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Clostridium fermentations have been developed for producing butanol and other value-added chemicals, but their development is constrained by some limitations, such as relatively high substrate cost and the need to maintain an anaerobic condition. Recently, co-culture is emerging as a popular way to address these limitations by introducing a partner strain with Clostridium. Generally speaking, the co-culture strategy enables the use of a cheaper substrate, maintains the growth of Clostridium without any anaerobic treatment, improves product yields, and/or widens the product spectrum. Herein, we review recent developments of co-culture strategies involving Clostridium species according to their partner stains' functions with representative examples. We also discuss research challenges that need to be addressed for the future development of Clostridium co-cultures. Clostridium co-culture has emerged as a solution to some problems faced by Clostridium monoculture. Co-culturing Clostridium with cellulolytic strains or acetogens enables the production of butanol from cheaper substrates such as lignocellulosic biomass and syngas. Aerotolerance is possible in anaerobic Clostridium fermentation in co-cultures with oxygen-consuming microbes. Co-culturing solventogenic Clostridium with acetogens can improve the production of butanol and its derivatives by increasing carbon recovery and relieving solvent toxicity. Other products, such as H 2 or fatty acids, can be efficiently produced by the co-culture of Clostridium and other H 2 -producing strains (such as PNS bacteria), or chain-elongating strains respectively. [ABSTRACT FROM AUTHOR]

Butanol and butyric acid produced from acetone-butanol-ethanol (ABE) fermentation can be used to produce butyl butyrate, an important fragrance ester. However, low levels of butanol and butyric acid need to be purified from culture media first with energy-intensive distillation processes. In this study, a triphasic (organic/aqueous/fluorous) system is developed to esterify butanol and butyric acid in spent culture media into butyl butyrate directly without purification. The produced butyl butyrate forms a distinct organic phase floating on top and can then be separated easily. In a model system containing 37.1 g/L of butanol and 44.1 g/L of butyric acid, 57% of the butanol is converted to butyl butyrate after 8 h of esterification. With multiple cycles of esterification and product removal, butanol conversion can be further increased to 86%. When spent culture medium containing 7.12 g/L of butanol and 4.81 g/L of butyric acid is used for esterification, 38% of butanol (0.36 mmol) is consumed and 0.33 mmol of butyl butyrate is produced. However, when ABE fermentation and esterification are carried out simultaneously, only 0.042 mmol of butyl butyrate is produced, probably due to the incompatible pH requirements for cell growth (pH 5–7) and esterification (pH 2–3). [ABSTRACT FROM AUTHOR]

Esterification of organic acids and alcohols in aqueous media is very inefficient due to thermodynamic constraints. However, fermentation processes used to produce organic acids and alcohols are often conducted in aqueous media. To produce esters in aqueous media, biphasic alginate beads with immobilized lipase are developed for in situ esterification of butanol and butyric acid. The biphasic beads contain a solid matrix of calcium alginate and hexadecane together with 5 mg/mL of lipase as the biocatalyst. Hexadecane in the biphasic beads serves as an organic phase to facilitate the esterification reaction. Under optimized conditions, the beads are able to catalyze the production of 0.16 mmol of butyl butyrate from 0.5 mmol of butyric acid and 1.5 mmol of butanol. In contrast, when monophasic beads (without hexadecane) are used, only trace amount of butyl butyrate is produced. One main application of biphasic beads is in simultaneous fermentation and esterification (SFE) because the organic phase inside the beads is very stable and does not leach out into the culture medium. SFE is successfully conducted with an esterification yield of 6.32% using biphasic beads containing iso-octane even though the solvent is proven toxic to the butanol-producing Clostridium spp. [ABSTRACT FROM AUTHOR]

Clostridium beijerinckii G117 produces hydrogen (∼4400 mL/L) from 15 g/L of native crude glycerol (CG). While increasing the native-CG to 20–80 g/L, strain G117 rejects 34–90% of CG and fails to support a relative enhancement in hydrogen production. Subsequently, to enhance the native-CG assimilation, and hydrogen production from strain G117, various agro-residual hydrolysates (ARH), and several metal-ions have individually tested as media-supplements. Among tested, rice bran hydrolysate (RBH) and ferrous chloride (Fe2+) are selected as media-supplements. By employing RSM-based central composite design, the medium composed of 37.63 g/L CG, 3.65 g/L RBH and 1.11 mM Fe2+ is optimized which results a favorable initial redox potential (−191.0 ± 1.5 mV), higher cellular growth (6.73 ± 0.12 OD 600), and 17350 ± 170 mL/L of hydrogen production from strain G117. The analysis of total-mass and electron balances suggest that this bioprocess is remarkably efficient (82–99%), and records the highest yield of hydrogen (2.27 ± 0.02 mol/mol glycerol) ever reported in literature. [Display omitted] • Clostridium beijerinckii G117 produces hydrogen from <15 g/L of crude glycerol (CG). • While raising CG to 20–80 g/L in the media, strain G117 rejects 34–90% of CG supplied. • Adding agro-residual hydrolysates/metal-ions alter CG-utilization and hydrogen yield. • Media of CG with rice bran hydrolysate as co-substrate and Fe2+ is optimized (OpM). • OpM supports G117 to produce 17350 mL/L hydrogen with yield of 2.27 mol H2 /mol Glycerol. [ABSTRACT FROM AUTHOR]

Butanol has been considered as a better alternative fuel and it can be produced from anaerobic Clostridial fermentation. Though several enzymes are involved in the biosynthesis of butanol in Clostridia, butanol dehydrogenase (BDH) is understood to play a major role, which catalyzes the conversion of butyraldehyde into butanol at the expenditure of a cofactor NAD(P)H. Recently, the strain Clostridium sp. BOH3 is reported to generate high level of butanol from monosugars. To investigate the BDH activity at various stages of fermentation, BOH3 was cultured in reinforced Clostridial medium with 30 g/l of glucose at 35 °C and the cells were harvested periodically from acid production and solvent production phases. During acid production, NADPH-dependent BDH activity is higher than NADH dependent BDH. Conversely, NADH-BDH activity is predominant during solvent production phase. The optimum pHs for NADH and NADPH-BDH are estimated as pH 6 and 8, respectively. By employing three steps of purification, NADH-BDH is purified to 102-fold with 36 % yield. Subsequent characterization reveals that NADH-BDH is a dimer composed of two subunits depicting the molecular weight of 44 kDa. The peptide finger printing analysis (MS/MS) suggests that the purified protein has higher homology with bifunctional acetaldehyde-CoA and alcohol dehydrogenase of Clostridium acetobutylicum. The extensive kinetic studies show that NADH-BDH follows an ordered sequential bi bi mechanism. The calculated values of K and K are 8.35 ± 0.25 and 0.076 ± 0.02 mM, respectively, whereas V is 4.02 ± 0.07 μmol/(mg protein. min). The purified NADH-BDH retains 70 % of its initial activity after 7 days at 4 °C. [ABSTRACT FROM AUTHOR]

Butanol toxicity, oxygen sensitivity, and high substrate cost limit wider applications of the traditional Clostridium acetone-butanol-ethanol (ABE) fermentation. In this study, a wild-type Clostridium beijerinckii strain was partnered with an engineered Escherichia coli strain to improve the ABE fermentation through a co-culture. Butanol and butyrate produced by C. beijerinckii were converted into butyl butyrate – a product that can be in situ removed to reduce product toxicity – by expressing a CoA transferase and an alcohol acyltransferase in E. coli. When integrated with a pretreatment technology and using commercial cellulase, the co-culture produced 1280 mg/L butyl butyrate from rice straw in bioreactors, without the need of maintaining a strict anaerobic condition. As a process harnessing both the genetic tractability of E. coli and the superior acid- and alcohol-producing ability of Clostridium , this co-culture fermentation will be useful in producing valuable esters from low-cost waste streams. [Display omitted] • E. coli was engineered to express a CoA transferase and an alcohol acyltransferase. • Butyl butyrate was produced by the co-culture from lignocellulosic biomass. • Aerobic co-culture of E. coli and Clostridium was achieved. [ABSTRACT FROM AUTHOR]

In this study, a fermentation process for production of butanol and butyl-butyrate by using Clostridium sp. strain BOH3 is developed. This strain is able to produce butyric acid and butanol when it ferments 60 g/L xylose. Meanwhile, it also excreted indigenous lipases (induced by olive oil) which naturally convert butyric acid and butanol into 1.2 g/L of butyl-butyrate. When Bio-OSR was used as both an inducer for lipase and extractant for butyl-butyrate, the butyl-butyrate concentration can reach 6.3 g/L. To further increase the yield, additional lipases and butyric acid are added to the fermentation system. Moreover, kerosene was used as an extractant to remove butyl-butyrate in situ . When all strategies are combined, 22.4 g/L butyl-butyrate can be produced in a fed-batch reactor spiked with 70 g/L xylose and 7.9 g/L butyric acid, which is 4.5-fold of that in a similar system (5 g/L) with hexadecane as the extractant. [ABSTRACT FROM AUTHOR]

Solventogenic Clostridium strains usually do not ferment lignocellulosic biomass directly due to insufficient expression of hydrolyzing enzymes. In this study, we show that a solventogenic Clostridium strain BOH3 is able to produce both butanol and hydrogen when it ferments mixtures of agricultural residues and oil cakes. In particular, fermentation of mixed rice bran and sesame oil cake gives the highest butanol and hydrogen concentrations. In an optimized agro-residual medium (OAM) containing 94.5 g/l of rice bran and 36.7 g/l of sesame oil cake, BOH3 produces 13.50 ± 0.12 g/l of butanol and 4.41 ± 0.04 l/l of hydrogen after 7 days. Because BOH3 excretes hydrolyzing enzymes such as cellulase (0.52 ± 0.08 U/ml), xylanase (4.10 ± 0.05 U/ml) and amylase (2.05 ± 0.12 U/ml) during fermentation, BOH3 is able to perform fermentation and saccharification simultaneously when rice bran and sesame oil cakes are used as substrates. This is the first report showing that direct fermentation of agricultural residues by using solventogenic Clostridia is possible. [ABSTRACT FROM AUTHOR]

Highlights: [•] Clostridium strain BOH3 is able to use xylan for its growth and ABE fermentation. [•] Optimized culture medium (OCM) enhances xylanase expression up to 1.6-fold. [•] In OCM, strain BOH3 can effectively utilize high concentration (30–50g/l) of xylan. [•] BOH3 produces >14g/l of butanol and >2.6l/l of hydrogen from xylan in OCM. [Copyright &y& Elsevier]

Abstract: A unique Clostridium species strain G117 was obtained in this study to be capable of producing dominant butanol from glucose. Butanol of 13.50g/L was produced when culture G117 was fed with 60g/L glucose, which is ∼20% higher than previously reported butanol production by wild-type Clostridium acetobutylicum ATCC 824 under similar conditions. Strain G117 also distinguishes itself by generating negligible amount of ethanol, but producing butanol and acetone as biosolvent end-products. A butanol dehydrogenase gene (bdh gene) was identified in strain G117, which demonstrated a ∼200-fold increase in transcription level measured by quantitative real-time PCR after 10h of culture growth. The high transcription suggests that this bdh gene could be a putative gene involved in butanol production. In all, Clostridium sp. strain G117 serves as a potential candidate for industrial biobutanol production while the absence of ethanol ensures an economic-efficient separation and purification of butanol. [Copyright &y& Elsevier]