C and pH 8.3. In this work, it was shown that wastewater from a wastewater treatment facility can be used as a low-cost alternative medium to cultivate M. wolfeii BSEL. Continuous gas fermentation fed with a synthetic biogas mimic along with H2 in a bubble column bioreactor using M. wolfeii BSEL as biocatalyst resulted in a CO2 conversion efficiency of 97% and a final methane (CH4) titer of 98.5%v, demonstrating the ability of the new strain for upgrading biogas to renewable natural gas.IMPORTANCEAs a methanogenic archaeon, Methanothermobacter wolfeii uses CO2 as electron acceptor, producing CH4 as final product. The metabolism of M. wolfeii can be harnessed to capture CO2 from industrial emissions, besides producing a drop-in renewable biofuel to substitute fossil natural gas. If used as biocatalyst in new-generation CO2 sequestration processes, M. wolfeii has the potential to accelerate the decarbonization of the energy generation sector, which is the biggest contributor of CO2 emissions worldwide. Nonetheless, the development of CO2 sequestration archaeal-based biotechnology is still limited by an uncertainty in the requirements to cultivate methanogenic archaea and the unknown longevity of archaeal cultures. In this study, we report the adaptation, isolation, and phenotypic characterization of a novel variant of M. wolfeii, which is capable of maximum growth with minimal nutrients input. Our findings demonstrate the potential of this variant for the production of renewable natural gas, paving the way for the development of more efficient and sustainable CO2 sequestration processes.

Assessing feasible H2-CO2 sources in the US as Feedstocks for Sustainable Aviation Fuel Precursors: Acetic Acid and Ethanol Production via Hydrogenotrophic Pathways.

The environmental impact of carbon dioxide emissions is significant, and research is focused on mitigating these emissions and developing eco-friendly technologies in line with green chemistry principles. Waste-to-energy technologies play a crucial role in converting waste into renewable energy and valuable biofuels and bioproducts. This study specifically explores the utilization of waste gas emissions, particularly carbon dioxide, from various sources in the United States for the production of sustainable aviation fuel (SAF) precursors, such as ethanol and acetic acid. The study categorizes and quantifies the volumes of carbon dioxide emissions into three types: non-biogenic, biogenic, and biogenic emissions from ethanol production facilities. Stoichiometric calculations are applied to compare the amounts of carbon dioxide from each category with the available hydrogen production capacity, determining if sufficient hydrogen is present for converting carbon dioxide into SAF precursors. The study reveals two key findings. Firstly, there is a significant reserve of carbon dioxide, approximately 1648 million metric tons per year (MMTy), combining all three categories, which would require a substantial increase of approximately 35-40 times in the existing hydrogen production capacity of 4.988 MMTy. This increased hydrogen production has the potential to yield approximately 1067.82 MMTy of acetic acid and 189.19 MMTy of ethanol annually. Secondly, upon analyzing the quality and application of the three sources of carbon dioxide with the currently available hydrogen production capacity, it is found that biogenic carbon dioxide from ethanol plants is the most suitable choice for immediate production of SAF precursors. This would theoretically result in an annual production of 1.36 MMTy of ethanol and 1.772 MMTy of acetic acid. The other two sources of carbon dioxide can be considered potential reserves for future utilization when additional hydrogen production facilities are established. The study provides a foundation for assessing the aggregation potential required for acetic acid and ethanol production. By optimizing the use of waste gases as raw materials, the study not only enables the production of SAF precursors but also contributes to the passive reduction of greenhouse gas emissions.

Acetate Production from Syngas Produced from Lignocellulosic Biomass Materials along with Gaseous Fermentation of the Syngas: A Review.

Biotransformation of lignocellulose-derived synthetic gas (syngas) into acetic acid is a promising way of creating biochemicals from lignocellulosic waste materials. Acetic acid has a growing market with applications within food, plastics and for upgrading into a wide range of biofuels and bio-products. In this paper, we will review the microbial conversion of syngas to acetic acid. This will include the presentation of acetate-producing bacterial strains and their optimal fermentation conditions, such as pH, temperature, media composition, and syngas composition, to enhance acetate production. The influence of syngas impurities generated from lignocellulose gasification will further be covered along with the means to alleviate impurity problems through gas purification. The problem with mass transfer limitation of gaseous fermentation will further be discussed as well as ways to improve gas uptake during the fermentation.

Strategies to overcome mass transfer limitations of hydrogen during anaerobic gaseous fermentations: A comprehensive review.

Fermentation of gaseous substrates such as carbon dioxide (CO2) has emerged as a sustainable approach for transforming greenhouse gas emissions into renewable fuels and biochemicals. CO2 fermentations are catalyzed by hydrogenotrophic methanogens and homoacetogens, these anaerobic microorganisms selectively reduce CO2 using hydrogen (H2) as electron donor. However, H2 possesses low solubility in liquid media leading to slow mass transport, limiting the reaction rates of CO2 reduction. Solving the problems of mass transport of H2 could boost the advance of technologies for valorizing industrial CO2-rich streams, like biogas or syngas. The application could further be extended to combustion flue gases or even atmospheric CO2. In this work, an overview of strategies for overcoming H2 mass transport limitations during methanogenic and acetogenic fermentation of H2 and CO2 is presented. The potential for using these strategies in future full-scale facilities and the knowledge gaps for these applications are discussed in detail.

Improved valorization of sewage sludge in the circular economy by anaerobic digestion: Impact of an innovative pretreatment technology.

Anaerobic digestion (AD) of sewage sludge shows low carbon conversion efficiency (CCE) due to the poor biodegradability of sewage sludge. The lack of digestibility is specifically linked to the waste-activated sludge (WAS) making up the majority of sewage sludge along with a smaller portion of primary sludge, depending on the wastewater treatment plant configuration. In this study, we examine the Advanced Wet Oxidation & Steam Explosion process (AWOEx) for improving the CCE of digested sewage sludge (DSS) by thermophilic AD. The effect of the pretreatment temperature in the range between 160 and 185 °C at a fixed residence time of 20 min with and without oxygen added at a dosage of 5 % of the organics present was tested. Methane yield improved by 97.92 % to 183.91 ± 4.93 mL/g vS over the untreated DSS (control), whose methane yield was 92.92 ± 9.07 mL/g vS We have demonstrated for the first time that 84 % of the organics in sewage sludge can successfully be transformed into biogas following AWOEx pretreatment, which can contribute significantly to the circular economy instead of greenhouse gas emissions from landfilling.

Bioaugmentation of Methanosarcina thermophila grown on biochar particles during semi-continuous thermophilic food waste anaerobic digestion under two different bioaugmentation regimes.

This study presents the effect of bioaugmentation of thermophilic anaerobic digestion of food waste with Methanosarcina thermophila grown on a wood-derived biochar. Two different supplementation regimes were tested, namely a single bioaugmentation (SBABC) in which 10% v/v of the microbes grown on biochar (1 g/L) is added at setup of the reactors, versus a routine bioaugmentation (RBABC) wherein the same amount of supplements were added over 10 feeding cycles. The optimally performing 'R' and 'S' reactors had increased methane yields by 37% and 32% over their respective controls while reactors SBABC 2 and 3 produced 21.89% and 56.09% higher average methane yield than RBABC 2 and 3, respectively. It appears that a single dose bioaugmentation is advantageous for improving AD as analysed in terms of average methane yield and VFA production. This study provides the basis for understanding how biochar and bioaugmentation can be used for engineering sustainable pilot-scale AD processes.

Degrading chlorinated aliphatics by reductive dechlorination of groundwater samples from the Santa Susana Field Laboratory.

Microbial reductive dechlorination is one of the chosen methods for remediation of chlorinated compounds in anaerobic environments. In this study we examined the degradation of chlorinated aliphatics in groundwater samples from the Santa Susana Field Laboratory (SSFL) containing a concentration of 0.228 mM trichloroethylene (TCE) and 0.279 mM 1,2 dichloroethylene (DCE). We tested the influence of adding different carbon sources on the dechlorinating activity in batch cultures with and without dechlorinating bacteria. In-situ microcosms were established using SSFL groundwater supplemented with EVO (5%) (vol/vol) SRS emulsion and with or without species of Dehalocococcoides (DCB-1, DCB-2 or DCB-3). Emulsified vegetable oil (EVO) gave the highest dechlorinating activity with DCB-1 added compared to any other substrate addition tested. All three bacterial cultures tested had significant dechlorinating activities while the native populations in the SSFL groundwater samples only showed limited degradation of trichloroethylene into intermediates in the form of DCE, vinyl chloride and ethane. The conversion of chlorinated ethylenes (CEs) was optimal in the bioreactors amended with DCB-1 followed by DCB-2, and DCB-3 all supplemented with EVO. We further analyzed the TCE degradation first order kinetics in batch cultures and found that the culture with DCB-1 supplemented with EVO showed 43.59% and 51.38% increased degradation rate compared to the same condition with cultures of DCB-2 or DCB-3 added. The microcosm studies further showed that with DCB-1 and EVO, reductive dechlorination of TCE in the SSFL converted 90% of the input TCE to ethane with a degradation rate of 0.0039 mM/day.

Influence of wet oxidation pretreatment with hydrogen peroxide and addition of clarified manure on anaerobic digestion of oil palm empty fruit bunches.

Food and energy requirements are increasing globally, and the challenge is to meet these demands in a sustainable manner. Oil palm has a relatively high productivity, but produces the lignocellulosic residue of empty fruit bunches (OPEFB). In this study, wet oxidation pretreatment is utilized to overcome the recalcitrance of OPEFB during semi-continuous anaerobic digestion (AD) with between 19.7 and 52.7% improvement over the control, and near total cellulose and hemicellulose content could be degraded. Clarified manure, the water phase of cattle and dairy manure after filtration, is further tested for its effect on methane production by providing necessary micronutrients and vitamins. An increase of 49% was found after addition of clarified manure to OPEFB compared to without this addition.

Increasing the Production of Volatile Fatty Acids from Corn Stover Using Bioaugmentation of a Mixed Rumen Culture with Homoacetogenic Bacteria.

Volatile fatty acids (VFA) are industrially versatile chemicals and have a major market. Although currently produced from petrochemicals, chemical industries are moving towards more bio-based VFA produced from abundant, cheap and renewable sources such as lignocellulosic biomass. In this study, we examined the effect of bioaugmentation with homoacetogenic bacteria for increasing VFA production in lignocellulose fermentation process. The central hypothesis of this study was that inhibition of methanogenesis in an in vitro rumen bioreactor fed with lignocellulosic biomass hydrolysate increases the hydrogen partial pressure, which can be redirected towards increased VFA production, particularly acetic acid, through targeted bioaugmentation with known homoacetogenic bacteria. In this study, methanogenesis during ruminal fermentation of wet exploded corn stover was initially inhibited with 10 mM of 2-bromoethanesulfonate (BES), followed by bioaugmentation with either Acetitomaculum ruminis and Acetobacterium woodii in two separate bioreactors. During the inhibition phase, we found that addition of BES decreased the acetic acid yield by 24%, while increasing headspace hydrogen from 1% to 60%. After bioaugmentation, the headspace hydrogen was consumed in both bioreactors and the concentration of acetic acids increased 45% when A. ruminis was added and 70% with A. woodii added. This paper demonstrates that mixed microbial fermentation can be manipulated to increase VFA production through bioaugmentation.

Sugar Production from Hybrid Poplar Sawdust: Optimization of Enzymatic Hydrolysis and Wet Explosion Pretreatment.

Wet explosion pretreatment of hybrid poplar sawdust (PSD) for the production of fermentable sugar was carried out in the pilot-scale. The effects of pretreatment conditions, such as temperature (170-190 °C), oxygen dosage (0.5-7.5% of dry matter (DM), w/w), residence time (10-30 min), on cellulose and hemicellulose digestibility after enzymatic hydrolysis were ascertained with a central composite design of the experiment. Further, enzymatic hydrolysis was optimized in terms of temperature, pH, and a mixture of CTec2 and HTec2 enzymes (Novozymes). Predictive modeling showed that cellulose and hemicellulose digestibility of 75.1% and 83.1%, respectively, could be achieved with a pretreatment at 177 °C with 7.5% O2 and a retention time of 30 min. An increased cellulose digestibility of 87.1% ± 0.1 could be achieved by pretreating at 190 °C; however, the hemicellulose yield would be significantly reduced. It was evident that more severe conditions were required for maximal cellulose digestibility than that of hemicellulose digestibility and that an optimal sugar yield demanded a set of conditions, which overall resulted in the maximum sugar yield.

Disruption and overexpression of 6-phosphofructo-2-kinase influence organic acid production in Aspergillus carbonarius ITEM 5010.

Aspergillus carbonarius is an efficient producer of organic acids with great potential for bio-based production of organic acids. In this study, we identified a gene f2kp encoding the enzyme 6-phosphofructo-2-kinase known as an allosteric regulator of the glycolytic flux and investigated its role in the production of organic acid. The strategy was to examine the impact of citric acid and malic acid production by overexpressing and disrupting f2kp, respectively. The overexpressing transformants expressed f2kp at higher level than the wild type, whereas no expression of f2kp was detected in the knockout transformants. Citric acid and malic acid production by the knockout strains decreased sharply along with a significant lower sugar consumption, though the overexpressing transformants produced similar amounts of citric acid and malic acid as the wild type. We conclude that 6-phosphofructo-2-kinase has an important regulatory role for the glycolytic flux and organic acid production in A. carbonarius.

Rheology of Polyacrylonitrile/Lignin Blends in Ionic Liquids under Melt Spinning Conditions.

Lignin, while economically and environmentally beneficial, has had limited success in use in reinforcing carbon fibers due to harmful chemicals used in biomass pretreatment along with the limited physical interactions between lignin and polyacrylonitrile (PAN) during the spinning process. The focus of this study is to use lignin obtained from chemical-free oxidative biomass pretreatment (WEx) for blending with PAN at melt spinning conditions to produce carbon fiber precursors. In this study, the dynamic rheology of blending PAN with biorefinery lignin obtained from the WEx process is investigated with the addition of 1-butyl-3-methylimidazolium chloride as a plasticizer to address the current barriers of developing PAN/lignin carbon fiber precursors in the melt-spinning process. Lignin was esterified using butyric anhydride to reduce its hydrophilicity and to enhance its interactions with PAN. The studies indicate that butyration of the lignin (BL) increased non-Newtonian behavior and decreased thermo-reversibility of blends. The slope of the Han plot was found to be around 1.47 for PAN at 150 °C and decreased with increasing lignin concentrations as well as temperature. However, these blends were found to have higher elasticity and solution yield stress (47.6 Pa at 20%wt BL and 190 °C) when compared to pure PAN (5.8 Pa at 190 °C). The results from this study are significant for understanding lignin-PAN interactions during melt spinning for lower-cost carbon fibers.

Identification of metabolite and protein explanatory variables governing microbiome establishment and re-establishment within a cellulose-degrading anaerobic bioreactor.

Proteins, metabolites, and 16S rRNA measurements were used to examine the community structure and functional relationships within a cellulose degrading anaerobic bioreactor. The bioreactor was seeded with bovine rumen fluid and operated with a 4 day hydraulic retention time on cellulose (avicel) as sole carbon and energy source. The reactor performance and microbial community structure was monitored during the establishment of the cellulose-degrading community. After stable operation was established in the bioreactor, the mixing intensity was increased in order to investigate the effect of a physical disruption of the microbial community structure. Finally, the original conditions were re-established to understand the stability of the microbial community after a perturbation. All factors measured were found to be inter-correlated during these three distinct phases of operation (establishment, perturbation and re-establishment). In particular, the return of community structure and function to pre-perturbed conditions suggests that propionate fermentation and acetate utilization were the explanatory factors for community establishment and re-establishment.

Anaerobic digestion of organic fraction from hydrothermal liquefied algae wastewater byproduct.

The wastewater stream from hydrothermal liquefaction (HTL) process used in biofuel production, contains a large amounts of organic compounds where several can be regarded as environmentally hazardous and requires significant treatment before disposal. In this study, semi-continuous anaerobic digestion is used to degrade the organic fraction of wastewater streams from HTL of the algae Tetraselmis (AgTet) and Chlorella (AgChlr). Results indicated high methane yields at 20-30% (v/v) HTL wastewater together with clarified manure, producing 327.2mL/gVSin (or volatile solids in feed) for AgTet and 263.4mL/gVSin for AgChlr. There was a significant reduction in methane production at concentrations higher than 40% (v/v) HTL wastewater in the feed, possibly due to the accumulation of chloride salts or inhibitory compounds such as pyridines, piperidines and pyrrolidines. This was further confirmed by comparing COD, salt and the ammonia concentrations of the effluents after anaerobic digestion at different concentrations of wastewater in manure.

A comparison of Agrobacterium-mediated transformation and protoplast-mediated transformation with CRISPR-Cas9 and bipartite gene targeting substrates, as effective gene targeting tools for Aspergillus carbonarius.

In recent years, versatile genetic tools have been developed and applied to a number of filamentous fungi of industrial importance. However, the existing techniques have limitations when it comes to achieve the desired genetic modifications, especially for efficient gene targeting. In this study, we used Aspergillus carbonarius as a host strain due to its potential as a cell factory, and compared three gene targeting techniques by disrupting the ayg1 gene involved in the biosynthesis of conidial pigment in A. carbonarius. The absence of the ayg1 gene leads to phenotypic change in conidia color, which facilitated the analysis on the gene targeting frequency. The examined transformation techniques included Agrobacterium-mediated transformation (AMT) and protoplast-mediated transformation (PMT). Furthermore, the PMT for the disruption of the ayg1 gene was carried out with bipartite gene targeting fragments and the recently adapted CRISPR-Cas9 system. All three techniques were successful in generating Δayg1 mutants, but showed different efficiencies. The most efficient method for gene targeting was AMT, but further it was shown to be dependent on the choice of Agrobacterium strain. However, there are different advantages and disadvantages of all three gene targeting methods which are discussed, in order to facilitate future approaches for fungal strain improvements.

Alkane biosynthesis by Aspergillus carbonarius ITEM 5010 through heterologous expression of Synechococcus elongatus acyl-ACP/CoA reductase and aldehyde deformylating oxygenase genes.

In this study we describe the heterologous expression of the recently identified cyanobacterial pathway for long chain alkane biosynthesis, involving the reduction of fatty acyl-ACP to fatty aldehyde and the subsequent conversion of this into alkanes, in the filamentous fungus Aspergillus carbonarius ITEM 5010. Genes originating from Synechococcus elongatus strain PCC7942, encoding acyl-ACP/CoA reductase and aldehyde deformylating oxygenase enzymes, were successfully expressed in A. carbonarius, which lead to the production of pentadecane and heptadecane, alkanes that have not been previously produced by this fungus. Titers of 0.2, 0.5 and 2.7 mg/l pentadecane and 0.8, 1.6 and 10.2 mg/l heptadecane were achieved using glucose, Yeast malt and oatmeal media, respectively. Besides producing alkanes, we found elevated levels of internal free fatty acids and triglycerides in the alkane producing transformant. These findings can indicate that a yet unidentified, native fatty aldehyde dehydrogenase channels back the fatty aldehydes into the fatty acid metabolism, thus competing for substrate with the heterologously expressed fatty aldehyde deformylating oxygenase. These findings will potentially facilitate the future application of robust, fungal cell factories for the production of advanced biofuels from various substrates.

Properties of plant nutrient: Comparison of two nutrient recovery techniques using liquid fraction of digestate from anaerobic digester treating pig manure.

Anaerobic digestate has valuable potential as organic fertilizer or soil amendment, given that it typically contains high amounts of plant nutrients, such as nitrogen, phosphate and plant hormones. In this study, ammonia stripping and vacuum evaporation were tested to compare their technical feasibilities and their effects on plant nutrient properties in the liquid fraction of digestate. Results of the batch experiments showed that the nutrient characteristics of liquid digestate, including total ammonia nitrogen (TAN), soluble P, gibberellic acid (GA), indoleacetic acid (IAA) and abscisic acid (ABA), were strongly dependent on the initial pH in both ammonia stripping and vacuum evaporation processes. A low plant nutrient concentration (TAN 137 mg · L(-1), soluble P 1.5 mg · L(-1), GA3/ABA 0.04) in the liquid digestate was achieved in the ammonia stripping process with Ca(OH)2 addition of 12 g · L(-1), whereas a high nutrient concentration (TAN 2998 mg · L(-1), soluble P 178.3 mg · L(-1), IAA 60.9 mg · L(-1) and GA3/ABA 0.4) was achieved in vacuum evaporation at a pH level of 6. According to the results, both ammonia stripping and vacuum evaporation can be used as an alternative of nutrient recovery techniques, which should be chosen based on the potential different applications of liquid digestate (e.g., soaking seed, increasing plant tolerance, and nutrients transportation).

Enhanced succinic acid production in Aspergillus saccharolyticus by heterologous expression of fumarate reductase from Trypanosoma brucei.

Aspergillus saccharolyticus exhibits great potential as a cell factory for industrial production of dicarboxylic acids. In the analysis of the organic acid profile, A. saccharolyticus was cultivated in an acid production medium using two different pH conditions. The specific activities of the enzymes, pyruvate carboxylase (PYC), malate dehydrogenase (MDH), and fumarase (FUM), involved in the reductive tricarboxylic acid (rTCA) branch, were examined and compared in cells harvested from the acid production medium and a complete medium. The results showed that ambient pH had a significant impact on the pattern and the amount of organic acids produced by A. saccharolyticus. The wild-type strain produced higher amount of malic acid and succinic acid in the pH buffered condition (pH 6.5) compared with the pH non-buffered condition. The enzyme assays showed that the rTCA branch was active in the acid production medium as well as the complete medium, but the measured enzyme activities were different depending on the media. Furthermore, a soluble NADH-dependent fumarate reductase gene (frd) from Trypanosoma brucei was inserted and expressed in A. saccharolyticus. The expression of the frd gene led to an enhanced production of succinic acid in frd transformants compared with the wild-type in both pH buffered and pH non-buffered conditions with highest amount produced in the pH buffered condition (16.2 ± 0.5 g/L). This study demonstrates the feasibility of increasing succinic acid production through the cytosolic reductive pathway by genetic engineering in A. saccharolyticus.

Pretreatment of forest residues of Douglas fir by wet explosion for enhanced enzymatic saccharification.

The logging and lumbering industry in the Pacific Northwest region generates huge amount of forest residues, offering an inexpensive raw material for biorefineries. Wet explosion (WEx) pretreatment was applied to the recalcitrant biomass to optimize process conditions including temperature (170-190 °C), time (10-30 min), and oxygen loading (0.5-7.5% of DM) through an experimental design. Optimal pH for enzymatic hydrolysis of the optimized samples and a complete mass balance have been evaluated. Results indicated that cellulose digestibility improved in all conditions tested with maximum digestibility achieved at 190 °C, time 30 min, and oxygen loading of 7.5%. Glucose yield at optimal pH of 5.5 was 63.3% with an excellent recovery of cellulose and lignin of 99.9% and 96.3%, respectively. Hemicellulose sugars recovery for xylose and mannose was found to be 69.2% and 76.0%, respectively, indicating that WEx is capable of producing relative high sugar yield even from the recalcitrant forest residues.

Making lignin accessible for anaerobic digestion by wet-explosion pretreatment.

Lignin is a major part of the recalcitrant fraction of lignocellulose and in nature its degradation occurs through oxidative enzymes along with microbes mediated oxidative chemical actions. Oxygen assisted wet-explosion pretreatment promotes lignin solubility and leads to an increase biodegradation of lignin during anaerobic digestion processes. The pretreatment of feedlot manure was performed in a 10L reactor at 170°C for 25min using 4bars oxygen and the material was fed to a continuous stirred tank reactor operated at 55°C for anaerobic digestion. Methane yield of untreated and pretreated material was 70±27 and 320±36L/kg-VS/Day, respectively, or 4.5 times higher yield as a result of the pretreatment. Aliphatic acids formed during the pretreatment were utilized by microbes. 44.4% lignin in pretreated material was actually converted in the anaerobic digestion process compared to 12.6% for untreated material indicating the oxygen assisted explosion promoted lignin degradation.