Thermoanaerobacterium Thermosaccharolyticum Research Articles

The intracellular accumulation of iron coincides with enhanced biohydrogen production by Thermoanaerobacterium thermosaccharolyticum

The aim of this study was to investigate the effects of different doses (10–200 mg/L) of magnetite (FeO·Fe2O3), Fe0, Ni0, and FeCl3 on the efficiency of dark fermentative H2 production from cheese whey using Thermoanaerobacterium thermosaccharolyticum SP-H2. Energy conversion efficiency and hydrogen yield increased significantly by 22.7 % and 34.8 %, respectively, when 10 mg/L of magnetite was added, and hydrogenase activity increased by 23.6 % when 100 mg/L of Fe0 was added compared to the control (p < 0.001). Using scanning electron microscopy and energy dispersive X-ray spectroscopy, it was found that during the early exponential phase of hydrogen evolution, T. thermosaccharolyticum cells accumulated up to 11.2 times more Fe (up to 2.8 wt%) in the presence of optimal concentrations of Fe additives compared to the control. Intracellular iron accumulation and hydrogen yield were positively correlated with acetate concentration (p < 0.001), suggesting an efficient acetate pathway for dark fermentation. During the stationary growth phase, with almost no H2 production, the intracellular iron content decreased by up to 67.9 %. The phenomenon of changes in the amount of metal in cells, depending on the phase of hydrogen production, was demonstrated for the first time. The findings suggest that intracellular metals can be used in cell metabolism to produce H2, and the mechanisms behind this process need further investigation. Overall, this study provides valuable insights into the optimal forms and doses of metals for enhancing biohydrogen production, as well as possible research directions for the potential use of metals by microorganisms during additive-assisted dark fermentation of carbohydrate-rich wastes.

Enhancement of biohythane production from palm oil mill effluent by Thermoanaerobacterium thermosaccharolyticum PSU-2 and methanogenic mixed cultures using a thermophilic two-ring bioreactor

A novel thermophilic two-ring bioreactor was employed to produce biohythane from palm oil mill effluent (POME) using Thermoanaerobacterium thermosaccharolyticum PSU-2 and thermophilic methanogenic mixed cultures. The reactor system demonstrated impressive performance, achieving hydrogen and methane yields of 113.3 ± 15.0 mL/g-VS and 473.0 ± 60.0 mL/g-VS, respectively, with a total biohythane composition of 4.3 % H2, 37.1 % CO2, and 58.6 % CH4. The process exhibited high treatment efficiency, with COD and VS removal efficiencies reaching 93.7 % and 84.3 %, respectively. Microbial community analysis revealed the crucial roles of various microorganisms in the biohythane production process. Thermoclostridium sp., Thermoanaerobacterium sp., and Anaerobranca sp. were identified as key players in hydrogen production, while Bacteroides sp. and Methanobacterium sp. were found to be essential for methane production. The optimization of operating conditions, including pH (5.0–8.0), temperature (55 °C), and hydraulic retention time (2 d for H2 and 10 d for CH4), significantly enhanced biohythane production efficiency. The techno-economic analysis demonstrated the economic viability of the thermophilic two-ring bioreactor system, with a net present value of 4,186,000 USD, an internal rate of return of 82 %, and a payback period of 1.4 years. These findings highlight the potential of this innovative technology as a sustainable and economically attractive solution for treating POME and simultaneously producing renewable energy in the form of biohythane, contributing to the sustainable development of the palm oil industry and the mitigation of greenhouse gas emissions.

Characterization of sugarcane bagasse solubilization and utilization by thermophilic cellulolytic and saccharolytic bacteria at increasing solid loadings

In Brazil the main feedstock used for ethanol production is sugarcane juice, resulting in large amounts of bagasse. Bagasse has high potential for cellulosic ethanol production, and consolidated bioprocessing (CBP) has potential for lowering costs. However, economic feasibility requires bioprocessing at high solids loadings, entailing engineering and biological challenges. This study aims to document and characterize carbohydrate solubilization and utilization by defined cocultures of Clostridium thermocellum and Thermoanaerobacterium thermosaccharolyticum at increasing loadings of sugarcane bagasse. Results show that fractional carbohydrate solubilization decreases as solids loading increases from 10 g/L to 80 g/L. Cocultures enhance solubilization and carbohydrate utilization compared to monocultures, irrespective of initial solids loading. Rinsing bagasse before fermentation slightly decreases solubilization. Experiments studying inhibitory effects using spent media and dilution of broth show that negative effects are temporary or reversible. These findings highlight the potential of converting sugarcane bagasse via CBP, pointing out performance limitations that must be addressed.

Solubilization of sugarcane bagasse by mono and cocultures of thermophilic anaerobes with and without cotreatment

Cotreatment, mechanical disruption of lignocellulosic biomass during microbial fermentation, is a potential alternative to thermochemical pretreatment as a means of increasing the accessibility of lignocellulose to biological attack. Successful implementation of cotreatment requires microbes that can withstand milling, while solubilizing and utilizing carbohydrates from lignocellulose. In this context, cotreatment with thermophilic, lignocellulose-fermenting bacteria has been successfully evaluated for a number of lignocellulosic feedstocks. Here, cotreatment was applied to sugarcane bagasse using monocultures of the cellulose-fermenting Clostridium thermocellum and cocultures with the hemicellulose-fermenting Thermoanaerobacterium thermosaccharolyticum. This resulted in 76 % carbohydrate solubilization (a 1.8-fold increase over non-cotreated controls) on 10 g/L solids loading, having greater effect on the hemicellulose fraction. With cotreatment, fermentation by wild-type cultures at low substrate concentrations increased cumulative product formation by 45 % for the monoculture and 32 % for the coculture. These findings highlight the potential of cotreatment for enhancing deconstruction of sugarcane bagasse using thermophilic bacteria.

Biological H2(g) Production and Modelling with Computational Fluid Dynamics (CFD)

In this study, bio-hydrogen gas [bio-H2(g)] production and modeling with a three-phase computational fluid dynamics (CFD) model, heat and mass transfer of bio-hydrogen production, reaction kinetics, and fluid dynamics; It was investigated by dark fermentation process in an anaerobic continuous plug flow reactor (ACPFR). The three-phase CFD model was used to determine the bio-H2(g) production in an ACPFR. The effect of different operating parameters, increasing hydrolic retention times (HRTs) (1, 2, 4, 8, and 12 days), different pH values (4.0, 5.0, 6.0, 7.0, and 8.0), and increasing feed rate as organic loading rates (OLRs) (0.5, 1.0, 2.0, 4.0, 8.0 and 10.0 g COD/l.d) on the bio-H2(g) production rates were operated in municipal sludge wastes (MSW) with Thermoanaerobacterium thermosaccharolyticum SP-H2 methane bacteria during dark fermentation for bio-H2(g) production. The effect of HRT, pH, and feed rate on the bioH2(g) efficiencies and H2(g) production rates were examined in the simulation stage. Production of volatile fatty acids (VFAs) namely, acetic acids, butyric acids, and propionic acids were important points influencing the bio-H2(g) production yields. The artificial neural network (ANN) model substrate inhibition on bio-H2(g) production to the methane (CH4) bacteria was also investigated. The reaction kinetics model used Thermotoga neapolitana microorganisms with the Andrews model of substrate inhibition. Furthermore, the ANN model was well-fitted to the experimental data to simulate the bio-H2(g) production from chemical oxygen demand (COD).

Metabolic engineering of Thermoanaerobacterium aotearoense strain SCUT27 for biofuels production from sucrose and molasses

BackgroundSucrose-rich sugarcane trash surpasses 28 million tons globally per year. Effective biorefinery systems could convert these biomasses to bioproducts, such as bioethanol from sugarcane sucrose in Brazil. Thermophilic microbes for biofuels have attracted great attention due to their higher fermentation temperature and wide substrate spectrum. However, few thermophiles using sucrose or molasses for biofuels production was reported. Thermoanaerobacterium aotearoense SCUT27 has been considered as an efficient ethanol producer, but it cannot directly utilize sucrose. In this study, various sucrose metabolic pathways were introduced and analyzed in Thermoanaerobaterium.ResultsThe sucrose-6-phosphate hydrolase (scrB), which was from a screened strain Thermoanaerobacterium thermosaccharolyticum G3-1 was overexpressed in T. aotearoense SCUT27 and endowed this strain with the ability to utilize sucrose. In addition, overexpression of the sucrose-specific PTS system (scrA) from Clostridium acetobutylicum accelerated the sucrose transport. To strengthen the alcohols production and substrates metabolism, the redox-sensing transcriptional repressor (rex) in T. aotearoense was further knocked out. Moreover, with the gene arginine repressor (argR) deleted, the ethanologenic mutant P8S10 showed great inhibitors-tolerance and finally accumulated ~ 34 g/L ethanol (a yield of 0.39 g/g sugars) from pretreated cane molasses in 5 L tank by fed-batch fermentation. When introducing butanol synthetic pathway, 3.22 g/L butanol was produced by P8SB4 with a yield of 0.44 g alcohols/g sugars at 50℃. This study demonstrated the potential application of T. aotearoense SCUT27 for ethanol and butanol production from low cost cane molasses.ConclusionsOur work provided strategies for sucrose utilization in thermophiles and improved biofuels production as well as stress tolerances of T. aotearoense SCUT27, demonstrating the potential application of the strain for cost-effective biofuels production from sucrose-based feedstocks.

Metabolic division of labor between Acetivibrio thermocellus DSM 1313 and Thermoanaerobacterium thermosaccharolyticum MJ1 enhanced hydrogen production from lignocellulose

In this consortium, DSM 1313 was responsible for degrading lignocellulose by cellulosome, while the highly efficient hydrogen-producing bacterium MJ1 consumed the sugar produced by DSM 1313 to grow and produce more hydrogen. The results showed that the maximum hydrogen production of 259.57 mL/g substrate was obtained at the inoculation ratio (OD600) of 2:1 (DSM 1313:MJ1) and substrate concentration of 10 g/L, 70.84 % higher than pure culture. Furthermore, MJ1 dominated the co-culture system by using various sugars resulting from the biodegradation of substrate, thereby relieving the inhibition of sugar on DSM 1313 and leading to more hydrogen production. In the co-culture system, the value of extracellular oxidation–reduction potential and the ratio of NAD+/NADH was lower than that of pure culture. Additionally, at the gene level, [NiFe]-hydrogenase and [FeFe]-hydrogenase related enzymes were significantly up-regulated, leading to a two-fold increase in hydrogenase activity of co-culture compared with pure culture.

Comparative genomic analysis reveals electron transfer pathways of Thermoanaerobacterium thermosaccharolyticum: Insights into thermophilic electroactive bacteria

Microbial extracellular respiration is an important energy metabolism on earth, which is significant for the elemental biogeochemical cycle. Herein, extracellular Fe(III) and electrode respiration were confirmed in Thermoanaerobacterium thermosaccharolyticum MJ2. The intra/extracellular electron transfer (IET/EET) mechanism of MJ2 was investigated by comparative genomic analysis for the first time. Morphological characterization and electrochemical properties of anode illustrated that MJ2 generated bio-electricity by forming a biofilm. The respiration chain inhibition and enzyme activity tests showed that hydrogenase with cytochrome c (Cyt-c) was involved in IET of MJ2. Noteworthily, the exogenous Cyt-c increased hydrogenase activity to promote bio-electricity generation by 92.84 %. The Cyt-c gene synteny between MJ2 and another well-known exoelectrogen (Thermincola potens JR) indicated that Cyt-c bound to the outer membrane mediated the formation of biofilm involved in EET of MJ2. This study broadened the understanding of microbial extracellular respiration diversity and provided new insights to explore the electron transfer pathways of exoelectrogens.

Biochemical hydrogen potential assay for predicting the patterns of the kinetics of semi-continuous dark fermentation

The performance and kinetics response of thermophilic semi-continuous dark fermentation (DF) of simulated complex carbohydrate-rich waste was investigated at various hydraulic retention times (HRT) (2, 2.5, and 3 d) and compared with data obtained from biochemical hydrogen potential assay (BHP). A culture of Thermoanaerobacterium thermosaccharolyticum was used as the inoculum and dominated both in BHP and semi-continuous reactor. Both the modified Gompertz and first-order models described the DF kinetics well (R2 = 0.97–1.00). HRT of 2.5 d was found to be optimal in terms of maximum hydrogen production rate and hydrogen potential, which were 3.97 and 1.26 times higher, respectively, than in BHP. The hydrolysis constant was highest at HRT of 3 d and was closest to the value obtained in the BHP. Overall, BHP has been shown to be a useful tool for predicting H2 potential and the hydrolysis constant, while the maximum H2 production rate is greatly underestimated.

Biochar prepared from human feces (HFBC) as a promoter in biohydrogen production using simulated feces as substrate

Human feces contain a lot of nutrients and organic matter, which can be used in many fields. In this study, biochar (HFBC) was prepared at 300, 500 and 800 °C using human feces as substrate, and the characteristics of HFBC prepared at different pyrolysis temperatures were investigated. It was confirmed that HFBC prepared at 800 ℃ (HFBC-800) had a better pH buffer capacity, more proportion of π-π shake-up satellites, better graphitization degree and higher conductivity, which could promote the efficiency of biohydrogen production of bacteria. Furthermore, the biohydrogen production of Thermoanaerobacterium thermosaccharolyticum M18 using different concentrations of simulated human feces (SHF) was further investigated. The best biohydrogen production was found for 40 wt%-SHF, with 128.54 mmol H2/L produced in 180 h. The highest biohydrogen production of 148.75 mmol H2/L was obtained by adding 6 g/L HFBC-800 to the biohydrogen production process. This study provides a feasibility for high-performance hydrogen production from human feces and presents an insight into the resource utilization of human waste.

Effects of adding Thermoanaerobacterium thermosaccharolyticum in the hydrogen production stage of a two-stage anaerobic digestion system on hydrogen-methane production and microbial communities

Microorganisms play a vital role in anaerobic digestion, however, bioaugmentation in a two-stage co-digestion system to directly regulate the microbial structure is limited. In this study, the different dosage of Thermoanaerobacterium thermosaccharolyticum was added in the hydrogen production stage of a two-stage anaerobic digestion system operating at a thermophilic temperature (55 °C). Results showed that Thermoanaerobacterium thermosaccharolyticum of 1.12 g had the best effect, with the cumulative hydrogen and methane yields reaching 81.54 mL/g VS and 550.98 mL/g VS respectively, which were 68.72 % and 84.45 % higher than the control group. According to the microbial analysis, the addition of Thermoanaerobacterium thermosaccharolyticum significantly changed the microbial community structure, and also its relative abundance increased in the hydrogen production stage, thereby increasing volatile fatty acids (VFAs) and hydrogen content. Although the methane production stage did not contain Thermoanaerobacterium thermosaccharolyticum, the microbial community structure changed, increasing the dominant species Anaerolineae and Limnochordia, thereby improving the efficiency of substrate decomposition and methane production. This study shows that Thermoanaerobacterium thermosaccharolyticum can promote not only hydrogen production but also the production of methane in two-stage anaerobic digestion.

Growth-uncoupled propanediol production in a Thermoanaerobacterium thermosaccharolyticum strain engineered for high ethanol yield

Cocultures of engineered thermophilic bacteria can ferment lignocellulose without costly pretreatment or added enzymes, an ability that can be exploited for low cost biofuel production from renewable feedstocks. The hemicellulose-fermenting species Thermoanaerobacterium thermosaccharolyticum was engineered for high ethanol yield, but we found that the strains switched from growth-coupled production of ethanol to growth uncoupled production of acetate and 1,2-propanediol upon growth cessation, producing up to 6.7 g/L 1,2-propanediol from 60 g/L cellobiose. The unique capability of this species to make 1,2-propanediol from sugars was described decades ago, but the genes responsible were not identified. Here we deleted genes encoding methylglyoxal reductase, methylglyoxal synthase and glycerol dehydrogenase. Deletion of the latter two genes eliminated propanediol production. To understand how carbon flux is redirected in this species, we hypothesized that high ATP levels during growth cessation downregulate the activity of alcohol and aldehyde dehydrogenase activities. Measurements with cell free extracts show approximately twofold and tenfold inhibition of these activities by 10 mM ATP, supporting the hypothesized mechanism of metabolic redirection. This result may have implications for efforts to direct and maximize flux through alcohol dehydrogenase in other species.

Feasibility of successive hydrogen and methane production in a single-reactor configuration of batch anaerobic digestion through bioaugmentation and stimulation of hydrogenase activity and direct interspecies electron transfer

Two-stage anaerobic digestion is a promising way to increase the efficiency of decomposition of organic waste. However, spatial separation of stages makes this technology less economically attractive. In this work, we studied a model of two-stage anaerobic digestion with stages separated in time in one reactor. To implement this model, we used a combination of (1) bioaugmentation with a hydrogen-producing Thermoanaerobacterium thermosaccharolyticum SP-H2, resistant to low pH, (2) setting a low initial pH (5.5) to reduce the activity of hydrogenotrophic methanogens, (3) supplementing additives in the form of soluble iron (II) and granular activated carbon (GAC) to stimulate hydrogenase activity and direct interspecies electron transfer (DIET), respectively. For comparison, two methanogenic inoculums with different dominant microbial groups and initial iron concentrations were used. With the simultaneous addition of GAC and iron (II), sequential production of hydrogen and methane was observed for both methanogenic inoculums. Without the addition of GAC, methane formation was practically not observed, which, apparently, indicated the impossibility of syntrophic degradation of acidogenesis products due to the high partial pressure of hydrogen and the absence of conductive material for activation of DIET. Significant differences in hydrogenase activity and the kinetics of hydrogen and methane formation according to the modified Gompertz equation depending on the iron content in the system may indicate the importance of this additive. Further studies should be aimed at determining the optimal initial pH, concentrations of stimulating additives, bioaugmentation and methanogenic cultures, as well as confirming the stability of the proposed single-reactor two-stage anaerobic digestion system in semi-continuous mode.

Thermophilic dark fermentation fast start-up of hydrogen production with substrate concentration regulation and moderate pretreatment inoculum

Accelerating start-up of thermophilic dark fermentation would improve hydrogen production efficiency was investigated. To find an efficient method for accelerating start-up and explore the effect of inoculum type for hydrogen production system, three thermophilic hydrogen production EGSB (expanded granular sludge bed) reactors equipped with flocculent or granular sludge using different startup modes were constructed in this study. Among three reactors under butyric acid type of fermentation, EGSB3 (startup mode: pretreated granular sludge as inoculum and regulated substrate concentration) was the most efficient one with shortest start-up time (13 days) and maximum hydrogen production (1.64 mol H2/mol glucose). EGSB1 (startup mode: pretreated flocculent sludge as inoculum and regulated substrate concentration) was started up in 27 days at 1.27 mol H2/mol glucose in maximum. While EGSB2 (startup mode: original granular sludge as inoculum and without regulating substrate concentration) took 46 days for start-up and the 1.6 mol H2/mol glucose hydrogen producing rate was obtained. After start-up, the dominant hydrogen production bacteria were Thermoanaerobacterium thermosaccharolyticum strain TG57 in three reactors. EGSB3 reactor owned the most proportion with 53.21 % than others. In all, mild pretreatment and regulating substrate concentration for granular sludge would be benefit for fast startup of thermophilic dark fermentation hydrogen production leading to shortest startup time, high hydrogen production efficiency and most functional microorganism.

Enhancing biohydrogen gas production in anaerobic system via comparative chemical pre-treatment on palm oil mill effluent (POME)

Biological hydrogen production using palm oil mill effluent (POME) as a carbon source through dark fermentation process has been suggested to be a promising bioenergy potential and enacts as alternative renewable energy source. Results have indicated that among various 1.5% (w/v) chemical pre-treatments (sodium hydroxide, NaOH; hydrochloric acid, HCl; sulphuric acid, H2SO4; phosphoric acid, H3PO4 and nitric acid, HNO3) on POME, using H3PO4 would generate maximum biohydrogen production of 0.193 mmol/L/h, which corresponded to a yield of 1.51 mol H2/mol TCconsumed with an initial total soluble carbohydrate concentration of 23.52 g/L. Among H3PO4 concentrations (1%–10%), the soluble carbohydrate content and the biohydrogen produced was highest and increased by 1.70-fold and 2.35-fold respectively at 2.5% (w/v), as compared to untreated POME. The batch fermentation maximum hydrogen production rate and yield of 0.208 mmol/L/h and 1.69 mol H2/mol TCconsumed were achieved at optimum pre-treatment conditions of pH 5.5 and thermophilic temperature (60 °C). This study suggests that chemical pre-treatment approaches manage to produce and improve the carbohydrate utilisation process further. Continuous fermentation in CSTR at the optimum conditions produce heightened 1.5-fold biohydrogen yield for 2.5% H3PO4 at 6 h HRT as compared to batch scale. Bacterial community via next-generation sequencing analysis at optimum HRT (6 h) revealed that Thermoanaerobacterium thermosaccharolyticum registered the highest relative frequency of 20.24%. At the class level, Clostridia, Bacilli, Bacteroidia, Thermoanaerobacteria, and Gammaproteobacteria were identified as the biohydrogen-producing bacteria in the continuous system. Insightful findings from this study suggest the substantial practical utility of dilute chemical pre-treatment in improving biohydrogen production.

Cloning, expression and characterization of a glycoside hydrolase family 51 α-l-arabinofuranosidase from Thermoanaerobacterium thermosaccharolyticum DSM 571.

The present study focused on the characterization of a glycoside hydrolase 51 family α-l-arabinofuranosidase named TtAbf51 from thermophile Thermoanaerobacterium thermosaccharolyticum DSM 571. The recombinant TtAbf51 with 497 amino acids was successfully expressed in Escherichia coli BL21(DE3) and purified via nickel affinity chromatography, and native protein was a dimer verified by size exclusion chromatography. The TtAbf51 showed an optimum pH and temperature of 5.5 and 55°C, and was relatively stable at pH 5.0-8.0 and up to 60°C for 2h of incubation. In addition, TtAbf51 was significantly inhibited by Cu2+, Zn2+ and 1mM or 10mM SDS. In the presence of 800mM arabinose, the residual activity remained over 40% of the initial activity. In addition, the recombinant enzyme possessed a good catalytic effect for both synthesized and natural substrates, and the specific enzyme activity toward CM-linear arabinan reached 426.5μmolmin-1mg-1. In summary, this study provides an α-l-arabinofuranosidase with potential in the synergistic hydrolysis of hemicellulose to fermentable sugars in applications such as liquid biofuels, food and beverages, and related industries.

Dark fermentative hydrogen production from simple sugars and various wastewaters by a newly isolated Thermoanaerobacterium thermosaccharolyticum SP-H2

The hydrogen-producing bacterium SP-H2 was isolated from a thermophilic acidogenic reactor inoculated with municipal sewage sludge and processing a carbohydrate-rich simulated food waste. Based on the 16S rRNA gene sequence, the bacterium was identified as Thermoanaerobacterium thermosaccharolyticum. The maximum growth rate was observed at 55–60 °C and pH 7.5. The H2-producing activity of the bacterium was studied using mono-, di- and tri-saccharides related to both hexoses (maltose, glucose, mannose, fructose, lactose, galactose, sucrose, raffinose, cellobiose) and pentoses (xylose and arabinose), as well as using real wastewaters (cheese whey, confectionery wastewater, sugar-beet processing wastewater). The highest H2 yield was observed during dark fermentation (DF) of maltose (1.91 mol H2/mol hexose or 77.8 mmol H2/L). The maximum H2 production rate was observed during DF of xylose (13.3 ml H2/g COD/h) and cellobiose (2.47 mmol H2/L/h). The main soluble metabolite products were acetate, ethanol and butyrate. The acetate concentration had a statistically significant positive correlation with the H2 content in biogas and the specific H2 yield. Based on the results of the correlation analysis, it was tentatively assumed that in the formic acid (mixed-acid) type fermentation, the rate of H2 production was higher than in the butyric acid type fermentation. With regard to real wastewater, cheese whey and confectionery wastewater were distinguished by a higher H2 yield (152 ml H2/g COD) and H2 production rate (0.57 mmol H2/L/h), respectively. The highest concentrations of confectionery wastewater and cheese whey, at which the DF process took place, were 5915 and 7311 mg COD/L, respectively. At the same time, SP-H2 dominated in the microbial community, despite the presence of indigenous microorganisms in wastewater. Thus, T. thermosaccharolyticum SP-H2 is a promising strain for DF of carbohydrate-rich unsterile wastewater under thermophilic conditions.

Declining carbohydrate solubilization with increasing solids loading during fermentation of cellulosic feedstocks by Clostridium thermocellum: documentation\xa0and diagnostic tests

BackgroundFor economically viable 2nd generation biofuels, processing of high solid lignocellulosic substrate concentrations is a necessity. The cellulolytic thermophilic anaerobe Clostridium thermocellum is one of the most effective biocatalysts for solubilization of carbohydrate harbored in lignocellulose. This study aims to document the solubilization performance of Clostridium thermocellum at increasing solids concentrations for two lignocellulosic feedstocks, corn stover and switchgrass, and explore potential effectors of solubilization performance.ResultsMonocultures of Clostridium thermocellum demonstrated high levels of carbohydrate solubilization for both unpretreated corn stover and switchgrass. However, fractional carbohydrate solubilization decreases with increasing solid loadings. Fermentation of model insoluble substrate (cellulose) in the presence of high solids lignocellulosic spent broth is temporarily affected but not model soluble substrate (cellobiose) fermentations. Mid-fermentation addition of cells (C. thermocellum) or model substrates did not significantly enhance overall corn stover solubilization loaded at 80 g/L, however cultures utilized the model substrates in the presence of high concentrations of corn stover. An increase in corn stover solubilization was observed when water was added, effectively diluting the solids concentration mid-fermentation. Introduction of a hemicellulose-utilizing coculture partner, Thermoanaerobacterium thermosaccharolyticum, increased the fractional carbohydrate solubilization at both high and low solid loadings. Residual solubilized carbohydrates diminished significantly in the presence of T. thermosaccharolyticum compared to monocultures of C. thermocellum, yet a small fraction of solubilized oligosaccharides of both C5 and C6 sugars remained unutilized.ConclusionDiminishing fractional carbohydrate solubilization with increasing substrate loading was observed for C. thermocellum-mediated solubilization and fermentation of unpretreated lignocellulose feedstocks. Results of experiments involving spent broth addition do not support a major role for inhibitors present in the liquid phase. Mid-fermentation addition experiments confirm that C. thermocellum and its enzymes remain capable of converting model substrates during the middle of high solids lignocellulose fermentation. An increase in fractional carbohydrate solubilization was made possible by (1) mid-fermentation solid loading dilutions and (2) coculturing C. thermocellum with T. thermosaccharolyticum, which ferments solubilized hemicellulose. Incomplete utilization of solubilized carbohydrates suggests that a small fraction of the carbohydrates is unaffected by the extracellular carbohydrate-active enzymes present in the culture.

Bioaugmentation combined with biochar to enhance thermophilic hydrogen production from sugarcane bagasse

In this study, Thermoanaerobacterium thermosaccharolyticum MJ2 and biochar were used to enhance thermophilic hydrogen production from sugarcane bagasse. MJ2 bioaugmentation notably increased the hydrogen production by 95.31%, which was further significantly improved by 158.10% by adding biochar. The addition of biochar promoted the degradation of substrate, improved the activities of hydrogenase and electron transfer system, and stimulated microbial growth and metabolism. Microbial community analysis showed that the relative abundance of Thermoanaerobacterium was significantly increased by bioaugmentation and further enriched by biochar. PICRUSt analysis showed that MJ2 combined with biochar promoted metabolic pathways related to substrate degradation and microbial metabolism. This study provides a novel enhancement method for hydrogen production of the cellulolytic microbial consortium by exogenous hydrogen-producing microorganism combined with biochar and deepens the understanding of its functional mechanism.

Lactic acid production from mesophilic and thermophilic fermentation of food waste at different pH

It is promising to recover lactic acid (LA) from fermentation of food waste (FW). In this study, pH and temperatures were investigated comprehensively to find their effects on LA fermentation, and microbial analyses were used to take insight to the variation of LA production. The results showed that mesophilic fermentation benefited hydrolysis and acidification, leading to a high yield of LA, while thermophilic conditions restricted other producers at low pH, leading to a high purity of LA. Lactobacillus amylolyticus was the main LA producer under thermophilic conditions, but Thermoanaerobacterium thermosaccharolyticum boomed at pH 5.0–6.0 and it converted LA partly to butyric acid. Simultaneously, Bacillus coagulans also increased and improved the optical purity (OP) of L-LA. From a series of this study, an operational condition of pH 5.5 and temperature of 52 °C would be potentially suitable for lactate fermentation of FW with high purity of 89%, while a stable LA production with an OP of 68% was achieved at 55 °C and pH 6.0.