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Abstract Background Butyl acetate has shown wide applications in food, cosmetics, medicine, and biofuel sectors. These short-chain fatty acid esters can be produced by either chemical or biological synthetic process with corresponding alcohols and acids. Currently, biosynthesis of short chain fatty acid esters, such as butyl butyrate, through microbial fermentation systems has been achieved; however, few studies regarding biosynthesis of butyl acetate were reported. Results In this study, three proof-of-principle strategies for the one-pot butyl acetate production from glucose through microbial fermentation were designed and evaluated. (1) 7.3 g/L of butyl acetate was synthesized by butanol-producing Clostridium acetobutylicum NJ4 with the supplementation of exogenous acetic acid; (2) With the addition of butanol, 5.76 g/L of butyl acetate can be synthesized by acetate-producing Actinobacillus succinogenes130z (ΔpflA); (3) Microbial co-culture of C. acetobutylicum NJ4 and A. succinogenes130z (ΔpflA) can directly produce 2.2 g/L of butyl acetate from glucose by using microbial co-culture system with the elimination of precursors. Through the further immobilization of A. succinogenes130z (ΔpflA), butyl acetate production was improved to 2.86 g/L. Conclusion Different microbial mono- and co-culture systems for butyl acetate biosynthesis were successfully constructed. These strategies may be extended to the biosynthesis of a wide range of esters, especially to some longer chain ones.

Abstract Background The global production of glycerol is increasing year by year since the demands of biodiesel is rising. It is benefit for high-yield succinate synthesis due to its high reducing property. A. succinogenes, a succinate-producing candidate, cannot grow on glycerol anaerobically, as it needs a terminal electron acceptor to maintain the balance of intracellular NADH and NAD+. Microbial fuel cell (MFC) has been widely used to release extra intracellular electrons. However, A. succinogenes is a non-electroactive strain which need the support of electron shuttle in MFC, and pervious research showed that acid-tolerant A. succinogenes has higher content of unsaturated fatty acids, which may be beneficial for the transmembrane transport of lipophilic electron shuttle. Results MFC-assisted succinate production was evaluated using neutral red as an electron shuttle to recover the glycerol utilization. First, an acid-tolerant mutant JF1315 was selected by atmospheric and room temperature plasma (ARTP) mutagenesis aiming to improve transmembrane transport of neutral red (NR). Additionally, MFC was established to increase the ratio of oxidized NR to reduced NR. By combining these two strategies, ability of JF1315 for glycerol utilization was significantly enhanced, and 23.92 g/L succinate was accumulated with a yield of 0.88 g/g from around 30 g/L initial glycerol, along with an output voltage above 300 mV. Conclusions A novel MFC-assisted system was established to improve glycerol utilization by A. succinogenes for succinate and electricity production, making this system as a platform for chemicals production and electrical supply simultaneously.

Abstract Background Rhamnolipids are the best known microbial-derived biosurfactants, which has attracted great interest as potential ‘‘green” alternative for synthetic surfactants. However, rhamnolipids are the major contributors to severe foam problems, which greatly inhibit the economics of industrial-scale production. In this study, a novel foam-control system was established for ex situ dealing with the massive overflowing foam. Based on the designed facility, foam reduction efficiency, rhamnolipids production by batch and repeated fed-batch fermentation were comprehensively investigated. Results An ex situ foam-control system was developed to control the massive overflowing foam and improve rhamnolipids production. It was found that the size of individual bubble in the early stage was much larger than that of late fermentation stage. The foam liquefaction efficiency decreased from 54.37% at the beginning to only 9.23% at the end of the fermentation. This difference of bubble stability directly resulted in higher foam reduction efficiency of 67.46% in the early stage, whereas the small uniform bubbles can only be reduced by 57.53% at the later fermentation stage. Moreover, reduction of secondary foam is very important for foam controlling. Two improved designs of the device in this study obtained about 20% improvement of foam reduction efficiency, respectively. The batch fermentation result showed that the average volume of the overflowing foam was reduced from 58–640 to 19–216 mL/min during the fermentation process, presenting a notable reduction efficiency ranging from 51.92 to 73.47%. Meanwhile, rhamnolipids production of batch fermentation reached 45.63 g/L, and the yield 0.76 g/g was significantly better than ever reported. Further, a repeated fed-batch fermentation based on the overall optimization was carried out. Total rhamnolipids concentration reached 48.67 g/L with the yield around of 0.67–0.83 g/g, which presented an improvement of 62% and 49% compared with conventional batch fermentation by using various kinds of defoamers, respectively. Conclusions The ex situ foam-control system presented a notable reduction efficiency, which helped greatly to easily solve the severe foaming problem without any defoamer addition. Moreover, rhamnolipids production and yield by repeated fed-batch fermentation obtained prominent improvement compared to conventional batch cultivation, which can further facilitate economical rhamnolipids production at large scales.

Abstract Microbial communities are ubiquitous in nature and exhibit several attractive features, such as sophisticated metabolic capabilities and strong environment robustness. Inspired by the advantages of natural microbial consortia, diverse artificial co-cultivation systems have been metabolically constructed for biofuels, chemicals and natural products production. In these co-cultivation systems, especially genetic engineering ones can reduce the metabolic burden caused by the complex of metabolic pathway through labor division, and improve the target product production significantly. This review summarized the most up-to-dated co-cultivation systems used for biofuels, chemicals and nature products production. In addition, major challenges associated with co-cultivation systems are also presented and discussed for meeting further industrial demands.

Abstract Background The co-production of single cell oil (SCO) with value-added products could improve the economic viability of industrial SCO production. The newly isolated oleaginous yeast Cryptococcus podzolicus DSM 27192 was able to co-produce SCO intracellularly and gluconic acid (GA) extracellularly. In this study, the metabolic regulation of carbon distribution between SCO and GA through process optimization was comprehensively investigated. Results The carbon flow distribution between SCO and GA was significantly influenced by the cultivation conditions, such as nitrogen sources, glucose concentration and dissolved oxygen concentration. It was found that organic nitrogen sources were beneficial for SCO accumulation, while GA production was decreased. Dissolved oxygen concentration (DOC) was found to enhance SCO accumulation, while high glucose concentration was more favorable for GA accumulation. Hence, a two-stage DOC or glucose concentration-controlled strategy was designed to improve cell growth and direct carbon distribution between SCO and GA. Moreover, C. podzolicus DSM 27192 could degrade its stored lipids to synthesize GA in the late stationary phase, although considerable amounts of glucose remained unconsumed in the culture medium, indicating the importance of fermentation time control in co-production systems. All these observations provide opportunity to favor either the production of SCO or GA or rather their simultaneous production. Conclusions Co-production of SCO and GA by C. podzolicus DSM 27192 can improve the economical value for microbial lipid-derived biodiesel production. Moreover, the results of the proposed co-production strategy might give guidance for other co-production systems.

Abstract Butanol has been regarded as an important bulk chemical and advanced biofuel; however, large scaling butanol production by solventogenic Clostridium sp. is still not economically feasible due to the high cost of substrates, low butanol titer and yield caused by the toxicity of butanol and formation of by-products. Renewed interests in biobutanol as biofuel and rapid development in genetic tools have spurred technological advances to strain modifications. Comprehensive reviews regarding these aspects have been reported elsewhere in detail. Meanwhile, more wild type butanol producers with unique properties were also isolated and characterized. However, few reviews addressed these discoveries of novel wild type solventogenic Clostridium sp. strains. Accordingly, this review aims to comprehensively summarize the most recent advances on wild type butanol producers in terms of fermentation patterns, substrate utilization et al. Future perspectives using these native ones as chassis for genetic modification were also discussed.

Abstract Methanol has become an attractive substrate for biotechnological applications due to its abundance and low-price. Chemicals production from methanol could alleviate the environmental concerns, costs, and foreign dependency associated with the use of petroleum feedstock. Recently, a growing fraction of research has focused on metabolites production using methanol as sole carbon and energy source or as co-substrate with carbohydrates by native or synthetic methylotrophs. In this review, we summarized the recent significant progress in native and synthetic methylotrophs and their application for methanol bioconversion into various products. Moreover, strategies for improvement of methanol metabolism and new perspectives on the generation of desired products from methanol were also discussed, which will benefit for the development of a methanol-based economy.

Abstract With the increased development in research, innovation, and policy interest in recent years, biosynthetic technology has developed rapidly, which combines engineering, electronics, computer science, mathematics, and other disciplines based on classical genetic engineering and metabolic engineering. It gives a wider perspective and a deeper level to perceive the nature of life via cell mechanism, regulatory networks, or biological evolution. Currently, synthetic biology has made great breakthrough in energy, chemical industry, and medicine industries, particularly in the programmable genetic control at multiple levels of regulation to perform designed goals. In this review, the most advanced and comprehensive developments achieved in biosynthetic technology were represented, including genetic engineering as well as synthetic genomics. In addition, the superiority together with the limitations of the current genome-editing tools were summarized.

Abstract Background Consolidated bioprocessing (CBP) has attracted increasing attention since it can accomplish hydrolytic enzymes production, lignocellulose degradation and microbial fermentation in one single step. Currently, biobutanol is mainly produced by mesophilic and solventogenic clostridia, such as Clostridium beijerinckii and C. acetobutylicum, which cannot directly utilize lignocellulose, an abundant, renewable and economic feedstock. Hence, metabolic construction or isolation of novel cellulolytic/hemicellulolytic and solventogenic bacteria to achieve direct butanol production from lignocellulose offers a promising alternative. Results In this study, a newly isolated Thermoanaerobacterium sp. M5 could directly produce butanol from xylan through CBP at 55 °C via the butanol–ethanol pathway. Further genomic and proteomic analysis showed that the capabilities of efficient xylan degradation and butanol synthesis were attributed to the efficient expression of xylanase, β-xylosidase and the bifunctional alcohol/aldehyde dehydrogenase (AdhE). Process optimization based on the characteristic of AdhE could further improve the final butanol titer to 1.17 g/L from xylan through CBP. Furthermore, a new co-cultivation system consisting of Thermoanaerobacterium sp. M5 which could release xylose from xylan efficiently and C. acetobutylicum NJ4 which possesses the capacity of high butanol production was established. This microbial co-cultivation system could improve the butanol titer to 8.34 g/L, representing the highest butanol titer from xylan through CBP. Conclusions A newly thermophilic and butanogenic bacterium Thermoanaerobacterium sp. M5 was isolated and key enzymes responsible for butanol production were characterized in this study. High butanol titer was obtained from xylan through process optimization. In addition, the newly set up microbial co-cultivation system, consisting of Thermoanaerobacterium sp. M5 and C. acetobutylicum NJ4, achieved the highest butanol production from xylan compared with the reported co-cultivation systems.

Abstract Background The formation of by-products, mainly acetone in acetone–butanol–ethanol (ABE) fermentation, significantly affects the solvent yield and downstream separation process. In this study, we genetically engineered Clostridium acetobutylicum XY16 isolated by our lab to eliminate acetone production and altered ABE to isopropanol–butanol–ethanol (IBE). Meanwhile, process optimization under pH control strategies and supplementation of calcium carbonate were adopted to investigate the interaction between the reducing force of the metabolic networks and IBE production. Results After successful introduction of secondary alcohol dehydrogenase into C. acetobutylicum XY16, the recombinant XY16 harboring pSADH could completely eliminate acetone production and convert it into isopropanol, indicating great potential for large-scale production of IBE mixtures. Especially, pH could significantly improve final solvent titer through regulation of NADH and NADPH levels in vivo. Under the optimal pH level of 4.8, the total IBE production was significantly increased from 3.88 to 16.09 g/L with final 9.97, 4.98 and 1.14 g/L of butanol, isopropanol, and ethanol. Meanwhile, NADH and NADPH levels were maintained at optimal levels for IBE formation compared to the control one without pH adjustment. Furthermore, calcium carbonate could play dual roles as both buffering agency and activator for NAD kinase (NADK), and supplementation of 10 g/L calcium carbonate could finally improve the IBE production to 17.77 g/L with 10.51, 6.02, and 1.24 g/L of butanol, isopropanol, and ethanol. Conclusion The complete conversion of acetone into isopropanol in the recombinant C. acetobutylicum XY16 harboring pSADH could alter ABE to IBE. pH control strategies and supplementation of calcium carbonate were effective in obtaining high IBE titer with high isopropanol production. The analysis of redox cofactor perturbation indicates that the availability of NAD(P)H is the main driving force for the improvement of IBE production.

Abstract Background High cost of traditional substrates and formation of by-products (such as acetone and ethanol) in acetone–butanol–ethanol (ABE) fermentation hindered the large-scale production of biobutanol. Here, we comprehensively characterized a newly isolated solventogenic and xylanolytic Clostridium species, which could produce butanol at a high ratio with elimination of ethanol and conversion of acetone to more value-added product, isopropanol. Ultimately, direct butanol production from hemicellulose was achieved with efficient expression of indigenous xylanase by the novel strain via consolidated bioprocessing. Results A novel wild-type Clostridium sp. strain NJP7 was isolated and characterized in this study, which was capable of fermenting monosaccharides, e.g., glucose into butanol via a fermentative acetone–isopropanol–butanol pathway. With enhancement of buffering capacity and alcohol dehydrogenase activities, butanol and isopropanol titer by Clostridium sp. strain NJP7 was improved to 12.21 and 1.92 g/L, respectively, and solvent productivity could be enhanced to 0.44 g/L/h. Furthermore, with in situ extraction with biodiesel, the amount of butanol and isopropanol was finally improved to 25.58 and 5.25 g/L in the fed-batch mode. Meanwhile, Clostridium sp. strain NJP7 shows capability of direct isopropanol–butanol production from hemicelluloses with expression of indigenous xylanase. 2.06 g/L of butanol and 0.54 g/L of isopropanol were finally achieved through the temperature-shift simultaneous saccharification and fermentation, representing the highest butanol production directly from hemicellulose. Conclusion The co-production of isopropanol with butanol by the newly isolated Clostridium sp. strain NJP7 would add on the economical values for butanol fermentation. Furthermore, the high isopropanol-butanol production with in situ extraction would also greatly enhance the economic feasibility for fermentative production of butanol–isopropanol in large scale. Meanwhile, its direct production of butanol–isopropanol from polysaccharides, hemicellulose through secretion of indigenous thermostable xylanase, shows great potential using lignocellulosic wastes for biofuel production.

Background: Low-cost feedstocks, a single product (butanol), and a high butanol titer are three key points for establishing a sustainable and economically viable process for biological butanol production. Here, we comprehensively investigated the butanol production from mono-substrates, mainly glycerol and polysaccharides, mainly starch and xylan by a newly identified wild-type Clostridium pasteurianum GL11. Results: Strain GL11 produced 14.7 g/L of butanol with a yield of 0.41 g/g from glycerol in the batch mode without formation of by-products of acetone and 1,3-propanediol (1,3-PDO). With in situ extraction with biodiesel, the amount of butanol was finally improved to 28.8 g/L in the fed-batch mode. Genomic and enzymatic analysis showed that the deficiency of key enzymes involved in acetone and 1,3-PDO pathway within strain GL11 led to the elimination of these by-products, which may also greatly simplify downstream separation. The elimination of acetone and 1,3-PDO and high butanol tolerance contributed to its high butanol production yield from glycerol. More importantly, strain GL11 could directly convert polysaccharides, such as xylan and starch to butanol with secretion of xylanase and amylase via consolidated bioprocessing. Conclusions: The wild-type strain GL11 was found to be particularly advantageous due to its capability of efficient butanol production from glycerol and polysaccharides with elimination of acetone and 1,3-PDO formation. And the high butanol production with in situ extraction by using biodiesel would significantly enhance the economic feasibility of fermentative production of butanol from glycerol. These unique features of C. pasteurianum GL11 open the door to the possibility of cost-effective biofuels production in large scale. [ABSTRACT FROM AUTHOR]