Prolonged conversion of n-butyrate to n-butanol with Clostridium saccharoperbutylacetonicum in a two-stage continuous culture with in-situ product removal

n‐Butanol was produced continuously in a two‐stage fermentor system with integrated product removal from a co‐feed of n‐butyric acid and glucose. Glucose was always required as a source of ATP and electrons for the conversion of n‐butyrate to n‐butanol and for biomass growth; for the latter it also served as a carbon source. The first stage generated metabolically active planktonic cells of Clostridium saccharoperbutylacetonicum strain N1‐4 that were continuously fed into the second (production) stage; the volumetric ratio of the two fermentors was 1:10. n‐Butanol was removed continuously from the second stage via gas stripping. Implementing a two‐stage process was observed to dramatically dampen metabolic oscillations (i.e., periodical changes of solventogenic activity). Culture degeneration (i.e., an irreversible loss of solventogenic activity) was avoided by periodical heat shocking and re‐inoculating stage 1 and by maintaining the concentration of undissociated n‐butyric acid in stage 2 at 3.4 mM with a pH‐auxostat. The system was successfully operated for 42 days during which 93% of the fed n‐butyrate was converted to n‐butanol at a production rate of 0.39 g/(L × h). The molar yields Y_{n‐butanol/n‐butyrate} and Y_{n‐butanol/glucose} were 2.0, and 0.718, respectively. For the same run, the molar ratio of n‐butyrate to glucose consumed was 0.358. The molar yield of carbon in n‐butanol produced from carbon in n‐butyrate and glucose consumed (Y_{n‐butanol/carbon}) was 0.386. These data illustrate that conversion of n‐butyrate into n‐butanol by solventogenic Clostridium species is feasible and that this can be performed in a continuous system operating for longer than a month. However, our data also demonstrate that a relatively large amount of glucose is required to supply electrons and ATP for this conversion and for cell growth in a continuous culture. Biotechnol. Bioeng. 2012; 109:913–921. © 2011 Wiley Periodicals, Inc.     

Optimization of redox reactions employing whole cell biocatalysis

The ability of Saccharomyces cerevisiae to catalyse the reduction reaction of carboxylic acids into alcohols is described. Earlier reports have led to the characterization of the reduction of carbonyl groups into alcohols mediated by the enzyme alcohol dehydrogenase. We investigated the ability of this organism to catalyse the said conversion using the carboxylic acids, acetic acid and butyric acid. In the absence of any previous characterization, whole cell catalysis proved effective. The uptake of these acids from the medium was estimated using a plate assay method involving litmus-agar. The plate assay was found to be a convenient and extremely adaptable method for quantitation of acids in organic as well as aqueous medium. The comparison of existing paradigms in pure protein catalysis with whole cells catalysis proved anomalous. We report that it is solvent toxicity rather than hydrophobic index that correlates with the activity observed in non-aqueous conditions for whole cell biocatalysis. Reduction of acetic acid as well as butyric acid occurred, with efficiency of reaction with butyric acid being marginally higher. The reduction therefore occurs for both the short chain carboxylic acids used in this study. We therefore illustrate the reduction route of acids into alcohols and propose a model two-step pathway for the reaction. Process optimization may be further attempted to enhance the presently moderate reaction efficiencies. Steps made in the direction by studying the pH dependency and use of sacrificial substrate have yielded encouraging results.     

Synthesis of flavor and fragrance esters using <Emphasis Type="Italic">Candida antarctica</Emphasis> lipase

Candida antarctica lipase fraction B (CAL-B) showed substrate specificity in the synthesis of esters in hexane involving reactions of short-chain acids having linear (acetic and butyric acids) and branched chain (isovaleric acid) structures, an unsaturated (tiglic acid) fatty acid, and phenylacetic acid with n-butanol and geraniol. The variation in the conversion to the esters was ca. 10%. Similar results were observed in a study of the alcohol specificity of the enzyme for esterification of acetic and butyric acids with four alcohols: n-butyl, isopentyl, 2-phenylethyl, and geraniol. Enantioselectivity of CAL-B in hexane with a range of chiral -substituted or -substituted carboxylic acids and n-butyl alcohol was analyzed. The results show that CAL-B can be employed as a robust biocatalyst in esterification reactions due to the high conversions obtained in the synthesis of short-chain flavor esters in an organic solvent, although this enzyme exhibited modest enantioselectivity with chiral short-chain carboxylic acids.     

Purification and characterization of a biodegradable plastic-degrading enzyme from Aspergillus oryzae

We used biodegradable plastics as fermentation substrates for the filamentous fungus Aspergillus oryzae. This fungus could grow under culture conditions that contained emulsified poly-(butylene succinate) (PBS) and emulsified poly-(butylene succinate-co-adipate) (PBSA) as the sole carbon source, and could digest PBS and PBSA, as indicated by clearing of the culture supernatant. We purified the PBS-degrading enzyme from the culture supernatant, and its molecular mass was determined as 21.6 kDa. The enzyme was identified as cutinase based on internal amino acid sequences. Specific activities against PBS, PBSA and poly-(lactic acid) (PLA) were determined as 0.42 U/mg, 11 U/mg and 0.067 U/mg, respectively. To obtain a better understanding of how the enzyme recognizes and hydrolyzes PBS/PBSA, we investigated the environment of the catalytic pocket, which is divided into carboxylic acid and alcohol recognition sites. The affinities for different substrates depended on the carbon chain length of the carboxylic acid in the substrate. Competitive inhibition modes were exhibited by carboxylic acids and alcohols that consisted of C₄-C₆ and C₃-C₈ chain lengths, respectively. Determination of the affinities for different chemicals indicated that the most preferred substrate for the enzyme would consist of butyric acid and n-hexanol.This revised version was published online in February 2005 with corrections to Table 1.     

Bio-electrochemical synthesis of commodity chemicals by autotrophic acetogens utilizing CO<Subscript>2</Subscript> for environmental remediation

Bio-electrochemical synthesis (BES) is a technique in which electro-autotrophic bacteria such as Clostridium ljungdahlii utilize electric currents as an electron source from the cathode to reduce CO₂ to extracellular, multicarbon, exquisite products through autotrophic conversion. The BES of volatile fatty acids and alcohols directly from CO₂ is a sustainable alternative for non-renewable, petroleum-based polymer production. This conversion of CO₂ implies reduction of greenhouse gas emissions. The synthesis of heptanoic acid, heptanol, hexanoic acid and hexanol, for the first time, by Clostridium ljungdahlii was a remarkable achievement of BES. In our study, these microorganisms were cultivated on the cathode of a bio-electrochemical cell at ?400 mV by a DC power supply at 37°C, pH 6.8, and was studied for both batch and continuous systems. Pre-enrichment of bio-cathode enhanced the electroactivity of cells and resulted in maximizing extracellular products in less time. The main aim of the research was to investigate the impact of low-cost substrate CO₂, and the longer cathode recovery range was due to bacterial reduction of CO₂ to multicarbon chemical commodities with electrons driven from the cathode. Reactor design was simplified for cost-effectiveness and to enhance energy efficiencies. The Columbic recovery of ethanoic acid, ethanol, ethyl butyrate, hexanoic acid, heptanoic acid and hexanol being in excess of 80% proved that BES was a remarkable technology.     

Online measurement of dissolved carbon monoxide concentrations reveals critical operating conditions in gas fermentation experiments

Syngas fermentation is one possible contributor to the reduction of greenhouse gas emissions. The conversion of industrial waste gas streams containing CO or H₂, which are usually combusted, directly reduces the emission of CO₂ into the atmosphere. Additionally, other carbon‐containing waste streams can be gasified, making them accessible for microbial conversion into platform chemicals. However, there is still a lack of detailed process understanding, as online monitoring of dissolved gas concentrations is currently not possible. Several studies have demonstrated growth inhibition of Clostridium ljungdahlii at high CO concentrations in the headspace. However, growth is not inhibited by the CO concentration in the headspace, but by the dissolved carbon monoxide tension (DCOT). The DCOT depends on the CO concentration in the headspace, CO transfer rate, and biomass concentration. Hence, the measurement of the DCOT is a superior method to investigate the toxic effects of CO on microbial fermentation. Since CO is a component of syngas, a detailed understanding is crucial. In this study, a newly developed measurement setup is presented that allows sterile online measurement of the DCOT. In an abiotic experiment, the functionality of the measurement principle was demonstrated for various CO concentrations in the gas supply (0%–40%) and various agitation rates (300–1100 min^?1). In continuous stirred tank reactor fermentation experiments, the measurement showed reliable results. The production of ethanol and 2,3‐butanediol increased with increasing DCOT. Moreover, a critical DCOT was identified, leading to the inhibition of the culture. Thus, the reported online measurement method is beneficial for process understanding. In future processes, it can be used for closed‐loop fermentation control.     

Production of biofuels from C1-gases with Clostridium and related bacteria—Recent advances

Clostridium spp. are suitable for the bioconversion of C₁-gases (e.g., CO₂, CO and syngas) into different bioproducts. These products can be used as biofuels and are reviewed here, focusing on ethanol, butanol and hexanol, mainly. The production of higher alcohols (e.g., butanol and hexanol) has hardly been reviewed. Parameters affecting the optimization of the bioconversion process and bioreactor performance are addressed as well as the pathways involved in these bioconversions. New aspects, such as mixotrophy and sugar versus gas fermentation, are also reviewed. In addition, Clostridia can also produce higher alcohols from the integration of the Wood-Ljungdahl pathway and the reverse ß-oxidation pathway, which has also not yet been comprehensively reviewed. In the latter process, the acetogen uses the reducing power of CO/syngas to reduce C₄ or C₆ fatty acids, previously produced by a chain elongating microorganism (commonly Clostridium kluyveri), into the corresponding bioalcohol.     

Accumulation of l-Glutamic Acid from Dibasic Carboxylic Acids by a Hydrocarbon Utilizing Bacterium

In order to know the substrate specificity in a hydrocarbon utilizing bacterium, the following materials were examined: n-alkanes, n-alkenes, monohydric alcohols, aldehydes, monobasic carboxylic acids, dihydric alcohols and dibasic carboxylic acids.

It was found that dibasic carboxylic acids were well utilized, and a great deal of l-glutamic acid was accumulated from them. Then suberic acid, which is C₈ dibasic carboxylic acid, was compared with n-dodecane in the effects of thiamine, penicillin, C/N ratio and substrate concentration on l-glutamic acid accumulation and cell growth.     

Effects of extractive fermentation on butyric acid production byClostridium acetobutylicum

Summary The addition of an oleyl alcohol extractant to a batch fermentation of glucose byClostridium acetobutylicum resulted in a concentration profile that was distinctly different from the non-extractive control fermentation. The concentration of butyric acid increased and subsequently decreased in the control fermentation. The concentration of butyric acid increased but did not subsequently decrease in the oleyl alcohol extractive fermentation. The production of butyric acid was found to have been prolonged into the solventogenic phase in the oleyl alcohol extractive fermentation. Butyric acid was continually replenished from glucose while it was being converted to butanol. Supplementation of exogenous acetic and butyric acids, the metabolic uncoupler carbonyl cyanide 3-chlorophenylhydrazone, or decanol to the oleyl alcohol extractive fermentation helped to reinstate the normal butyric acid concentration profile. These findings are discussed with respect to the effects of these additives on the pH ofC. acetobutylicum and its importance with regard to the production of butyric acid.     

Online monitoring applying the anaerobic respiratory monitoring system reveals iron(II) limitation in YTF medium for Clostridium ljungdahlii

Online monitoring of microbial cultures is an effective approach for studying both aerobic and anaerobic microorganisms. Especially in small‐scale cultivations, several parallel online monitored experiments can generate a detailed understanding of the cultivation, compared to a situation where a few data points are generated from time course sampling and offline analysis. However, the availability of small‐scale online monitoring devices for acetogenic organisms is limited. In this study, the previously reported anaerobic Respiration Activity MOnitoring System (anaRAMOS) device was adapted for online monitoring of Clostridium ljungdahlii (C. ljungdahlii) cultures with fructose as the carbon source. The anaRAMOS was applied to identify conversion of different carbon sources present in commonly used YTF medium. An iron(II) deficiency was discovered in this medium for C. ljungdahlii. Addition of iron(II) to the YTF medium reduced the cultivation time and increased biomass yield of C. ljungdahlii cultures by 50% and 40%, respectively. The measurement of the carbon dioxide transfer rate was used to calculated the iron(II) contained in complex components. By demonstrating the application of the anaRAMOS device for medium optimization, it is proven that the described online monitoring device has potential for use in process development.     

Enhanced butyric acid tolerance and bioproduction by Clostridium tyrobutyricum immobilized in a fibrous bed bioreactor

Repeated fed‐batch fermentation of glucose by Clostridium tyrobutyricum immobilized in a fibrous bed bioreactor (FBB) was successfully employed to produce butyric acid at a high final concentration as well as to adapt a butyric‐acid‐tolerant strain. At the end of the eighth fed‐batch fermentation, the butyric acid concentration reached 86.9 ± 2.17 g/L, which to our knowledge is the highest butyric acid concentration ever produced in the traditional fermentation process. To understand the mechanism and factors contributing to the improved butyric acid production and enhanced acid tolerance, adapted strains were harvested from the FBB and characterized for their physiological properties, including specific growth rate, acid‐forming enzymes, intracellular pH, membrane‐bound ATPase and cell morphology. Compared with the original culture used to seed the bioreactor, the adapted culture showed significantly reduced inhibition effects of butyric acid on specific growth rate, cellular activities of butyric‐acid‐forming enzyme phosphotransbutyrylase (PTB) and ATPase, together with elevated intracellular pH, and elongated rod morphology. Biotechnol. Bioeng. 2011; 108:31–40. © 2010 Wiley Periodicals, Inc.     

In silico metabolic engineering of Clostridium ljungdahlii for synthesis gas fermentation

Synthesis gas fermentation is one of the most promising routes to convert synthesis gas (syngas; mainly comprised of H₂ and CO) to renewable liquid fuels and chemicals by specialized bacteria. The most commonly studied syngas fermenting bacterium is Clostridium ljungdahlii, which produces acetate and ethanol as its primary metabolic byproducts. Engineering of C. ljungdahlii metabolism to overproduce ethanol, enhance the synthesize of the native byproducts lactate and 2,3-butanediol, and introduce the synthesis of non-native products such as butanol and butyrate has substantial commercial value. We performed in silico metabolic engineering studies using a genome-scale reconstruction of C. ljungdahlii metabolism and the OptKnock computational framework to identify gene knockouts that were predicted to enhance the synthesis of these native products and non-native products, introduced through insertion of the necessary heterologous pathways. The OptKnock derived strategies were often difficult to assess because increase product synthesis was invariably accompanied by decreased growth. Therefore, the OptKnock strategies were further evaluated using a spatiotemporal metabolic model of a syngas bubble column reactor, a popular technology for large-scale gas fermentation. Unlike flux balance analysis, the bubble column model accounted for the complex tradeoffs between increased product synthesis and reduced growth rates of engineered mutants within the spatially varying column environment. The two-stage methodology for deriving and evaluating metabolic engineering strategies was shown to yield new C. ljungdahlii gene targets that offer the potential for increased product synthesis under realistic syngas fermentation conditions.     

Propionic acid fermentation from glycerol: comparison with conventional substrates

Instead of the conventional carbon sources used for propionic acid biosynthesis, the utilization of glycerol is considered here, since the metabolic pathway involved in the conversion of glycerol to propionic acid is redox-neutral and energetic. Three strains, Propionibacterium acidipropionici, Propionibacterium acnes and Clostridium propionicum were tested for their ability to convert glycerol to propionic acid during batch fermentation with initially 20 g/l glycerol. P. acidipropionici showed higher efficiency in terms of fermentation time and conversion yield than did the other strains. The fermentation profile of this bacterium consisted in propionic acid as the major product (0.844 mol/mol), and in minimal by-products: succinic (0.055 mol/mol), acetic (0.023 mol/mol) and formic (0.020 mol/mol) acids and n-propanol (0.036 mol/mol). The overall propionic acid productivity was 0.18 g l⁻¹h⁻¹. A comparative study with glucose and lactic acid as carbon sources showed both less diversity in end-product composition and a 17% and 13% lower propionic acid conversion yield respectively than with glycerol. Increasing the initial glycerol concentration resulted in an enhanced productivity up to 0.36 g l⁻¹h⁻¹ and in a maximal propionic acid concentration of 42 g/l, while a slight decrease of the conversion yield was noticed. Such a propionic acid production rate was similar or higher than the values obtained with lactic acid (0.35 g l⁻¹h⁻¹) or glucose (0.28 g l⁻¹h⁻¹). These results demonstrated that glycerol is a carbon source of interest for propionic acid production. Received: 15 July 1996 / Received revision: 11 November 1996 / Accepted: 11 November 1996     

A novel isolate of <Emphasis Type="Italic">Clostridium butyricum</Emphasis> for efficient butyric acid production by xylose fermentation

Bacterial fermentation of lignocellulose has been regarded as a sustainable approach to butyric acid production. However, the yield of butyric acid is hindered by the conversion efficiency of hydrolysate xylose. A mesophilic alkaline-tolerant strain designated as Clostridium butyricum B10 was isolated by xylose fermentation with acetic and butyric acids as the principal liquid products. To enhance butyric acid production, performance of the strain in batch fermentation was evaluated with various temperatures (20–47 °C), initial pH (5.0–10.0), and xylose concentration (6–20 g/L). The results showed that the optimal temperature, initial pH, and xylose concentration for butyric acid production were 37 °C, 9.0, and 8.00 g/L, respectively. Under the optimal condition, the yield and specific yield of butyric acid reached about 2.58 g/L and 0.36 g/g xylose, respectively, with 75.00% butyric acid in the total volatile fatty acids. As renewable energy, hydrogen was also collected from the xylose fermentation with a yield of about 73.86 mmol/L. The kinetics of growth and product formation indicated that the maximal cell growth rate (μ _m ) and the specific butyric acid yield were 0.1466 h^?1 and 3.6274 g/g cell (dry weight), respectively. The better performance in xylose fermentation showed C. butyricum B10 a potential application in efficient butyric acid production from lignocellulose.     

Engineering Clostridium ljungdahlii as the gas-fermenting cell factory for the production of biofuels and biochemicals

Clostridium ljungdahlii is a representative autotrophic gas-fermenting acetogen capable of converting CO₂ and CO into biomass and multiple metabolites. The carbon fixation and conversion based on C. ljungdahlii have great potential for the sustainable production of bulk biochemicals and biofuels using industrial syngas and waste gases. With substantial recent advances in genetic manipulation tools, it has become possible to study and improve the metabolic capability of C. ljungdahlii in gas fermentation. The product scope of C. ljungdahlii has been expanded through the introduction of heterologous production pathways followed by the modification of native metabolic networks. In addition, progress has been made in understanding the physiological and metabolic mechanisms of this anaerobe, contributing to strain designs for expected phenotypes. In this review, we highlight the latest research progresses regarding C. ljungdahlii and discuss the next steps to comprehensively understand and engineer this bacterium for an improved bacterial gas bioconversion platform.     

Effects of Acetate and Butyrate During Glycerol Fermentation by Clostridium butyricum

The effects of acetate and butyrate during glycerol fermentation to 1,3-propanediol at pH 7.0 by Clostridium butyricum CNCM 1211 were studied. At pH 7.0, the calculated quantities of undissociated acetic and butyric acids were insufficient to inhibit bacterial growth. The initial addition of acetate or butyrate at concentrations of 2.5 to 15 gL⁻¹ had distinct effects on the metabolism and growth of Clostridium butyricum. Acetate increased the biomass and butyrate production, reducing the lag time and 1,3-propanediol production. In contrast, the addition of butyrate induced an increase in 1,3-propanediol production (yield: 0.75 mol/mol glycerol, versus 0.68 mol/mol in the butyrate-free culture), and reduced the biomass and butyrate production. It was calculated that reduction of butyrate production could provide sufficient NADH to increase 1,3-propanediol production. The effects of acetate and butyrate highlight the metabolic flexibility of Cl. butyricum CNCM 1211 during glycerol fermentation. Received: 2 January 2001 / Accepted: 6 February 2001     

Electron availability in CO2, CO and H2 mixtures constrains flux distribution,energy management and product formation in Clostridium ljungdahlii

Acetogens such as Clostridium ljungdahlii can play a crucial role reducing the human CO₂ footprint by converting industrial emissions containing CO₂, CO and H₂ into valuable products such as organic acids or alcohols. The quantitative understanding of cellular metabolism is a prerequisite to exploit the bacterial endowments and to fine-tune the cells by applying metabolic engineering tools. Studying the three gas mixtures CO₂ + H₂, CO and CO + CO₂ + H₂ (syngas) by continuously gassed batch cultivation experiments and applying flux balance analysis, we identified CO as the preferred carbon and electron source for growth and producing alcohols. However, the total yield of moles of carbon (mol-C) per electrons consumed was almost identical in all setups which underlines electron availability as the main factor influencing product formation. The Wood–Ljungdahl pathway (WLP) showed high flexibility by serving as the key NAD⁺ provider for CO₂ + H_2, whereas this function was strongly compensated by the transhydrogenase-like Nfn complex when CO was metabolized. Availability of reduced ferredoxin (Fd_red) can be considered as a key determinant of metabolic control. Oxidation of CO via carbon monoxide dehydrogenase (CODH) is the main route of Fd_red formation when CO is used as substrate, whereas Fd_red is mainly regenerated via the methyl branch of WLP and the Nfn complex utilizing CO₂ + H₂. Consequently, doubled growth rates, highest ATP formation rates and highest amounts of reduced products (ethanol, 2,3-butanediol) were observed when CO was the sole carbon and electron source.     

The Formation of Higher Alcohols in the Fermentation of Amino Acids by Yeast

We have found that some straight-chained α-amino acids are converted by yeast to the alcohols with correspondingly longer carbon chains in the alcoholic fermentation contrary to F. Ehrlich’s scheme, i.e., isobutyl alcohol from alanine and active amyl alcohol from α-amino-n-butyric acid or threonine.

In this report, we confirmed this fact in the alcoholic fermentation of many aliphatic amino acids by 2 yeast strains using gas chromatography. Moreover, n-propyl alcohol was proved to come from α-amino-n-butyric acid or threonine. Small quantities of n-propyl, isobutyl, active amyl and isoamyl alcohols were found in all the fermented solutions. There was some difference in the composition of higher alcohols of the alcoholic solutions fermented by different yeasts.     

Acidolysis between Triolein and Short-Chain Fatty Acid by Lipase in Organic Solvents

Ten kinds of lipases were examined as biocatalysts for the incorporation of short-chain fatty acids (acetic, propionic, and butyric acids) into triolein in order to produce one kind of reduced-calorie structured lipids. Trans-esterification (acidolysis) was successfully done in n-hexane by several microbial lipases. Among them, lipase from Aspergillus oryzae was used to investigate the effects of incubation time, substrate molar ratio, and water content on acidolysis. Finally, more than 80% of triolein was incorporated by butyric acid (molar ratio of triolein to butyric acid, 1:10) in the dried n-hexane at 52 °C for 72 h. More than 90% of the products was monosubstituent, which was esterified with this short chain fatty acid at the 1-position of the glycerol moiety of triolein. These results suggest that A. oryzae lipase would be a powerful biocatalyst for the synthesis of low caloric oil, such as triacylglycerol containing a mixture of long- and short-chain aliphatic acids.