Recent advances and state-of-the-art strategies in strain and process engineering for biobutanol production by Clostridium acetobutylicum

Butanol as an advanced biofuel has gained great attention due to its environmental benefits and superior properties compared to ethanol. However, the cost of biobutanol production via conventional acetone-butanol-ethanol (ABE) fermentation by Clostridium acetobutylicum is not economically competitive, which has hampered its industrial application. The strain performance and downstream process greatly impact the economics of biobutanol production. Although various engineered strains with carefully orchestrated metabolic and sporulation-specific pathways have been developed, none of them is ideal for industrial biobutanol production. For further strain improvement, it is necessary to develop advanced genome editing tools and a deep understanding of cellular functioning of genes in metabolic and regulatory pathways. Processes with integrated product recovery can increase fermentation productivity by continuously removing inhibitory products while generating butanol (ABE) in a concentrated solution. In this review, we provide an overview of recent advances in C. acetobutylicum strain engineering and process development focusing on in situ product recovery. With deep understanding of systematic cellular bioinformatics, the exploration of state-of-the-art genome editing tools such as CRISPR-Cas for targeted gene knock-out and knock-in would play a vital role in Clostridium cell engineering for biobutanol production. Developing advanced hybrid separation processes for in situ butanol recovery, which will be discussed with a detailed comparison of advantages and disadvantages of various recovery techniques, is also imperative to the economical development of biobutanol.     

Metaproteomics of cellulose methanisation under thermophilic conditions reveals a surprisingly high proteolytic activity

Cellulose is the most abundant biopolymer on Earth. Optimising energy recovery from this renewable but recalcitrant material is a key issue. The metaproteome expressed by thermophilic communities during cellulose anaerobic digestion was investigated in microcosms. By multiplying the analytical replicates (65 protein fractions analysed by MS/MS) and relying solely on public protein databases, more than 500 non-redundant protein functions were identified. The taxonomic community structure as inferred from the metaproteomic data set was in good overall agreement with 16S rRNA gene tag pyrosequencing and fluorescent in situ hybridisation analyses. Numerous functions related to cellulose and hemicellulose hydrolysis and fermentation catalysed by bacteria related to Caldicellulosiruptor spp. and Clostridium thermocellum were retrieved, indicating their key role in the cellulose-degradation process and also suggesting their complementary action. Despite the abundance of acetate as a major fermentation product, key methanogenesis enzymes from the acetoclastic pathway were not detected. In contrast, enzymes from the hydrogenotrophic pathway affiliated to Methanothermobacter were almost exclusively identified for methanogenesis, suggesting a syntrophic acetate oxidation process coupled to hydrogenotrophic methanogenesis. Isotopic analyses confirmed the high dominance of the hydrogenotrophic methanogenesis. Very surprising was the identification of an abundant proteolytic activity from Coprothermobacter proteolyticus strains, probably acting as scavenger and/or predator performing proteolysis and fermentation. Metaproteomics thus appeared as an efficient tool to unravel and characterise metabolic networks as well as ecological interactions during methanisation bioprocesses. More generally, metaproteomics provides direct functional insights at a limited cost, and its attractiveness should increase in the future as sequence databases are growing exponentially.     

<Emphasis Type="Italic">In situ</Emphasis> fermentation dynamics during production of <Emphasis Type="Italic">gundruk</Emphasis> and <Emphasis Type="Italic">khalpi</Emphasis>, ethnic fermented vegetable products of the Himalayas

Gundruk is a fermented leafy vegetable and khalpi is a fermented cucumber product, prepared and consumed in the Himalayas. In situ fermentation dynamics during production of gundruk and khalpi was studied. Significant increase in population of lactic acid bacteria (LAB) was found during first few days of gundruk and khlapi fermentation, respectively. Gundruk fermentation was initiated by Lactobacillus brevis, Pediococcus pentosaceus and finally dominated by Lb. plantarum. Similarly in khalpi fermentation, heterofermentative LAB such as Leuconostoc fallax, Lb. brevis and P. pentosaceus initiated the fermentation and finally completed by Lb. plantarum. Attempts were made to produce gundruk and khalpi using mixed starter culture of LAB previously isolated from respective products. Both the products prepared under lab condition had scored higher sensory-rankings comparable to market products.     

Microbial production of a biofuel (acetone–butanol–ethanol) in a continuous bioreactor: impact of bleed and simultaneous product removal

Acetone butanol ethanol (ABE) was produced in an integrated continuous one-stage fermentation and gas stripping product recovery system using Clostridium beijerinckii BA101 and fermentation gases (CO₂ and H₂). In this system, the bioreactor was fed with a concentrated sugar solution (250–500 g L^?1 glucose). The bioreactor was bled semi-continuously to avoid accumulation of inhibitory chemicals and products. The continuous system was operated for 504 h (21 days) after which the fermentation was intentionally terminated. The bioreactor produced 461.3 g ABE from 1,125.0 g total sugar in 1 L culture volume as compared to a control batch process in which 18.4 g ABE was produced from 47.3 g sugar. These results demonstrate that ABE fermentation can be operated in an integrated continuous one-stage fermentation and product recovery system for a long period of time, if butanol and other microbial metabolites in the bioreactor are kept below threshold of toxicity.     

In situ biobutanol recovery from clostridial fermentations: a critical review

Butanol is a precursor of many industrial chemicals, and a fuel that is more energetic, safer and easier to handle than ethanol. Fermentative biobutanol can be produced using renewable carbon sources such as agro-industrial residues and lignocellulosic biomass. Solventogenic clostridia are known as the most preeminent biobutanol producers. However, until now, solvent production through the fermentative routes is still not economically competitive compared to the petrochemical approaches, because the butanol is toxic to their own producer bacteria, and thus, the production capability is limited by the butanol tolerance of producing cells. In order to relieve butanol toxicity to the cells and improve the butanol production, many recovery strategies (either in situ or downstream of the fermentation) have been attempted by many researchers and varied success has been achieved. In this article, we summarize in situ recovery techniques that have been applied to butanol production through Clostridium fermentation, including liquid–liquid extraction, perstraction, reactive extraction, adsorption, pervaporation, vacuum fermentation, flash fermentation and gas stripping. We offer a prospective and an opinion about the past, present and the future of these techniques, such as the application of advanced membrane technology and use of recent extractants, including polymer solutions and ionic liquids, as well as the application of these techniques to assist the in situ synthesis of butanol derivatives.     

Sequential production of two biopolymers-levan and poly-ε-lysine by microbial fermentation

Sequential fermentation for the production of two invaluable biopolymers, levan and poly-ε-lysine (ε-PL), has been successfully developed. It involves fermentation of Bacillus subtilis (natto) Takahashi in sucrose medium to produce levan, separation of levan product from small remaining sugar molecules by ultrafiltration and fermentation of the remnant from levan production by Streptomyces albulus to produce ε-PL. In the process, 50-60 g/L of levan was produced (100% recovery after precipitation by ethanol). The remnant from levan production with glucose adjusted to 30 g/L and with combined use of yeast extract (10 g/L), (NH₄)₂SO₄ (2 g/L) and basal salts was proven to be suitable for ε-PL production. 4.37 g/L of ε-PL accumulation (85% recovery after purification) was reached in 72 h using two-stage fermentation with control of pH. The process of using remnant (waste) from levan fermentation for the second biopolymer (ε-PL) production is unprecedented and the products obtained are environmental-friendly.     

Biotechnological production of enantiomerically pure <Emphasis Type="Italic">d</Emphasis>-lactic acid

The fermentation process of l-lactic acid is well known. Little importance was attached to d-lactic acid, but in the past 10 years, d-lactic acid gained significantly in importance. d-Lactic acid is an interesting precursor for manufacturing heat-resistant polylactic acid (PLA) bioplastics which can be widely used, for example as packaging material, coatings, for textiles or in the automotive industry.This review provides a comprehensive overview of the most recent developments, including a spectrum of studied microorganisms and their capabilities for the production of d-lactic acid. Additionally, the technological achievements in biotechnological d-lactic acid production including fermentation techniques like fed batch, simultaneous saccharification, and fermentation and continuous techniques are presented. Attention is also turned to suitable alternative substrates and their applicability in fermentation processes. Furthermore, advantages and disadvantages of product recovery and purification are discussed. Economic aspects of PLA are pointed out, and the present industrial producers of lactic acid are briefly introduced.     

Routine manufacture of recombinant proteins using expanded bed adsorption chromatography

Two different recombinant human proteins were purified directly from Pichia pastoris whole cell fermentation broth, containing 30–44% biomass (wet weight percent), by strong cation exchange expanded bed adsorption chromatography. Expanded bed adsorption chromatography provided clarification, product purification and product concentration in a single unit operation at large scale (2000-l nominal fermentation volume). The efficiency of expanded bed adsorption chromatography resulted in a short process time, high process yield, and limited proteolytic degradation of the target proteins. The separations were operated using a 60-cm (d) column run at 14 l/min. For one protein, expanded bed adsorption chromatography resulted in an average product recovery of 113% (relative to fermentation supernatant) and a purity of 89% (n=10). For the other protein, the average product recovery was 99% (relative to fermentation supernatant) and the purity was 62.1 (n=10). Laboratory experiments showed that biomass reduced product dynamic binding capacity for protein 2.     

Two-phase systems: potential for in situ extraction of microalgal products

Algae are currently used for production of niche products and are becoming increasingly interesting for the production of bulk commodities, such as biodiesel. For the production of these goods to become economically feasible, production costs will have to be lowered by one order of magnitude. The application of two-phase systems could be used to lower production costs. These systems circumvent the costly step of cell harvesting, whilst the product is extracted and prepared for downstream processing. The mechanism of extraction is a fundamental aspect of the practical question whether two-phase systems can be applied for in situ extraction, viz, simultaneous growth, product formation and extraction, or as a separate downstream processing step. Three possible mechanisms are discussed; 1) product excretion 2) cell permeabilization, and 3) cell death. It was shown that in the case of product excretion, the application of two-phase systems for in situ extraction can be very valuable. With permeabilization and cell death, in situ extraction is not ideal, but the application of two-phase systems as downstream extraction steps can be part of a well-designed biorefinery process. In this way, processing costs can be decreased while the product is mildly and selectively extracted.Thus far none of the algal strains used in two-phase systems have been shown to excrete their product; the output has always been the result of cell death. Two-phase systems can be a good approach as a downstream processing step for these species. For future applications of two-phase in situ extraction in algal production processes, either new species that show product excretion should be discovered, or existing species should be modified to induce product excretion.     

Biobutanol production in a Clostridium acetobutylicum biofilm reactor integrated with simultaneous product recovery by adsorption

Background

Clostridium acetobutylicum can propagate on fibrous matrices and form biofilms that have improved butanol tolerance and a high fermentation rate and can be repeatedly used. Previously, a novel macroporous resin, KA-I, was synthesized in our laboratory and was demonstrated to be a good adsorbent with high selectivity and capacity for butanol recovery from a model solution. Based on these results, we aimed to develop a process integrating a biofilm reactor with simultaneous product recovery using the KA-I resin to maximize the production efficiency of biobutanol.

Results

KA-I showed great affinity for butanol and butyrate and could selectively enhance acetoin production at the expense of acetone during the fermentation. The biofilm reactor exhibited high productivity with considerably low broth turbidity during repeated batch fermentations. By maintaining the butanol level above 6.5 g/L in the biofilm reactor, butyrate adsorption by the KA-I resin was effectively reduced. Co-adsorption of acetone by the resin improved the fermentation performance. By redox modulation with methyl viologen (MV), the butanol-acetone ratio and the total product yield increased. An equivalent solvent titer of 96.5 to 130.7 g/L was achieved with a productivity of 1.0 to 1.5 g?·?L^-1?·?h^-1. The solvent concentration and productivity increased by 4 to 6-fold and 3 to 5-fold, respectively, compared to traditional batch fermentation using planktonic culture.

Conclusions

Compared to the conventional process, the integrated process dramatically improved the productivity and reduced the energy consumption as well as water usage in biobutanol production. While genetic engineering focuses on strain improvement to enhance butanol production, process development can fully exploit the productivity of a strain and maximize the production efficiency.     

Illumina sequencing-based analyses of bacterial communities during short-chain fatty-acid production from food waste and sewage sludge fermentation at different pH values

Short-chain fatty acids (SCFAs) can be produced by primary and waste activated sludge anaerobic fermentation. The yield and product spectrum distribution of SCFAs can be significantly affected by different initial pH values. However, most studies have focused on the physical and chemical aspects of SCFA production by waste activated sludge fermentation at different pH values. Information on the bacterial community structures during acidogenic fermentation is limited. In this study, comparisons of the bacterial communities during the co-substrate fermentation of food wastes and sewage sludge at different pH values were performed using the barcoded Illumina paired-end sequencing method. The results showed that different pH environments harbored a characteristic bacterial community, including sequences related to Lactobacillus, Prevotella, Mitsuokella, Treponema, Clostridium, and Ureibacillus. The most abundant bacterial operational taxonomic units in the different pH environments were those related to carbohydrate-degrading bacteria, which are associated with constituents of co-substrate fermentation. Further analyses showed that during organic matter fermentation, a core microbiota composed of Firmicutes, Proteobacteria, and Bacteroidetes existed. Comparison analyses revealed that the bacterial community during fermentation was significantly affected by the pH, and that the diverse product distribution was related to the shift in bacterial communities.     

Comparison of solid-state and submerged-state fermentation for the bioprocessing of switchgrass to ethanol and acetate by Clostridium phytofermentans

The conversion of sustainable energy crops using microbiological fermentation to biofuels and bioproducts typically uses submerged-state processes. Alternatively, solid-state fermentation processes have several advantages when compared to the typical submerged-state processes. This study compares the use of solid-state versus submerged-state fermentation using the mesophilic anaerobic bacterium Clostridium phytofermentans in the conversion of switchgrass to the end products of ethanol, acetate, and hydrogen. A shift in the ratio of metabolic products towards more acetate and hydrogen production than ethanol production was observed when C. phytofermentans was grown under solid-state conditions as compared to submerged-state conditions. Results indicated that the end product concentrations (in millimolar) obtained using solid-state fermentation were higher than using submerged-state fermentation. In contrast, the total fermentation products (in weight of product per weight of carbohydrates consumed) and switchgrass conversion were higher for submerged-state fermentation. The conversion of xylan was greater than glucan conversion under both fermentation conditions. An initial pH of 7 and moisture content of 80 % resulted in maximum end products formation. Scanning electron microscopy study showed the presence of biofilm formed by C. phytofermentans growing on switchgrass under submerged-state fermentation whereas bacterial cells attached to surface and no apparent biofilm was observed when grown under solid-state fermentation. To our knowledge, this is the first study reporting consolidated bioprocessing of a lignocellulosic substrate by a mesophilic anaerobic bacterium under solid-state fermentation conditions.     

Acetyl-coenzyme a can regulate activity of the mitochondrial pyruvate dehydrogenase complex in situ

In vitro, the pyruvate dehydrogenase complex is sensitive to product inhibition by NADH and acetyl-coenzyme A (CoA). Based upon K_m and K_i relationships, it was suggested that NADH can play a primary role in control of pyruvate dehydrogenase complex activity in vivo (JA Miernyk, DD Randall [1987] Plant Physiol 83:306-310). We have now extended the in vitro studies of product inhibition by assaying pyruvate dehydrogenase complex activity in situ, using purified intact mitochondria from green pea (Pisum sativum) seedlings. In situ activity of the pyruvate dehydrogenase complex is inhibited when mitochondria are incubated with malonate. In some instances, isolated mitochondria show an apparent lack of coupling during pyruvate oxidation. The inhibition by malonate, and the apparent lack of coupling, can both be explained by an accumulation of acetyl-CoA. Inhibition could be alleviated by addition of oxalacetate, high levels of malate, or l-carnitine. The CoA pool in nonrespiring mitochondria was approximately 150 micromolar, but doubled during pyruvate oxidation, when 60 to 95% of the total was in the form of acetyl-CoA. Our results indicate that in situ activity of the mitochondrial pyruvate dehydrogenase complex can be controlled in part by acetyl-CoA product inhibition.     

Fermentation of Insoluble Cellulose by Continuous Cultures of Ruminococcus albus

The hydrolysis and fermentation of insoluble cellulose (Avicel) by continuous cultures of Ruminococcus albus 7 was studied. An anaerobic continuous culture apparatus was designed which permitted gas collection, continuous feeding, and wasting at different retention times. The operation of the apparatus was controlled by a personal computer. Cellulose destruction ranged from ca. 30 to 70% for hydraulic retention times of 0.5 to 2.0 days. Carbon recovery in products was 92 to 97%, and the oxidation-reduction ratios ranged from 0.91 to 1.15. The total product yield (biomass not included) per gram of cellulose (expressed as glucose) was 0.83 g g⁻¹, and the ethanol yield was 0.41 g g⁻¹. The product yield was constant, indicating that product formation was growth linked.     

Enhanced anthraquinones production from adsorbent-treated Morinda elliptica cell suspension cultures in production medium strategy

Continuous removal of anthraquinones (AQ) by Amberlite polymeric adsorbents (XAD-4, XAD-7 and XAD-16) through in situ adsorption in Morinda elliptica cell suspension cultures is studied for product recovery and improvement of the overall titre. Ethanol was the best eluting solvent for effective recovery of AQ from all adsorbents. Pre-treatment of XAD-4 with sodium acetate not only enhanced intracellular AQ, but also AQ release and subsequent recovery from the adsorbent. The addition of sodium acetate pretreated XAD-4 on day 18 for 6-day contact period, achieved comparable cell growth to control (41 g/L), but with 1.3-fold higher intracellular AQ (124 mg/g DW) and two-fold increase in extracellular AQ (14.3 mg/L). High amount of adsorbent and longer contact period for the cultures entering stationary growth phase, stimulated AQ release and recovery but at the expense of cell growth. With 5–8.3 g XAD-4 adsorbent per litre M. elliptica culture in production (P) medium, between 60 and 90% AQ was recovered from extracellular AQ after 24–26 days of culture period.     

Changes in microflora and biochemical composition during the Baceman stage of traditional indonesian Kecap (soy sauce) production

Indonesian soy sauce (kecap) is made from black soybeans in a traditional way which involves two microbiological stages: a solid-state fermentation and a brine fermentation. This study is concerned with the brine fermentation, called baceman. Samples from different kecap producers were analyzed for (bio)chemical content and micro-organisms. It was found that the final composition of the baceman differed from manufacturer to manufacturer, and even within companies large differences were found in microflora and the amounts of fermentation products, formol nitrogen and salt concentration. The main fermentation products were lactate, acetate, glycerol and ethanol. Pediococcus halophilus, staphylococci, a coryneform bacterium and yeasts belonging to Candida, Debaromyces and Sterigmatomyces were isolated from the brines. Compared to Japanese soy sauce production, fermentation by yeasts does not play an important role in Indonesian kecap production. This is due to the fact that kecap is made from whole soybeans only, which are poor in sugars. After fermentation by P. halophilus no substrates are left for growth and ethanol production by yeasts. The presence of film forming yeasts can even lead to spoilage of the product.     

Improved cellobiose utilization in E. coli by including both hydrolysis and phosphorolysis mechanisms

Cellobiose is a major intermediate from cellulase hydrolysis of pretreated plant biomass. Engineering biocatalysts for direct use of cellobiose could eliminate the need for exogenous β-glucosidase. Additionally, rapid removal of cellobiose in a simultaneous saccharification and fermentation facilitates enzymatic hydrolysis as cellobiose is a potent inhibitor for cellulases. We report here improved cellobiose utilization by engineering Escherichia coli to assimilate the disaccharide both hydrolytically and phosphorolytically (shorter fermentation time). Additionally, we demonstrate that engineering intracellular cellobiose utilization circumvents catabolite repression allowing simultaneous fermentation of xylose and cellobiose. Using meso-2,3-butanediol as model product, we further demonstrate that the accelerated carbon metabolism led to improved product formation (higher titers and shorter fermentation times), illustrating the utility of the engineered biocatalysts in biorefinery applications.     

Simultaneous solid–liquid separation and primary purification of clavulanic acid from fermentation broth of Streptomyces clavuligerus using salting‐out extraction system

Clavulanic acid (CA) is usually used together with other β‐lactam antibiotics as combination drugs to inhibit bacterial β‐lactamases, which is mainly produced from the fermentation of microorganism such as Streptomyces clavuligerus. Recently, it is still a challenge for downstream processing of low concentration and unstable CA from fermentation broth with high solid content, high viscosity, and small cell size. In this study, an integrated process was developed for simultaneous solid–liquid separation and primary purification of CA from real fermentation broth of S. clavuligerus using salting‐out extraction system (SOES). First, different SOESs were investigated, and a suitable SOES composed of ethanol/phosphate was chosen and further optimized using the pretreated fermentation broth. Then, the optimal system composed of 20% ethanol/15% K₂HPO₄ and 10% KH₂PO₄ w/w was used to direct separation of CA from untreated fermentation broth. The result showed that the partition coefficient (K) and recovery yield (Y) of CA from untreated fermentation broth were 29.13 and 96.8%, respectively. Simultaneously, the removal rates of the cells and proteins were 99.8% and 63.3%, respectively. Compared with the traditional method of membrane filtration or liquid–liquid extraction system, this developed SOES showed the advantages of simple operation, shorter operation time, lower process cost and higher recovery yield of CA. These results demonstrated that the developed SOES could be used as an attractive alternative for the downstream processing of CA from real fermentation broth.     

Fermentation of soybean oil deodorizer distillate with Candida tropicalis to concentrate phytosterols and to produce sterols-rich yeast cells

Phytosterols have been recovered from the deodorizer distillate produced in the final deodorization step of vegetable oil refining by various processes. The deodorizer distillate contains mainly free fatty acids (FFAs), phytosterols, and tocopherols. The presence of FFAs hinders recovery of phytosterols. In this study, fermentation of soybean oil deodorizer distillate (SODD) with Candida tropicalis 1253 was carried out. FFAs were utilized as carbon source and converted into cellular components as the yeast cells grew. Phytosterols concentration in SODD increased from 15.2 to 28.43 % after fermentation. No significant loss of phytosterols was observed during the process. Microbial fermentation of SODD is a potential approach to concentrate phytosterols before the recovery of phytosterols from SODD. During SODD fermentation, sterols-rich yeast cells were produced and the content of total sterols was as high as 6.96 %, but its major sterol was not ergosterol, which is the major sterol encountered in Saccharomyces cerevisiae. Except ergosterol, other sterols synthesized in the cells need to be identified.