Biotechnological Prospects of Thermoanerobacter AK15: End-Product Formation from Carbohydrates, Amino Acids, and Lignocellulosic and Macroalgae Hydrolysates.

The conversion of lignocellulosic and algal biomass by thermophilic bacteria has been an area of active investigation. Thermoanaerobacter species have proven to be particularly capable in the production of bioethanol and biohydrogen from lignocellulosic biomass, although detailed studies of their abilities to utilize the full gamut of carbohydrate, amino acids, and proteins encountered in biomass hydrolysates are seldom comprehensively examined. Here, we re-evaluate the ability of Thermoanaerobacter strain AK15, a highly ethanologenic strain previously isolated from a hot spring in Iceland. Similar to other Thermoanaerobacter species, the strain degraded a wide range of mono- and di-saccharides and produced a maximum of 1.57 mol ethanol per mol of glucose degraded at high liquid-gas phase ratios. The ability of strain AK15 to utilize amino acids in the presence of thiosulfate is limited to the branched-chain amino acids as well as serine and threonine. Similar to other Thermoanaerobacter species, strain AK15 produces a mixture of branched-chain fatty acids and alcohols, making the strain of interest as a potential source of longer-chain alcohols. Finally, the strain was also shown to use butyrate as an electron sink during glucose degradation resulting in the reduced product butanol, in addition to end-products produced from glucose. Thus, strain AK15 is a promising candidate for ethanol and higher-order alcohols from a range of lignocellulosic and algal biomass.

Branched-chain amino acid catabolism of Thermoanaerobacter pseudoethanolicus reveals potential route to branched-chain alcohol formation.

The fermentation of branched-chain amino acids (BCAAs) to branched-chain fatty acids (BCFAs) and branched-chain alcohols (BCOHs) is described using Thermoanaerobacter pseudoethanolicus. BCAAs were not degraded without an electron scavenging system but were degraded to a mixture of their BCFA (major) and BCOH (minor) when thiosulfate was added to the culture. Various environmental parameters were investigated using isoleucine as the substrate which ultimately demonstrated that at higher liquid-gas phase ratios the formation of 2-methyl-1-butanol from isoleucine achieved a maximal titer of 3.4 mM at a 1:1 liquid-gas ratio suggesting that higher partial pressure of hydrogen influences the BCOH/BCFA ratio but did not increase further with higher L-G phase ratios. Alternately, increasing the thiosulfate concentration decreased the BCOH to BCFA ratio. Kinetic monitoring of BCAA degradation revealed that the formation of BCOHs occurs slowly after the onset of BCFA formation. 13C2-labeled studies of leucine confirmed the production of a mixture of 3-methyl-1-butyrate and 3-methyl-1-butanol, while experiments involving 13C1-labeled 3-methyl-1-butyrate in fermentations containing leucine demonstrated that the carboxylic acid is reduced to the corresponding alcohol. Thus, the role of carboxylic acid reduction is likely of importance in the production of BCOH formation during the degradation of BCAA such as leucine.

Branched-chain amino acid catabolism of Thermoanaerobacter strain AK85 and the influence of culture conditions on branched-chain alcohol formation.

The bioprocessing of amino acids to branched-chain fatty acids and alcohols is described using Thermoanaerobacter strain AK85. The amino acid utilization profile was evaluated without an electron scavenger, with thiosulfate, and in a co-culture with a methanogen. There was an emphasis on the production of branched-chain alcohols and fatty acids from the branched-chain amino acids, particularly the influence of culture conditions which was investigated using isoleucine, which revealed that the concentration of thiosulfate was of great importance for the branched-chain alcohols/fatty acid ratio produced. Kinetic studies show that branched-chain amino acid fermentation is relatively slow as compared to glucose metabolism with the concentrations of the branched-chain alcohol increasing over time. To understand the flow of electrons and to investigate if the branched-chain fatty acid was being converted to branched-chain alcohol, enzyme assays and fermentation studies using 13C-labeled leucine and 3-methyl-1-butyrate were performed which indeed suggest that carboxylic acid reduction is a source of branched-chain alcohols when Thermoanaerobacter strain AK85 was cultivated with thiosulfate as an electron scavenger.

Biotransformation of organic acids to their corresponding alcohols by Thermoanaerobacter pseudoethanolicus.

Higher order alcohols, such as 1-butanol and 1-hexanol, have a large number of applications but are currently prepared from non-renewable feedstocks. Here, the ability of Thermoanaerobacter pseudoethanolicus to reduce short-chain fatty acids to their corresponding alcohols using reducing potential generated by glucose catabolism with yields between 21.0 and 61.0%. 13C-labelled acetate, 1-propionate and 1-butyrate demonstrates that exogenously added fatty acids are indeed reduced to their corresponding alcohols. This mode of producing primary alcohols from fatty acids using a thermophilic anaerobe opens the door for the production of such alcohols from low-value materials using an inexpensive source of reducing potential.

Production of (S)-1,2-propanediol from l-rhamnose using the moderately thermophilic Clostridium strain AK1.

Clostridium strain AK1 was investigated for its capacity of producing 1,2-propanediol from l-rhamnose but not l-fucose. The maximum yields of 1,2-propanediol from rhamnose was 0.81 mol 1,2-PD/mol l-rhamnose. The influence of different initial substrate concentrations as well as the effect of temperature and pH on 1,2-PD production was investigated.

Evaluation of the genus of Caldicellulosiruptor for production of 1,2-propanediol from methylpentoses.

Caldicellulosiruptor species degrade l-rhamnose and l-fucose to 1,2-propanediol. Six of the nine species within the genus produced 1,2-propanediol from l-rhamnose and three utilized l-fucose to produce the compound. Yields of 1,2-propanediol up to 40.5% of the theoretical yield were observed from methylpentoses catabolism.

Branched-chain alcohol formation by thermophilic bacteria within the genera of Thermoanaerobacter and Caldanaerobacter.

Fifty-six thermophilic strains including members of Caldanaerobacter, Caldicellulosiruptor, Caloramator, Clostridium, Thermoanaerobacter, and Thermoanaerobacterium, were investigated for branched-chain amino acid degradation in the presence of thiosulfate in batch culture. All of the Thermoanaerobacter and Caldanaerobacter strains (24) degraded the branched-chain amino acids (leucine, isoleucine, and valine) to a mixture of their corresponding branched-chain fatty acids and branched-chain alcohols. Only one Caloramator strain degraded the branched-chain amino acids to the corresponding branched-chain fatty acids. The ratio of branched-chain fatty acid production over branched-chain alcohol production for Thermoanaerobacter was 7.15, 6.61, and 11.53 for leucine, isoleucine, and valine, respectively. These values for Caldanaerobacter were 3.49, 4.13, and 7.31, respectively. This indicates that members within Caldanaerobacter produce proportionally more of the alcohols as compared with Thermoanaerobacter. No species within other genera investigated produced branched-chain alcohols from branched-chain amino acids in the presence of thiosulfate.

Branched-chain alcohol formation from branched-chain amino acids by Thermoanaerobacter brockii and Thermoanaerobacter yonseiensis.

Thermoanaerobacter species degrade branched-chain amino acids to a mixture of their corresponding branched-chain fatty acids and alcohols in the presence of thiosulfate; only acid formation occurred when Thermoanaerobacter strains were cultivated in co-culture with a hydrogenotrophic methanogen. Increased pH2 at high liquid-gas phase ratios increases the relative concentration of branched-chain alcohol.

Dataset describing the influence of culture conditions on the bioreduction of organic acids to alcohols by Thermoanaerobacter pseudethanolicus.

The dataset describes the influence of culture conditions on the bioreduction of organic acids by Thermoanaerobacter pseudethanolicus as reported in [1]. The data shows that during glucose fermentation of Thermoanaerobacter pseudethanolicus the reducing equivalents are not only converted to ethanol and hydrogen but also, in the presence of carboxylic acids (C2-C6), to its corresponding alcohol. To maximize the alcohol production produced from their carboxylic acid, several experiments were performed to investigate the effect of various environmental factors (initial glucose concentration, pH, liquid-gas phase ratio, and inhibitory effects of alcohols) on growth. A kinetic experiment of glucose in the absence and presence of selected fatty acids are also presented as are data on selected enzyme activities related to alcohols and aldehydes and a time course study of the reduction of 13C1 labeled butyrate using glucose as a carbon source.

Dataset describing the amino acid catabolism of Thermoanaerobacter pseudethanolicus.

The dataset depicts the catabolism of branched-chain amino acids by Thermoanaerobacter pseudethanolicus in the presence of thiosulfate under different culture conditions. The results reveal that the strain can degrade all three branched-chain amino acids resulting in the production of their corresponding branched-chain fatty acids and branched-chain alcohols with the fatty acids always being the dominant product. The highest amounts of 2-methyl-1-butanol from isoleucine were at pH 6.5, liquid-gas ratio of 0.98, and at 20 mM thiosulfate concentration. A kinetic experiment of the branched-chain amino acids was done in the presence of thiosulfate as are data on selected enzyme activities related to alcohols and aldehydes. Finally, an NMR study using 13C1 methyl-1-butyrate during the degradation of leucine in the presence of thiosulfate was done to prove that the 13C1-methyl-1-butanol was indeed from its corresponding fatty acid.

Metabolic engineering of Thermoanaerobacterium AK17 for increased ethanol production in seaweed hydrolysate.

Sustainably produced renewable biomass has the potential to replace fossil-based feedstocks, for generation of biobased fuels and chemicals of industrial interest, in biorefineries. In this context, seaweeds contain a large fraction of carbohydrates that are a promising source for enzymatic and/or microbial biorefinery conversions. The thermoanaerobe Thermoanaerobacterium AK17 is a versatile fermentative bacterium producing ethanol, acetate and lactate from various sugars. In this study, strain AK17 was engineered for more efficient production of ethanol by knocking out the lactate and acetate side-product pathways. This was successfully achieved, but the strain reverted to acetate production by recruiting enzymes from the butyrate pathway. Subsequently this pathway was knocked out and the resultant strain AK17_M6 could produce ethanol close to the maximum theoretical yield (90%), leading to a 1.5-fold increase in production compared to the wild-type strain. Strain AK17 was also shown to successfully ferment brown seaweed hydrolysate from Laminaria digitata to ethanol in a comparatively high yield of 0.45 g/g substrate, with the primary carbon sources for the fermentations being mannitol, laminarin-derived glucose and short laminari-oligosaccharides. As strain AK17 was successfully engineered and has a wide carbohydrate utilization range that includes mannitol from brown seaweed, as well as hexoses and pentoses found in both seaweeds and lignocellulose, the new strain AK17_M6 obtained in this study is an interesting candidate for production of ethanol from both second and third generations biomass.

Effect of various factors on ethanol yields from lignocellulosic biomass by Thermoanaerobacterium AK17.

The ethanol production capacity from sugars and lignocellulosic biomass hydrolysates (HL) by Thermoanaerobacterium strain AK(17) was studied in batch cultures. The strain converts various carbohydrates to, acetate, ethanol, hydrogen, and carbon dioxide. Ethanol yields on glucose and xylose were 1.5 and 1.1 mol/mol sugars, respectively. Increased initial glucose concentration inhibited glucose degradation and end product formation leveled off at 30 mM concentrations. Ethanol production from 5 g L(-1) of complex biomass HL (grass, hemp, wheat straw, newspaper, and cellulose) (Whatman paper) pretreated with acid (0.50% H(2) SO(4)), base (0.50% NaOH), and without acid/base (control) and the enzymes Celluclast and Novozyme 188 (0.1 mL g(-1) dw; 70 and 25 U g(-1) of Celluclast and Novozyme 188, respectively) was investigated. Highest ethanol yields (43.0 mM) were obtained on cellulose but lowest on hemp leafs (3.6 mM). Chemical pretreatment increased ethanol yields substantially from lignocellulosic biomass but not from cellulose. The influence of various factors (HL, enzyme, and acid/alkaline concentrations) on end-product formation from 5 g L(-1) of grass and cellulose was further studied to optimize ethanol production. Highest ethanol yields (5.5 and 8.6 mM ethanol g(-1) grass and cellulose, respectively) were obtained at very low HL concentrations (2.5 g L(-1)); with 0.25% acid/alkali (v/v) and 0.1 mL g(-1) enzyme concentrations. Inhibitory effects of furfural and hydroxymethylfurfural during glucose fermentation, revealed a total inhibition in end product formation from glucose at 4 and 6 g L(-1), respectively.

Production of ethanol from sugars and lignocellulosic biomass by Thermoanaerobacter J1 isolated from a hot spring in Iceland.

Thermophilic bacteria have gained increased attention as candidates for bioethanol production from lignocellulosic biomass. This study investigated ethanol production by Thermoanaerobacter strain J1 from hydrolysates made from lignocellulosic biomass in batch cultures. The effect of increased initial glucose concentration and the partial pressure of hydrogen on end product formation were examined. The strain showed a broad substrate spectrum, and high ethanol yields were observed on glucose (1.70 mol/mol) and xylose (1.25 mol/mol). Ethanol yields were, however, dramatically lowered by adding thiosulfate or by cocultivating strain J1 with a hydrogenotrophic methanogen with acetate becoming the major end product. Ethanol production from 4.5 g/L of lignocellulosic biomass hydrolysates (grass, hemp stem, wheat straw, newspaper, and cellulose) pretreated with acid or alkali and the enzymes Celluclast and Novozymes 188 was investigated. The highest ethanol yields were obtained on cellulose (7.5 mM·g(-1)) but the lowest on straw (0.8 mM·g(-1)). Chemical pretreatment increased ethanol yields substantially from lignocellulosic biomass but not from cellulose. The largest increase was on straw hydrolysates where ethanol production increased from 0.8 mM·g(-1) to 3.3 mM·g(-1) using alkali-pretreated biomass. The highest ethanol yields on lignocellulosic hydrolysates were observed with hemp hydrolysates pretreated with acid, 4.2 mM·g(-1).

Hydrogenophilus islandicus sp. nov., a thermophilic hydrogen-oxidizing bacterium isolated from an Icelandic hot spring.

A novel chemolithotrophic bacterium, strain 16C(T), was isolated from a hot spring in Graendalur, south-west Iceland. Cells of this organism were Gram-negative, rod-shaped and motile. The isolate was aerobic and capable of chemolithotrophic growth on hydrogen and carbon dioxide, heterotrophic growth on butyrate and several other organic compounds, and mixotrophic growth on butyrate, hydrogen and carbon dioxide. Heterotrophic growth was generally enhanced in the presence of yeast extract. Autotrophic growth on hydrogen was observed at pH values between 6.0 and 10.0 and temperatures between 35 and 60 °C; optimum growth conditions were pH 7.0 and 55 °C. The DNA G+C content was 63.9 mol%. 16S rRNA gene sequence analysis showed that strain 16C(T) was a member of a distinct species belonging to the class Betaproteobacteria and was most closely related to Hydrogenophilus thermoluteolus NBRC 14978(T) and Hydrogenophilus hirschii DSM 11420(T). The major cellular fatty acids were straight-chain C(16 : 0) (44.98 %) and C(18 : 1)ω7c (17.93 %), as well as cyclic C(17 : 0) (13.90 %) and C(19 : 0)ω8c (4.67 %) fatty acids. Based on its physiological and molecular properties, it is concluded that strain 16C(T) represents a novel species within the genus Hydrogenophilus, for which the name Hydrogenophilus islandicus is proposed; the type strain is 16C(T) (=DSM 21442(T)=JCM 16106(T)).

Thiomonas islandica sp. nov., a moderately thermophilic, hydrogen- and sulfur-oxidizing betaproteobacterium isolated from a hot spring.

A novel, hydrogen- and sulfur-oxidizing bacterium, designated strain 6C(T), was isolated from a hot spring in Graendalur, south-western Iceland. Cells of this organism were Gram-reaction-negative, rod-shaped and motile. The strain grew aerobically and was capable of chemolithotrophic growth on thiosulfate and hydrogen, heterotrophic growth on pyruvate, oxalate, acetate and on glutamate in the presence of yeast extract and mixotrophic growth on several organic compounds, thiosulfate and/or hydrogen. During growth on thiosulfate, the final product was sulfate, resulting in a drop in pH from 6.8 to 2.7. Heterotrophic growth on pyruvate was observed at pH 4-7 (optimum pH 4) and 35-50 °C (optimum 45 °C). The DNA G+C content was 65.2 mol%. As determined by 16S rRNA gene sequence analysis, strain 6C(T) represents a distinct species belonging to the class Betaproteobacteria and is most closely related to Thiomonas intermedia DSM 18155(T) and Thiomonas perometabolis DSM 18570(T). DNA-DNA hybridization between strain 6C(T) and Thiomonas intermedia DSM 18155(T) and Thiomonas perometabolis DSM 18570(T) gave relatedness values below 32 %. These results, together with physiological characteristics, showed that strain 6C(T) represents a novel species within the genus Thiomonas, for which the name Thiomonas islandica sp. nov. is proposed. The type strain is 6C(T) (=DSM 21436(T) =JCM 16107(T)).

Influence of Culture Conditions on the Bioreduction of Organic Acids to Alcohols by Thermoanaerobacter pseudoethanolicus.

Thermoanaerobacter species have recently been observed to reduce carboxylic acids to their corresponding alcohols. The present investigation shows that Thermoanaerobacter pseudoethanolicus converts C2-C6 short-chain fatty acids (SCFAs) to their corresponding alcohols in the presence of glucose. The conversion yields varied from 21% of 3-methyl-1-butyrate to 57.9% of 1-pentanoate being converted to their corresponding alcohols. Slightly acidic culture conditions (pH 6.5) was optimal for the reduction. By increasing the initial glucose concentration, an increase in the conversion of SCFAs reduced to their corresponding alcohols was observed. Inhibitory experiments on C2-C8 alcohols showed that C4 and higher alcohols are inhibitory to T. pseudoethanolicus suggesting that other culture modes may be necessary to improve the amount of fatty acids reduced to the analogous alcohol. The reduction of SCFAs to their corresponding alcohols was further demonstrated using 13C-labelled fatty acids and the conversion was followed kinetically. Finally, increased activity of alcohol dehydrogenase (ADH) and aldehyde oxidation activity was observed in cultures of T. pseudoethanolicus grown on glucose as compared to glucose supplemented with either 3-methyl-1-butyrate or pentanoate, using both NADH and NADPH as cofactors, although the presence of the latter showed higher ADH and aldehyde oxidoreductase (ALDH) activity.

Biotransformation of Carboxylic Acids to Alcohols: Characterization of Thermoanaerobacter Strain AK152 and 1-Propanol Production via Propionate Reduction.

Thermoanaerobacter strains have recently gained interest because of their ability to convert short chain fatty acids to alcohols using actively growing cells. Thermoanaerobacter thermohydrosulfuricus strain AK152 was physiologically investigated for its ethanol and other alcohol formation. The temperature and pH optimum of the strain was 70 °C and pH 7.0 and the strain degraded a variety of compounds present in lignocellulosic biomass like monosaccharides, disaccharides, and starch. The strain is highly ethanologenic, producing up to 86% of the theoretical ethanol yield form hexoses. Strain AK152 was inhibited by relatively low initial substrate (30 mM) concentration, leading to inefficient degradation of glucose and levelling up of all end-product formation. The present study shows that the strain produces alcohols from most of the tested carboxylic acids, with the highest yields for propionate conversion to propanol (40.7%) with kinetic studies demonstrating that the maximum conversion happens within the first 48 h of fermentation. Various physiological tests were performed to maximize the acid conversion to the alcohol which reveals that the optimum pH for propionate conversion is pH 6.7 which affords a 57.3% conversion. Kinetic studies reveal that propionate conversion is rapid, achieving a maximum conversion within the first 48 h of fermentation. Finally, by using 13C NMR, it was shown that the addition of propionate indeed converted to propanol.

Dataset describing the amino acid catabolism of Thermoanaerobacter strain AK85: The influence of culture conditions on end product formation.

The dataset describes the catabolism of the 20 proteogenics amino acids and their end products by Thermoanaerobacter strain AK85 under different electron scavenging conditions with an emphasis on the branched-chain amino acids as reported in Scully and Orlygsson, 2019.

Ethanol and hydrogen production by two thermophilic, anaerobic bacteria isolated from Icelandic geothermal areas.

Microbial fermentations are potential producers of sustainable energy carriers. In this study, ethanol and hydrogen production was studied by two thermophilic bacteria (strain AK15 and AK17) isolated from geothermal springs in Iceland. Strain AK15 was affiliated with Clostridium uzonii (98.8%), while AK17 was affiliated with Thermoanaerobacterium aciditolerans (99.2%) based on the 16S rRNA gene sequence analysis. Both strains fermented a wide variety of sugar residues typically found in lignocellulosic materials, and some polysaccharides. In the batch cultivations, strain AK17 produced ethanol from glucose and xylose fermentations of up to 1.6 mol-EtOH/mol-glucose (80% of the theoretical maximum) and 1.1 mol-EtOH/mol-xylose (66%), respectively. The hydrogen yields by AK17 were up to 1.2 mol-H2/ mol-glucose (30% of the theoretical maximum) and 1.0 mol-H2/mol-xylose (30%). The strain AK15 produced hydrogen as the main fermentation product from glucose (up to 1.9 mol-H2/mol-glucose [48%]) and xylose (1.1 mol-H2/mol-xylose [33%]). The strain AK17 tolerated exogenously added ethanol up to 4% (v/v). The ethanol and hydrogen production performance from glucose by a co-culture of the strains AK15 and AK17 was studied in a continuous-flow bioreactor at 60 degrees C. Stable and continuous ethanol and hydrogen co-production was achieved with ethanol yield of 1.35 mol-EtOH/mol-glucose, and with the hydrogen production rate of 6.1 mmol/h/L (H2 yield of 0.80 mol-H2/mol-glucose). PCR-DGGE analysis revealed that the AK17 became the dominant bacterium in the bioreactor. In conclusion, strain AK17 is a promising strain for the co-production of ethanol and hydrogen with a wide substrate utilization spectrum, relatively high ethanol tolerance, and ethanol yields among the highest reported for thermoanaerobes.

High-efficiency hydrogen production by an anaerobic, thermophilic enrichment culture from an Icelandic hot spring.

Dark fermentative hydrogen production from glucose by a thermophilic culture (33HL), enriched from an Icelandic hot spring sediment sample, was studied in two continuous-flow, completely stirred tank reactors (CSTR1, CSTR2) and in one semi-continuous, anaerobic sequencing batch reactor (ASBR) at 58 degrees C. The 33HL produced H2 yield (HY) of up to 3.2 mol-H2/mol-glucose along with acetate in batch assay. In the CSTR1 with 33HL inoculum, H2 production was unstable. In the ASBR, maintained with 33HL, the H2 production enhanced after the addition of 6 mg/L of FeSO4 x H2O resulting in HY up to 2.51 mol-H2/mol-glucose (H2 production rate (HPR) of 7.85 mmol/h/L). The H2 production increase was associated with an increase in butyrate production. In the CSTR2, with ASBR inoculum and FeSO4 supplementation, stable, high-rate H2 production was obtained with HPR up to 45.8 mmol/h/L (1.1 L/h/L) and HY of 1.54 mol-H2/mol-glucose. The 33HL batch enrichment was dominated by bacterial strains closely affiliated with Thermobrachium celere (99.8-100%). T. celere affiliated strains, however, did not thrive in the three open system bioreactors. Instead, Thermoanaerobacterium aotearoense (98.5-99.6%) affiliated strains, producing H2 along with butyrate and acetate, dominated the reactor cultures. This culture had higher H2 production efficiency (HY and specific HPR) than reported for mesophilic mixed cultures. Further, the thermophilic culture readily formed granules in CSTR and ASBR systems. In summary, the thermophilic culture as characterized by high H2 production efficiency and ready granulation is considered very promising for H2 fermentation from carbohydrates.