Thermoanaerobacterium Thermosaccharolyticum Research Articles

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.

Enhanced biohydrogen production with low graphene oxide content using thermophilic bioreactors

Modern society envisions hydrogen (H2) fuel to drive the transportation, industrial, and domestic sectors. Here, we explore use of graphene oxide nanoparticles (GO NPs) for greatly enhancing bio-H2 production by a consortium based on Thermoanaerobacterium thermosaccharolyticum spp. Thermophilic batch bioreactors were set up at 60 OC and initial pH of 8.5 to assess the effects of GO NPs supplements on biohydrogen production. Under optimal GO NPs loading of 10 mg/L, the supplemented system yielded ∼ 300% higher H2 yield (6.78 mol H2/mol sucrose) than control. Such an optimized system offered 73% H2 purity and 85% conversion efficiency by promoted the desirable acetate fermentation pathway. Miseq Illumina sequencing tests revealed that the optimal levels of GO NPs did not alter the microbial composition of consortium.

Theoretical and Experimental Investigation of the Thermal Inactivation of Thermoanaerobacterium Thermosaccharolyticum and Geobacillus Stearothermophilus in Different Canned Food Matrices

Abstract In the canning industry, thermal preservation processes typically are designed based on Clostridium botulinum thermal destruction kinetics. However, some bacteria can still survive, necessitating implementation of stricter timetemperature regimen for sterilization process. The aim of this study was to compare processing effectiveness at F0 (sterilization value) 8 ±1 min from the perspective of the vegetable-based product canning facility, while analyzing the inactivation, viability, and recovery of thermophilic bacteria. Four commercial products [tomato soup and rassolnik soup - acidified food (AF), and mushroom soup and pea porridge - low-acid food (LACF)] with different heat transfer characteristics (convection and conduction) were inoculated with 6.6 log10 spores/ml Geobacillus stearothermophilus LMKK 244 (reported as DSM 6790 and ATCC 10149 in other collections) and 4.810 log spores/ml Thermoanaerobacterium thermosaccharolyticum DSM 571 spore suspensions. Food samples contaminated with bacterial spores were processed in a steam-air retort at 118 °C for 75 min. G. stearothermophilus and T. thermosaccharolyticum growth was not detected in AF samples (pH = 4.4 and 4.5), but was observed in LACF samples (pH = 5.1 and 5.8). Practical evaluation showed that T. thermosaccharolyticum did not survive thermal processing, which was verified using a presence/absence test after incubation at 55 °C. G. stearothermophilus did not survive thermal processing, but recovered in pea porridge (pH = 5.8) during incubation. Our observations showed that food pH is a crucial factor determining microorganism survival during heat treatment and may be used by the vegetable-based product canning facilities to improve the food sterilization conditions.

Biohydrogen production from food processing wastewater by a newly isolated thermophilic bacterium

The aim of this work was a comparative study of biohydrogen production from cheese whey and confectionary wastewater by a newly isolated thermophilic microbial strain Thermoanaerobacterium thermosaccharolyticum SP-H2. Experimental results showed that the fermentative hydrogen was successfully produced with the highest hydrogen yield of 3.9 mL H2/mL cheese whey or 80 mL H2/g chemical oxygen demand. The profile of soluble metabolite products showed that hydrogen generation by a new isolate was mainly acetate-type fermentation in the case of confectionary wastewater and mixed ethanol-acetate-lactate type fermentation in the case of cheese whey. The more optimal metabolic pathway of confectionary wastewater fermentation was confirmed by the better kinetic characteristics according to the Gompertz model.

Co-production of hydrogen and ethanol by Thermoanaerobacterium thermosaccharolyticum KKU-ED1 from alpha-cellulose and cellulose fraction of sugarcane bagasse

This research aims to evaluate the co-production of hydrogen and ethanol from lignocellulosic materials by Thermoanaerobacterium thermosaccharolyticum KKU-ED1. The optimum conditions for co-production of hydrogen and ethanol in batch fermentation was determined using alpha-cellulose as a basic model substrate to mimic the natural cellulose part of a lignocellulosic material. The optimal conditions attained were an initial pH of 7.0, a temperature of 55 °C, and a cellulose content of 20 g/L, which resulted in the maximum hydrogen production and ethanol (EtOH) yield of 6113 mL H2/L and 1.71 mmol EtOH/g-celluloseconsumed, respectively. The potential of the KKU-ED1 strain to produce hydrogen and ethanol from natural cellulose in sugarcane bagasse was further evaluated under the optimum conditions. Hydrogen production and ethanol yield of 218 mL H2/L, and 0.26 mmol EtOH/g-celluloseconsumed were obtained. Results indicated that the strain KKU-ED1 is a thermophilic cellulolytic bacterium capable of co-producing hydrogen and ethanol from lignocellulosic materials.

Co-digestion of Oil Palm Trunk Hydrolysate and Slaughterhouse Wastewater for Biohydrogen Production in a Fixed Bed Reactor by Immobilized Thermoanaerobacterium thermosaccharolyticum KKU19 on Expanded Clay

The goal of this study was to evaluate the use of expanded clay as a support material for Thermoanaerobacterium thermosaccharolyticum KKU19 to produce hydrogen from oil palm trunk hydrolysate (OPT) and slaughterhouse wastewater (SHW) in a fixed-bed reactor (FBR) under non-sterile conditions. The effects of hydraulic retention time (HRT) on the performance of the FBR were also investigated. The FBR was operated at an OPT hydrolysate to SHW ratio of 2.55:1 (v:v), 60°C, initial pH 6.5, and 1.2 mg (as total volatile solids/g expanded clay) of T. thermosaccharolyticum KKU19 immobilized on expanded clay. A maximum hydrogen production rate (HPR) and hydrogen yield (HY) of 7.15 ± 0.22 L/L day and 234.45 ± 5.14 mL H2/g-COD, respectively, were obtained at an HRT of 6 h. Long-term operation of FBR at 6 h HRT indicated that expanded clay efficiently immobilizes T. thermosaccharolyticum KKU19, for which an HPR of 6.82 ± 0.56 L H2/L day, and an HY of 231.99 ± 19.59 mL H2/g-COD were obtained. Furthermore, the COD removal efficiency of 30% obtained under long-term operation was comparable to that under short-term operation at an HRT of 6 days. Butyric and acetic acids were the main soluble metabolite products, thereby indicating a butyrate–acetate type fermentation. Our findings indicate that expanded clay is an effective support material that contributes to the protection of microbial cells and can be used for long-term operation.

Coculture with hemicellulose-fermenting microbes reverses inhibition of corn fiber solubilization by Clostridium thermocellum at elevated solids loadings

BackgroundThe cellulolytic thermophile Clostridium thermocellum is an important biocatalyst due to its ability to solubilize lignocellulosic feedstocks without the need for pretreatment or exogenous enzyme addition. At low concentrations of substrate, C. thermocellum can solubilize corn fiber > 95% in 5 days, but solubilization declines markedly at substrate concentrations higher than 20 g/L. This differs for model cellulose like Avicel, on which the maximum solubilization rate increases in proportion to substrate concentration. The goal of this study was to examine fermentation at increasing corn fiber concentrations and investigate possible reasons for declining performance.ResultsThe rate of growth of C. thermocellum on corn fiber, inferred from CipA scaffoldin levels measured by LC–MS/MS, showed very little increase with increasing solids loading. To test for inhibition, we evaluated the effects of spent broth on growth and cellulase activity. The liquids remaining after corn fiber fermentation were found to be strongly inhibitory to growth on cellobiose, a substrate that does not require cellulose hydrolysis. Additionally, the hydrolytic activity of C. thermocellum cellulase was also reduced to less-than half by adding spent broth. Noting that > 15 g/L hemicellulose oligosaccharides accumulated in the spent broth of a 40 g/L corn fiber fermentation, we tested the effect of various model carbohydrates on growth on cellobiose and Avicel. Some compounds like xylooligosaccharides caused a decline in cellulolytic activity and a reduction in the maximum solubilization rate on Avicel. However, there were no relevant model compounds that could replicate the strong inhibition by spent broth on C. thermocellum growth on cellobiose. Cocultures of C. thermocellum with hemicellulose-consuming partners—Herbinix spp. strain LL1355 and Thermoanaerobacterium thermosaccharolyticum—exhibited lower levels of unfermented hemicellulose hydrolysis products, a doubling of the maximum solubilization rate, and final solubilization increased from 67 to 93%.ConclusionsThis study documents inhibition of C. thermocellum with increasing corn fiber concentration and demonstrates inhibition of cellulase activity by xylooligosaccharides, but further work is needed to understand why growth on cellobiose was inhibited by corn fiber fermentation broth. Our results support the importance of hemicellulose-utilizing coculture partners to augment C. thermocellum in the fermentation of lignocellulosic feedstocks at high solids loading.

Comparison of thermophilic and hyperthermophilic dark fermentation with subsequent mesophilic methanogenesis in expanded granular sludge bed reactors

Herein, dark fermentation (DF, V = 5.5 L) and subsequent mesophilic methanogenesis (V = 43.5 L) are run as expanded granular sludge bed reactors (EGSB) at thermophilic (υDF = 60 °C) and hyperthermophilic (υDF = 80 °C) temperatures. A synthetic glucose wastewater is run with a 22.5 g/L chemical oxygen demand (COD) and 48–9 h hydraulic retention times (HRTs), giving organic loading rates (OLRs) of 11–60 g COD/L/d for DF. The maximum hydrogen production rate (HPR) is HPR = 3.0 m³/m³/d for HRT = 9 h with a 50 L/kg COD hydrogen yield (HY) and 40 vol% H2. Methane production rate (MPR) reaches MPR = 2.6 m³/m³/d with 70 vol% CH4 at HRT = 2.8 d. The highest H2 yields are HY = 180 L/kg COD with 53 vol% H2 (thermophilic, HRT = 48 h). Hyperthermophilic temperatures led to lower HPRs (0.7 m³/m³/d) and MPRs (1.6 m³/m³/d). 53% of Thermoanaerobacterium thermosaccharolyticum as an H2 producer are found. Discoloration of granular sludge from black to white and granule stability was observed in DF.

Anaerobic wastewater treatment and reuse enabled by thermophilic bioprocessing integrated with a bioelectrochemical/ultrafiltration module

The current study aimed to develop an anaerobic wastewater treatment and reuse module enabled by thermophilic bioprocessing, a microbial fuel cell (MFC) and ultrafiltration (UF) treatment. A previously unexplored consortium based on Thermoanaerobacterium thermosaccharolyticum and Arcobacter sp. was used to remove ~73% of chemical oxygen demand (COD) from wastewater under anaerobic conditions (CODi = 200 mg/L). The subsequent MFC and UF treatment removed the COD remnants to meet the secondary treatment standards and reuse criteria. The energy efficiency of polyethersulfone UF membranes was improved by modifying their surfaces with coatings based on self-polymerized dopamine, mixtures of dopamine and poly(2-dimethylamino) ethyl methacrylate methyl, and dopamine analog norepinephrine. The resulting hydrophilic, anti-fouling layers were found to reduce interactions between rejected species and the membrane surface. Finally, this study presents a comparative treatment performance and energy efficiency of the wastewater treatment and reuse modules arranged in six different configurations.

Consolidated bioprocessing performance of a two-species microbial consortium for butanol production from lignocellulosic biomass.

Consolidated bioprocessing (CBP) by using microbial consortium was considered as a promising approach to achieve direct biofuel production from lignocellulose. In this study, the interaction mechanism of microbial consortium consisting of Thermoanaerobacterium thermosaccharolyticum M5 and Clostridium acetobutylicum NJ4 was analyzed, which could achieve efficient butanol production from xylan through CBP. Strain M5 possesses efficient xylan degradation capability, as 19.73 g/L of xylose was accumulated within 50 hr. The efficient xylose utilization capability of partner strain NJ4 could relieve the substrate inhibition to hydrolytic enzymes of xylanase and xylosidase secreted by strain M5. In addition, the earlier solventogenesis of strain NJ4 was observed due to the existence of butyrate generated by strain M5. The mutual interaction of these two strains finally gave 13.28 g/L of butanol from 70 g/L of xylan after process optimization, representing a relatively high butanol production from hemicellulose. Moreover, 7.61 g/L of butanol was generated from untreated corncob via CBP. This successfully constructed microbial consortium exhibits efficient cooperation performance on butanol production from lignocellulose, which could provide a platform for the emerging butanol production from lignocellulose.

Genomic and metabolic insights into solvent production by Thermoanaerobacterium thermosaccharolyticum GSU5

Thermoanaerobacterium thermosaccharolyticum GSU5 was isolated from animal dung collected in a pasture plain in Buenos Aires, Argentina. This thermophilic and anaerobic microorganism was able to produce butanol and ethanol, but not acetone, using sugars such as xylose, arabinose, glucose, galactose, fructose, sucrose, and cellobiose. Key metabolic enzymes leading to solvent production were identified in its genome. A detailed analysis of the solvent and organic acid biosynthetic pathway genes of sequenced strains revealed new insights into the unique metabolic features of this species. Genes required for the synthesis of acetone are absent in the genomes of all sequenced Thermoanaerobacterium, suggesting that it is a general trait of the genus. Strains able to produce butanol synthesize butyrate through the one step pathway catalyzed by the butyryl-CoA:acetate-CoA transferase (But). The large range of fermentable substrates and the ability to produce both ethanol and butanol without acetone makes this species an interesting candidate for second generation biofuel production.

Unraveling hydrogen production potential by glucose and xylose co‐fermentation of Thermoanaerobacterium thermosaccharolyticum W16 and its metabolisms through transcriptomic sequencing

Unraveling hydrogen production potential by glucose and xylose co‐fermentation of <i>Thermoanaerobacterium thermosaccharolyticum</i> <scp>W16</scp> and its metabolisms through transcriptomic sequencing

Development of a thermophilic coculture for corn fiber conversion to ethanol

The fiber in corn kernels, currently unutilized in the corn to ethanol process, represents an opportunity for introduction of cellulose conversion technology. We report here that Clostridium thermocellum can solubilize over 90% of the carbohydrate in autoclaved corn fiber, including its hemicellulose component glucuronoarabinoxylan (GAX). However, Thermoanaerobacterium thermosaccharolyticum or several other described hemicellulose-fermenting thermophilic bacteria can only partially utilize this GAX. We describe the isolation of a previously undescribed organism, Herbinix spp. strain LL1355, from a thermophilic microbiome that can consume 85% of the recalcitrant GAX. We sequence its genome, and based on structural analysis of the GAX, identify six enzymes that hydrolyze GAX linkages. Combinations of up to four enzymes are successfully expressed in T. thermosaccharolyticum. Supplementation with these enzymes allows T. thermosaccharolyticum to consume 78% of the GAX compared to 53% by the parent strain and increases ethanol yield from corn fiber by 24%.

Exploring the Relationship Between Clostridium thermocellum JN4 and Thermoanaerobacterium thermosaccharolyticum GD17

Characterizing and engineering microbial communities for lignocellulosic biofuel production has received widespread attention. Previous research has established that Clostridium thermocellum JN4 and Thermoanaerobacterium thermosaccharolyticum GD17 coculture significantly improves overall cellulosic biofuel production efficiency. Here, we investigated this interaction and revealed the mechanism underlying the improved efficiency observed. In contrast to the previously reported mutualistic relationship, a harmful effect toward C. thermocellum JN4 was observed in these microbial consortia. Although T. thermosaccharolyticum GD17 relieves the carbon catabolite repression of C. thermocellum JN4 regarding obtaining more cellobiose or glucose released from lignocellulose, T. thermosaccharolyticum GD17 significantly hampers the growth of C. thermocellum JN4 in coculture. The increased formation of end products is due to the strong competitive metabolic advantage of T. thermosaccharolyticum GD17 over C. thermocellum JN4 in the conversion of glucose or cellobiose into final products. The possibility of controlling and rebalancing these microbial consortia to modulate cellulose degradation was achieved by adding T. thermosaccharolyticum GD17 stimulants into the system. As cellulolytic bacteria are usually at a metabolic disadvantage, these discoveries may apply to a large proportion of cellulosic biofuel-producing microbial consortia. These findings provide a reference for engineering efficient and modular microbial consortia for modulating cellulosic conversion.

Thermophilic biohydrogen production from palm oil mill effluent: Effect of immobilized cells on granular activated carbon in fluidized bed reactor

In this study, the performance of immobilized cells on granular activated carbon (GAC) for thermophilic biohydrogen production is determined using POME as a fermentation substrate. The immobilized cells are formed at different pH medium using sugar composition characterized in the POME. The pH 6 revealed the optimum pH used for biofilm development with HPR of 2.8mmol H2/Lh. The effect of sugar utilization by the immobilized cells on GAC are determined at different sugar concentration using the Monod model prior validated the performance of the cells in the fluidized bed reactor (FBR). From the model, 0.316±0.013h−1 of maximum specific growth rate was obtained at 20g/L sugar used and was keep increasing to the maximum of 30g/L of sugar used with HPR 2.6–2.8mmol H2/Lh. Lastly, the POME-enriched nutrients are used as the carbon source in the fluidized bed reactor (FBR). The highest HPR obtained was at HRT 12h, (5.2mmol H2/Lh) and HY of 1.24mol H2/mol sugar. The screening of the microbial population by DGGE revealed that the Thermoanaerobacterium thermosaccharolyticum sp. was dominant for all the HRTs, thereby indicating that this bacterium is resilient towards environmental disturbances.

Specify the individual and synergistic effects of lignocellulose-derived inhibitors on biohydrogen production and inhibitory mechanism research

The present study evaluated the individual and synergistic effects of eight lignocellulose-derived inhibitors on biohydrogen fermentation and their inhibitory mechanisms. The growth of Thermoanaerobacterium thermosaccharolyticum MJ1 (T. thermosaccharolyticum MJ1) was completely inhibited by 1.5 g/L ferulic acid and 0.5 g/L p-coumaric acid, while other inhibitors (≤2 g/L) exhibited the weaker inhibition. The inhibition on biohydrogen by vanillin and syringaldehyde was much stronger than growth, which was different with other inhibitors. At 2 g/L vanillin and syringaldehyde, the relative maximum OD600 values were 64.7% and 78.2% respectively, however the relative biohydrogen productions were only 12.6% and 33.6% respectively. The lag phase of biohydrogen production was significantly prolonged by 0.3 g/L p-coumaric acid (7.5 times higher than control). Metabolite analysis showed that the metabolic flux had been redirected by vanillin and syringaldehyde. The increased lactate production and decreased production of acetate and butyrate contributed to a lower biohydrogen production. The synergistic effects of inhibitors on growth and biohydrogen production were studied. The main functional inhibitors in mixture were vanillin and syringaldehyde, which were further demonstrated by metabolite analysis. The present work provided a comprehensive insight of inhibitors on biohydrogen fermentation.

Bioaugmentation with Thermoanaerobacterium thermosaccharolyticum W16 to enhance thermophilic hydrogen production using corn stover hydrolysate

In the present work, with corn stover hydrolysate as the substrate, an efficient hydrogen-producing thermophile, Thermoanaerobacterium thermosaccharolyticum W16, was added to three kinds of seed sludge (rotten corn stover (RCS), cow dung compost (CDC), and sludge from anaerobic digestion (SAD)) to investigate the effect of bioaugmentation on thermophilic hydrogen production. Batch test results indicate that the bioaugmentation with a small amount of the strain T. thermosaccharolyticum W16 (5% of total microbes) increased the hydrogen yield to varying degrees (RCS: from 8.78 to 9.90 mmol H2/g utilized sugar; CDC: from 8.18 to 8.42 mmol H2/g utilized sugar; SAD: from 8.55 to 9.17 mmol H2/g utilized sugar). The bioaugmentation process also influenced the soluble metabolites composition towards more acetate and less butyrate production for RCS, and more acetate and less ethanol accumulation for SAD. Microbial community analysis indicates that Thermoanaerobacterium spp. and Clostridium spp. dominated microbial community in all situations and might be mainly responsible for thermophilic hydrogen generation. For RCS and SAD, the bioaugmentation obviously increased the relative abundance of the strain T. thermosaccharolyticum W16 in microbial community, which might be the main reason for the improvement of hydrogen production in these cases.

Mikrobiologie pivovarské výroby - bakterie rodu Clostridium

In most cases, the Clostridium species rank among the sporogenic obligate anaerobes, which are sensitive to bitter hop substances and acidic pH. Thus they rank among the less dangerous contaminants in brewing operations. Their increased incidence in breweries can be linked to the modernization of technologies, which reduces access to oxygen in individual production phases. The endospores of Clostridium bacteria were isolated, e.g., from the storage vessels for liquid malt substitutes (glucose, starch syrups). They are able to survive the conditions of mashing and wort boiling, but they can not germinate in the following phases of beer production. However, their ability to be present in the vegetative form until the wort preparation presents a risk of damage to finished beer – vegetative cells form a number of undesirable organic acids, which may pass into the finished product. We provide here an overview of basic morphological and physiological properties of these bacteria and describe their importance in the brewing process. Particular attention is given to species Clostridium acetobutylicum, C. butyricum, C. cellulovorans, C. pasteurianum, C. thermocellum, „C. thermosaccharolyticum“(Thermoanaerobacterium thermosaccharolyticum) and C. tyrobutyricum, which were detected in brewing operations.

Consolidated Bioprocessing Using Clostridium thermocellum and Thermoanaerobacterium thermosaccharolyticum Co-culture for Enhancing Ethanol Production from Corn Straw

A co-culture consisting of Clostridium thermocellum ATCC 27405 and Thermoanaerobacterium thermosaccharolyticum DSM 571 was employed to improve the ethanol yield from microcrystalline cellulose (MCC) and corn straw substrates. An ethanol concentration of 1.29 g/L (26.1% ethanol yield) was obtained with 98.6% cellulose degradation when MCC was used as substrate in fermenter tanks. The ethanol yield obtained in fermenter tanks was 13.9% higher than that obtained via an anaerobic process performed in bottles. An ethanol concentration of 0.45 g/L, corresponding to 55.6% cellulose degradation and 11.2% ethanol yield, was achieved with corn straw as substrate in fermenter tanks. This ethanol yield was 28.2% higher than that formed in anaerobic bottles. Surprisingly, a 40.7% hemicellulose degradation was achieved via fermentation tanks, which was 127% higher than that obtained from anaerobic bottles.

Analysis of the Active Site Cysteine Residue of the Sacrificial Sulfur Insertase LarE from Lactobacillus plantarum

LarE from Lactobacillus plantarum is an ATP-dependent sulfur transferase that sacrifices its Cys176 sulfur atom to form a dehydroalanine (Dha) side chain during biosynthesis of the covalently linked nickel-pincer nucleotide (NPN) cofactor (pyridinium 3-thioamide-5-thiocarboxylic acid mononucleotide) of lactate racemase. Coenzyme A (CoA) stabilizes LarE and forms a CoA-Cys176 mixed disulfide with the protein. This study presents the crystal structure of the LarE/CoA complex, revealing protein interactions with CoA that mimic those for binding ATP. CoA weakly inhibits LarE activity, and the persulfide of CoA is capable of partially regenerating functional LarE from the Dha176 form of the protein. The physiological relevance of this cycling reaction is unclear. A new form of LarE was discovered, an NPN-LarE covalent adduct, explaining prior results in which activation of the lactate racemase apoprotein required only the isolated LarE. The crystal structure of the inactive C176A variant revealed a fold essentially identical to that of wild-type LarE. Additional active site variants of LarE were created and their activities characterized, with all LarE variants analyzed in terms of the structure. Finally, the L. plantarum LarE structure was compared to a homology model of Thermoanaerobacterium thermosaccharolyticum LarE, predicted to contain three cysteine residues at the active site, and to other proteins with a similar fold and multiple active site cysteine residues. These findings suggest that some LarE orthologs may not be sacrificial but instead may catalyze sulfur transfer by using a persulfide mechanism or from a labile site on a [4Fe-4S] cluster at this position.