Enhanced isopropanol and <Emphasis Type="Italic">n</Emphasis>-butanol production by supplying exogenous acetic acid via co-culturing two clostridium strains from cassava bagasse hydrolysate

The focus of this study was to produce isopropanol and butanol (IB) from dilute sulfuric acid treated cassava bagasse hydrolysate (SACBH), and improve IB production by co-culturing Clostridium beijerinckii (C. beijerinckii) with Clostridium tyrobutyricum (C. tyrobutyricum) in an immobilized-cell fermentation system. Concentrated SACBH could be converted to solvents efficiently by immobilized pure culture of C. beijerinckii. Considerable solvent concentrations of 6.19 g/L isopropanol and 12.32 g/L butanol were obtained from batch fermentation, and the total solvent yield and volumetric productivity were 0.42 g/g and 0.30 g/L/h, respectively. Furthermore, the concentrations of isopropanol and butanol increased to 7.63 and 13.26 g/L, respectively, under the immobilized co-culture conditions when concentrated SACBH was used as the carbon source. The concentrations of isopropanol and butanol from the immobilized co-culture fermentation were, respectively, 42.62 and 25.45 % higher than the production resulting from pure culture fermentation. The total solvent yield and volumetric productivity increased to 0.51 g/g and 0.44 g/L/h when co-culture conditions were utilized. Our results indicated that SACBH could be used as an economically favorable carbon source or substrate for IB production using immobilized fermentation. Additionally, IB production could be significantly improved by co-culture immobilization, which provides extracellular acetic acid to C. beijerinckii from C. tyrobutyricum. This study provided a technically feasible and cost-efficient way for IB production using cassava bagasse, which may be suitable for industrial solvent production.     

Use of <Emphasis Type="Italic">Cupriavidus basilensis</Emphasis>-aided bioabatement to enhance fermentation of acid-pretreated biomass hydrolysates by <Emphasis Type="Italic">Clostridium beijerinckii</Emphasis>

Lignocellulose-derived microbial inhibitors (LDMICs) prevent efficient fermentation of Miscanthus giganteus (MG) hydrolysates to fuels and chemicals. To address this problem, we explored detoxification of pretreated MG biomass by Cupriavidus basilensis ATCC^®BAA-699 prior to enzymatic saccharification. We document three key findings from our test of this strategy to alleviate LDMIC-mediated toxicity on Clostridium beijerinckii NCIMB 8052 during fermentation of MG hydrolysates. First, we demonstrate that growth of C. basilensis is possible on furfural, 5-hydroxymethyfurfural, cinnamaldehyde, 4-hydroxybenzaldehyde, syringaldehyde, vanillin, and ferulic, p-coumaric, syringic and vanillic acid, as sole carbon sources. Second, we report that C. basilensis detoxified and metabolized ~98 % LDMICs present in dilute acid-pretreated MG hydrolysates. Last, this bioabatement resulted in significant payoffs during acetone-butanol-ethanol (ABE) fermentation by C. beijerinckii: 70, 50 and 73 % improvement in ABE concentration, yield and productivity, respectively. Together, our results show that biological detoxification of acid-pretreated MG hydrolysates prior to fermentation is feasible and beneficial.     

Enhanced butanol production in a microbial electrolysis cell by <Emphasis Type="Italic">Clostridium beijerinckii</Emphasis> IB4

Reducing power such as NADH is an essential factor for acetone/butanol/ethanol (ABE) fermentation using Clostridium spp. The objective of this study was to increase available NADH in Clostridium beijerinckii IB4 by a microbial electrolysis cell (MEC) with an electron carrier to enhance butanol production. First of all, a MEC was performed without electron carrier to study the function of cathodic potential applying. Then, various electron carriers were tested, and neutral red (NR)-amended cultures showed an increase of butanol concentration. Optimal NR concentration (0.1 mM) was used to add in a MEC. Electricity stimulated the cell growth obviously and dramatically diminished the fermentation time from 40 to 28 h. NR and electrically reduced NR improved the final butanol concentration and inhibited the acetone generation. In the MEC with NR, the butanol concentration, yield, proportion and productivity were increased by 12.2, 17.4, 7.2 and 60.3 %, respectively. To further understand the mechanisms of NR, cathodic potential applying and electrically reduced NR, NADH and NAD⁺ levels, ATP levels and hydrogen production were determined. NR and electrically reduced NR also improved ATP levels and the ratio of NADH/NAD⁺, whereas they decreased hydrogen production. Thus, the MEC is an efficient method for enhancing the butanol production.     

Improvements of metabolites tolerance in <Emphasis Type="Italic">Clostridium acetobutylicum</Emphasis> by micronutrient zinc supplementation

Micronutrient zinc is of great importance for acetone-butanol-ethanol (ABE) fermentation by Clostridium acetobutylicum. The effect of zinc supplementation on toxic metabolites (formic, acetic, butyric acid and butanol) tolerance during ABE fermentation was investigated under various stress-shock conditions without pH control. Great improvements on cell growth, glucose utilization and butanol production were achieved. In the presence of 0.45 g/L formic acid, zinc contributed to 11.28 g/L butanol produced from 55.24 g/L glucose compared to only 5.27 g/L butanol from 29.49 g/L glucose in the control without zinc supplementation. More importantly, relatively higher levels of 7.5 g/L acetic acid, 5.5 g/L butyric acid and 18 g/L butanol could be tolerated by C. acetobutylicum with zinc supplementation while no fermentation was observed under the same stress-shock condition respectively, suggesting that the acids and butanol tolerance in C. acetobutylicum could be significantly facilitated by pleiotropic regulation of micronutrient zinc. Thus, this paper provides an efficient bioprocess engineering strategy for improving stress tolerance in Clostridium species.     

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.     

Bioproduction of butanol in bioreactors: new insights from simultaneous in situ butanol recovery to eliminate product toxicity

Simultaneous acetone butanol ethanol (ABE) fermentation by Clostridium beijerinckii P260 and in situ product recovery was investigated using a vacuum process operated in two modes: continuous and intermittent. Integrated batch fermentations and ABE recovery were conducted at 37 °C using a 14-L bioreactor (7.0 L fermentation volume) containing initial substrate (glucose) concentration of 60 g/L. The bioreactor was connected in series with a condensation system and vacuum pump. Vacuum was applied continuously or intermittently with 1.5 h vacuum sessions separated by 4, 6, and 8 h intervals. A control ABE fermentation experiment was characterized by incomplete glucose utilization due to butanol toxicity to C. beijerinckii P260, while fermentation coupled with in situ recovery by both continuous and intermittent vacuum modes resulted in complete utilization of glucose, greater productivity, improved cell growth, and concentrated recovered ABE stream. These results demonstrate that vacuum technology can be applied to integrated ABE fermentation and recovery even though the boiling point of butanol is greater than that of water.     

Production of butanol and isopropanol with an immobilized <Emphasis Type="Italic">Clostridium</Emphasis>

Clostridium beijerinckii optinoii is a Clostridium species that produces butanol, isopropanol and small amounts of ethanol. This study compared the performances of batch and continuous immobilized cell fermentations, investigating how media flow rates and nutritional modification affected solvent yields and productivity. In 96-h batch cultures, with 80 % of the 30 g L^?1 glucose consumed in synthetic media, solvent concentration was 9.45 g L^?1 with 66.0 % as butanol. In a continuous fermentation using immobilized C. beijerinckii optinoii cells, also with 80 % of 30 g L^?1 glucose utilization, solvent productivity increased to 1.03 g L^?1 h^?1. Solvent concentration reached 12.14 g L^?1 with 63.0 % as butanol. Adjusting the dilution rate from 0.085 to 0.050 h^?1 to allow extended residence time in column was required when glucose concentration in fresh media was increased from 30 to 50 g L^?1. When acetate was used to improve the buffer capacity in media, the solvent concentration reached 12.70 on 50 g L^?1 glucose. This continuous fermentation using immobilized cells showed technical feasibility for solvent production.     

Enhancement of ABE fermentation through regulation of ammonium acetate and D–xylose uptake from acid-pretreated corncobs

Clostridium acetobutylicum TISTR 1462 and Clostridium beijerinckii TISTR 1461 were chosen to optimize acetone–butanol–ethanol (ABE) fermentation by using glucose as a carbon source. The enhancement in its productivity by adding various concentrations of ammonium acetate was studied. Then, the variation of glucose/xylose ratios in the pre-grown medium was investigated. The results showed that both increased ammonium acetate in the production medium and D–xylose in the pre-grown medium could produce more ABE. With these conditions, using corncob hydrolysate as a substrate, 20.58 g/L ABE was produced from C. beijerinckii TISTR 1461 with 0.44 g/L/h and 0.45 of ABE productivity and yield, respectively.     

Precultivation of <Emphasis Type="Italic">Bacillus coagulans</Emphasis> DSM2314 in the presence of furfural decreases inhibitory effects of lignocellulosic by-products during <Emphasis Type="SmallCaps">l</Emphasis>(+)-lactic acid fermentation

By-products resulting from thermo-chemical pretreatment of lignocellulose can inhibit fermentation of lignocellulosic sugars to lactic acid. Furfural is such a by-product, which is formed during acid pretreatment of lignocellulose. pH-controlled fermentations with 1 L starting volume, containing YP medium and a mixture of lignocellulosic by-products, were inoculated with precultures of Bacillus coagulans DSM2314 to which 1 g/L furfural was added. The addition of furfural to precultures resulted in an increase in l(+)-lactic acid productivity by a factor 2 to 1.39 g/L/h, an increase in lactic acid production from 54 to 71 g and an increase in conversion yields of sugar to lactic acid from 68 to 88 % W/W in subsequent fermentations. The improved performance was not caused by furfural consumption or conversion, indicating that the cells acquired a higher tolerance towards this by-product. The improvement coincided with a significant elongation of B. coagulans cells. Via RNA-Seq analysis, an upregulation of pathways involved in the synthesis of cell wall components such as bacillosamine, peptidoglycan and spermidine was observed in elongated cells. Furthermore, the gene SigB and genes promoted by SigB, such as NhaX and YsnF, were upregulated in the presence of furfural. These genes are involved in stress responses in bacilli.     

Isolation and characterisation of non-anaerobic butanol-producing symbiotic system TSH06

Butanol-producing microorganisms are all obligate anaerobes. In this study, a unique symbiotic system TSH06 was isolated to be capable of producing butanol under non-anaerobic condition. Denaturing gradient gel electrophoresis (DGGE) analysis of 16S ribosomal RNA (rRNA) revealed that two strains coexist in TSH06. The two strains were identical to Clostridium acetobutylicum and Bacillus cereus, respectively. They were isolated individually and named as C. acetobutylicum TSH1 and B. cereus TSH2. C. acetobutylicum TSH1 is a butanol-producing, obligate anaerobic strain. Facultative anaerobic B. cereus TSH2 did not possess the ability of butanol production; however, it offered C. acetobutylicum TSH1 the viability under non-anaerobic condition. Moreover, B. cereus TSH2 enhanced butanol yield and speed of fermentation. TSH06 produced 12.97 g/L butanol and 15.39 g/L total solvent under non-anaerobic condition, which is 25 and 24 %, respectively, higher than those of C. acetobutylicum TSH1. In addition, TSH06 produced butanol faster under non-anaerobic condition than under anaerobic condition. Butanol accounted for more than 80 % of total solvent, which is higher than the known report. TSH06 was stable during passage. In all, TSH06 is a promising candidate for industrialisation of biobutanol with high yield, high butanol proportion, easy-handling and time-saving system. These results demonstrated the potential advantage of symbiosis. This study also provides a promising strategy for butanol fermentation.     

Enhanced fed-batch production of pyrroloquinoline quinine in <Emphasis Type="Italic">Methylobacillus</Emphasis> sp. CCTCC M2016079 with a two-stage pH control strategy

The effects of pH control strategy and fermentative operation modes on the biosynthesis of pyrroloquinoline quinine (PQQ) were investigated systematically with Methylobacillus sp. CCTCC M2016079 in the present work. Firstly, the shake-flask cultivations and benchtop fermentations at various pH values ranging from 5.3 to 7.8 were studied. Following a kinetic analysis of specific cell growth rate (μ _x ) and specific PQQ formation rate (μ _p ), the discrepancy in optimal pH values between cell growth and PQQ biosynthesis was observed, which stimulated us to develop a novel two-stage pH control strategy. During this pH-shifted process, the pH in the broth was controlled at 6.8 to promote the cell growth for the first 48 h and then shifted to 5.8 to enhance the PQQ synthesis until the end of fermentation. By applying this pH-shifted control strategy, the maximum PQQ production was improved to 158.61 mg/L in the benchtop fermenter, about 44.9% higher than that under the most suitable constant pH fermentation. Further fed-batch study showed that PQQ production could be improved from 183.38 to 272.21 mg/L by feeding of methanol at the rate of 11.5 mL/h in this two-stage pH process. Meanwhile, the productivity was also increased from 2.02 to 2.84 mg/L/h. In order to support cell growth during the shifted pH stage, the combined feeding of methanol and yeast extract was carried out, which brought about the highest concentration (353.28 mg/L) and productivity (3.27 mg/L/h) of PQQ. This work has revealed the potential of our developed simple and economical strategy for the large-scale production of PQQ.     

Very high gravity ethanol and fatty acid production of <Emphasis Type="Italic">Zymomonas mobilis</Emphasis> without amino acid and vitamin

Very high gravity (VHG) fermentation is the mainstream technology in ethanol industry, which requires the strains be resistant to multiple stresses such as high glucose concentration, high ethanol concentration, high temperature and harsh acidic conditions. To our knowledge, it was not reported previously that any ethanol-producing microbe showed a high performance in VHG fermentations without amino acid and vitamin. Here we demonstrate the engineering of a xylose utilizing recombinant Zymomonas mobilis for VHG ethanol fermentations. The recombinant strain can produce ethanol up to 136 g/L without amino acid and vitamin with a theoretical yield of 90 %, which is significantly superior to that produced by all the reported ethanol-producing strains. The intracellular fatty acids of the bacterial were about 16 % of the bacterial dry biomass, with the ratio of ethanol:fatty acids was about 273:1 (g/g). The recombinant strain was achieved by a multivariate-modular strategy tackles with the multiple stresses which are closely linked to the ethanol productivity of Z. mobilis. The over-expression of metB/yfdZ operon enabled the growth of the recombinant Z. mobilis in a chemically defined medium without amino acid and vitamin; and the fatty acids overproduction significantly increased ethanol tolerance and ethanol production. The coupled production of ethanol with fatty acids of the Z. mobilis without amino acid and vitamin under VHG fermentation conditions may permit a significant reduction of the production cost of ethanol and microbial fatty acids.     

Low-Input Fermentations of <Emphasis Type="Italic">Agave tequilana</Emphasis> Leaf Juice Generate High Returns on Ethanol Yields

During tequila production, up to 75 % w/w of the Agave plant is discarded when leaves are removed from the stem. The discarded leaves represent an extensive amount of unexploited biomass that was used here for bioethanol production in no-input fermentations, where no acid or enzymatic hydrolysis, supplementation of nutrients or standardization of carbohydrate content occur. Ethanol yield from Agave leaf juice is unaffected by sterilization but reduced if fermentation is reliant solely on endogenous microorganisms. Non-Saccharomyces yeasts, including Kluyveromyces marxianus and Candida akabanensis, proved to be more robust than standard Saccharomyces spp. and yielded up to 88 % of the theoretical maximum ethanol from leaf juice. Combining leaf and stem juice, as from a whole plant, was predicted to maximize yield at up to 19,439 L/ha of ethanol from mature plants.     

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.     

In Situ Enzymatic Conversion of <Emphasis Type="Italic">Nannochloropsis oceanica</Emphasis> IMET1 Biomass into Fatty Acid Methyl Esters

Conventionally, production of methyl ester fuels from microalgae occurs through an energy-intensive two-step chemical extraction and transesterification process. To improve the energy efficiency, we performed in situ enzymatic conversion of whole algae biomass from an oleaginous heterokont microalga Nannochloropsis oceanica IMET1 with the immobilized lipase from Candida antarctica. The fatty acid methyl ester yield reached 107.7% for dry Nannochloropsis biomass at biomass to t-butanol to methanol weight ratio of 1:2:0.5 and a reaction time of 12 h at 25 °C, representing the first report of efficient whole algae biomass conversion into fatty acid methyl esters at room temperature. Different forms of algal biomass including wet Nannochloropsis biomass were tested. The maximum yield of wet biomass was 81.5%. Enzyme activity remained higher than 95% after 55 days of treatment (equal to 110 cycles of reaction) under the conditions optimized for dry algae biomass conversion. The low reaction temperature, high enzyme stability, and high yield from this study indicate in situ enzymatic conversion of dry algae biomass may potentially be used as an energy-efficient method for algal methyl ester fuel production while allowing co-product recovery.     

Construction of heterologous gene expression cassettes for the development of recombinant <Emphasis Type="Italic">Clostridium beijerinckii</Emphasis>

Gene-expression cassettes for the construction of recombinant Clostridium beijerinckii were developed as potential tools for metabolic engineering of C. beijerinckii. Gene expression cassettes containing ColE1 origin and pAMB origin along with the erythromycin resistance gene were constructed, in which promoters from Escherichia coli, Lactococcus lactis, Ralstonia eutropha, C. acetobutylicum, and C. beijerinckii are examined as potential promoters in C. beijerinckii. Zymogram analysis of the cell extracts and comparison of lipase activities of the recombinant C. beijerinckii strains expressing Pseudomonas fluorescens tliA gene suggested that the tliA gene was functionally expressed by all the examined promoters with different expression level. Also, recombinant C. beijerinckii expressing C. beijerinckii secondary alcohol dehydrogenase by the constructed expression cassettes successfully produced 2-propanol from glucose. The best promoter for TliA expression was the R. eutropha phaP promoter while that for 2-propanol production was the putative C. beijerinckii pta promoter. Gene expression cassettes developed in this study may be useful tools for the construction of recombinant C. beijerinckii strains as host strains for the valuable chemicals and fuels from renewable resources.     

Engineering cofactor flexibility enhanced 2,3-butanediol production in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Enzymatic reduction of acetoin into 2,3-butanediol (2,3-BD) typically requires the reduced nicotinamide adenine dinucleotide (NADH) or its phosphate form (NADPH) as electron donor. Efficiency of 2,3-BD biosynthesis, therefore, is heavily influenced by the enzyme specificity and the cofactor availability which varies dynamically. This work describes the engineering of cofactor flexibility for 2,3-BD production by simultaneous overexpression of an NADH-dependent 2,3-BD dehydrogenase from Klebsiella pneumoniae (KpBudC) and an NADPH-specific 2,3-BD dehydrogenase from Clostridium beijerinckii (CbAdh). Co-expression of KpBudC and CbAdh not only enabled condition versatility for 2,3-BD synthesis via flexible utilization of cofactors, but also improved production stereo-specificity of 2,3-BD without accumulation of acetoin. With optimization of medium and fermentation condition, the co-expression strain produced 92 g/L of 2,3-BD in 56 h with 90% stereo-purity for (R,R)-isoform and 85% of maximum theoretical yield. Incorporating cofactor flexibility into the design principle should benefit production of bio-based chemical involving redox reactions.     

Increasing butanol/acetone ratio and solvent productivity in ABE fermentation by consecutively feeding butyrate to weaken metabolic strength of butyrate loop

In this study, we attempted to increase butanol/acetone ratio and total solvent productivity in ABE fermentations with corn- and cassava-based media, by consecutively feeding a small amount of butyrate/acetate during solventogenic phase to weaken the metabolic strengths in butyrate/acetate closed-loops. Consecutively feeding a small amount of butyrate (a total of 3.0 g/L-broth) is most effective in improving performance of corn-based ABE fermentations, as it simultaneously increased average butanol/acetone ratio by 23 % (1.92–2.36) and total solvent productivity by 16 % (0.355–0.410 g/L/h) as compared with those of control. However, the butyrate feeding strategy could not improve butanol/acetone ratio and total solvent productivity in cassava-based ABE fermentations, where the metabolic strength of butyrate closed-loop had already been very low.