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.

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.

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.

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.

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.

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.

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.

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.

Dataset describing ethanol and 1,2-propanediol production by a stenothermal moderately thermophilic anaerobe, Clostridium strain AK1.

The dataset details the fermentation of D-glucose, L-rhamnose, and L-fucose and their end-product formation by the moderate thermophile Clostridium strain AK1 (DSM 18778) as related to the work described in "Propanediol from L-rhamnose using the moderately thermophilic Clostridium strain AK1" [1]. The influence of culture conditions on end product formation from D-glucose and L-rhamnose by AK1 was investigated in batch culture. Strain AK1 was cultivated at initial substrate concentrations varying from 0 to 60 mM and initial pH values varying from 4.5 to 8.5. Additionally different cultivation temperatures (30-65 °C), the influence of liquid-gas phase ratio as well as different phosphate concentrations on growth were investigated.

Fermentation of Mannitol Extracts From Brown Macro Algae by Thermophilic Clostridia.

Mannitol-containing macro algae biomass, such as Ascophyllum nodosum and Laminaria digitata, are a potential feedstock for the production of biofuels such as bioethanol. The purpose of this work was to evaluate the ability of thermophilic anaerobes within Class Clostridia to ferment mannitol and mannitol-containing algal extracts. Screening of the type strains of six genera, Caldanaerobius, Caldanaerobacter, Caldicellulosiruptor, Thermoanaerobacter, Thermobrachium, and Thermoanaerobacterium) was conducted on 20 mM mannitol and revealed that 11 of 41 strains could utilize mannitol with ethanol being the dominant end-product. Mannitol utilization seems to be most common within the genus of Thermoanaerobacter (7 of 16 strains) with yields up to 88% of the theoretical yield in the case of Thermoanaerobacter pseudoethanolicus. Six selected mannitol-degrading strains (all Thermoanaerobacter species) were grown on mannitol extracts prepared from A. nodosum and L. digitata. Five of the strains produced similar amounts of ethanol as compared with ethanol yields from mannitol only. Finally, T. pseudoethanolicus was kinetically monitored using mannitol and mannitol extracts made from two macro algae species, A. nodosum and L. digitata for end-product formation.

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.

Amino Acid Metabolism of Thermoanaerobacter Strain AK90: The Role of Electron-Scavenging Systems in End Product Formation.

The catabolism of the 20 amino acids by Thermoanaerobacter strain AK90 (KR007667) was investigated under three different conditions: as single amino acids without an electron-scavenging system, in the presence of thiosulfate, and in coculture with a hydrogenotrophic methanogen. The strain degraded only serine without an alternative electron acceptor but degraded 11 amino acids (alanine, cysteine, isoleucine, leucine, lysine, methionine, phenylalanine, serine, threonine, tyrosine, and valine) under both of the electron-scavenging systems investigated. Acetate was the dominant end product from alanine, cysteine, lysine, serine, and threonine under electron-scavenging conditions. The branched-chain amino acids, isoleucine, leucine, and valine, were degraded to their corresponding fatty acids under methanogenic conditions and to a mixture of their corresponding fatty acids and alcohols in the presence of thiosulfate. The partial pressure of hydrogen seems to be of importance for the branched-chain alcohol formation. This was suggested by low but detectable hydrogen concentrations at the end of cultivation on the branched-chain amino acid in the presence of thiosulfate but not when cocultured with the methanogen. A more detailed examination of the role of thiosulfate as an electron acceptor was performed with Thermoanaerobacter ethanolicus (DSM 2246) and Thermoanaerobacter brockii (DSM 1457).

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.

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).

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.

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)).