Physiological characteristics of Corynebacterium glutamicum as a cell factory under anaerobic conditions.

Corynebacterium glutamicum, a gram-positive and facultative anaerobic bacterium, is widely used for the industrial production of amino acids, such as L-glutamate and L-lysine. C. glutamicum grows and produces amino acids under aerobic conditions. When restricted under anaerobic conditions, it produces organic acids, such as L-lactate and succinate, through metabolic shift. With the increasing threat of global warming, these organic acids have drawn considerable attention as bio-based plastic monomers. In addition to the organic acids, the anaerobic bioprocess is also used to produce other value-added compounds, including isobutanol, ethanol, 3-methyl-1-butanol, 2,3-butanediol, L-alanine, and L-valine. Therefore, C. glutamicum is now a versatile cell factory for producing a wide variety of useful chemicals under both aerobic and anaerobic conditions. The growth and metabolism of the bacterium depend on the oxygen levels, which modulate the rearrangement of the carbon flux by reprogramming gene expression patterns and intracellular redox states. Anaerobic cell growth and L-lysine production as well as aerobic succinate production have been demonstrated by engineering the metabolic pathways or supplying a terminal electron acceptor instead of oxygen. In this review, we discuss the physiological and metabolic changes in C. glutamicum associated with its application as a cell factory under different oxygen states. Physiological switching in bacteria is initiated with the sensing of oxygen availability. While such a sensor has not been identified in C. glutamicum yet, the molecular mechanism for oxygen sensing in related bacteria is also discussed. KEY POINTS: • C. glutamicum produces a wide variety of useful compounds under anaerobic conditions. • C. glutamicum is a versatile cell factory under both aerobic and anaerobic conditions. • Metabolic fate can be overcome by engineering metabolic pathways.

Elevated, non-proliferative temperatures change the profile of fermentation products in Corynebacterium glutamicum.

Although temperature is a crucial factor affecting enzymatic activity on biochemical and biofuel production, the reaction temperature for the generation of these products is usually set at the optimal growth temperature of the host strain, even under non-proliferative conditions. Given that the production of fermentation products only requires a fraction of the cell's metabolic pathways, the optimal temperatures for microbial growth and the fermentative production of a target compound may be different. Here, we investigated the effect of temperature on lactic and succinic acids production, and related enzymatic activities, in wild-type and succinic acid-overproducing strains of Corynebacterium glutamicum. Interestingly, fermentative production of lactic acid increased with the temperature in wild-type: production was 69% higher at 42.5 °C, a temperature that exceeded the upper limit for growth, than that at the optimal growth temperature (30 °C). Conversely, succinic acid production was decreased by 13% under the same conditions in wild-type. The specific activity of phosphoenolpyruvate carboxylase decreased with the increase in temperature. In contrast, the other glycolytic and reductive TCA cycle enzymes demonstrated increased or constant activity as the temperature was increased. When using a succinic acid over-producing strain, succinic acid production was increased by 34% at 42.5 °C. Our findings demonstrate that the profile of fermentation products is dependent upon temperature, which could be caused by the modulation of enzymatic activities. Moreover, we report that elevated temperatures, exceeding the upper limit for cell growth, can be used to increase the production of target compounds in C. glutamicum. KEY POINTS: • Lactate productivity was increased by temperature elevation. • Succinate productivity was increased by temperature elevation when lactate pathway was deleted. • Specific activity of phosphoenolpyruvate carboxylase was decreased by temperature elevation.

Recent progress in production of amino acid-derived chemicals using Corynebacterium glutamicum.

Green chemical production by microbial processes is critical for the development of a sustainable society in the twenty-first century. Among the important industrial microorganisms, the gram-positive bacterium Corynebacterium glutamicum has been utilized for amino acid fermentation, which is one of the largest microbial-based industries. To date, several amino acids, including L-glutamic acid, L-lysine, and L-threonine, have been produced by C. glutamicum. The capability to produce substantial amounts of amino acids has gained immense attention because the amino acids can be used as a precursor to produce other high-value-added chemicals. Recent developments in metabolic engineering and synthetic biology technologies have enabled the extension of metabolic pathways from amino acids. The present review provides an overview of the recent progress in the microbial production of amino acid-derived bio-based monomers such as 1,4-diaminobutane, 1,5-diaminopentane, glutaric acid, 5-aminolevulinic acid, L-pipecolic acid, 4-amino-1-butanol, and 5-aminolevulinic acid, as well as building blocks for healthcare products and pharmaceuticals such as ectoine, L-theanine, and gamma-aminobutyric acid by metabolically engineered C. glutamicum.

Requirement of de novo synthesis of pyruvate carboxylase in long-term succinic acid production in Corynebacterium glutamicum.

Protein turnover through de novo synthesis is critical for sustainable cellular functions. We previously found that glucose consumption rate in Corynebacterium glutamicum under anaerobic conditions increased at temperature higher than the upper limit of growth temperature. Here, we showed that production of lactic and succinic acids increased at higher temperature for long-term (48 h) anaerobic reaction in metabolically engineered strains. At 42 °C, beyond the upper limit of growth temperature range, biomass-specific lactic acid production rate was 8% higher than that at 30 °C, the optimal growth temperature. In contrast, biomass-specific succinic acid production rate was highest at 36 °C, 28% higher than that at 30 °C, although the production at 42 °C was still 23% higher than that at 30 °C. As enzymes are usually unstable at high temperatures, we investigated whether protein turnover of metabolic enzymes is required for the production of lactic and succinic acids under these conditions. Interestingly, when de novo protein synthesis was inhibited by addition of chloramphenicol, after 6 h, only succinic acid production was inhibited. Because glycolytic enzymes are involved in both lactic and succinic acids synthesis, enzymes in the anaplerotic pathway and the tricarboxylic acid (TCA) cycle leading to succinic acid synthesis were likely to be responsible for its decreased production. Among the five enzymes examined, the specific activity of only pyruvate carboxylase was drastically decreased after 48 h at 42 °C. Thus, the de novo synthesis of pyruvate carboxylase is required for long-term production of succinic acid. Graphical abstract KEY POINTS: • Long-term reaction for organic acids can be improved at temperature beyond ideal growth conditions. • De novo synthesis of pyruvate carboxylase is required for long-term succinic acid production.

Anaerobic glucose consumption is accelerated at non-proliferating elevated temperatures through upregulation of a glucose transporter gene in Corynebacterium glutamicum.

Cell proliferation is achieved through numerous enzyme reactions. Temperature governs the activity of each enzyme, ultimately determining the optimal growth temperature. The synthesis of useful chemicals and fuels utilizes a fraction of available metabolic pathways, primarily central metabolic pathways including glycolysis and the tricarboxylic acid cycle. However, it remains unclear whether the optimal temperature for these pathways is correlated with that for cell proliferation. Here, we found that wild-type Corynebacterium glutamicum displayed increased glycolytic activity under non-growing anaerobic conditions at 42.5 °C, at which cells do not proliferate under aerobic conditions. At this temperature, glucose consumption was not inhibited and increased by 28% compared with that at the optimal growth temperature of 30 °C. Transcriptional analysis revealed that a gene encoding glucose transporter (iolT2) was upregulated by 12.3-fold compared with that at 30 °C, with concomitant upregulation of NCgl2954 encoding the iolT2-regulating transcription factor. Deletion of iolT2 decreased glucose consumption rate at 42.5 °C by 28%. Complementation of iolT2 restored glucose consumption rate, highlighting the involvement of iolT2 in the accelerating glucose consumption at an elevated temperature. This study shows that the optimal temperature for glucose metabolism in C. glutamicum under anaerobic conditions differs greatly from that for cell growth under aerobic conditions, being beyond the upper limit of the growth temperature. This is beneficial for fuel and chemical production not only in terms of increasing productivity but also for saving cooling costs. KEY POINTS: • C. glutamicum accelerated anaerobic glucose consumption at elevated temperature. • The optimal temperature for glucose consumption was above the upper limit for growth. • Gene expression involved in glucose transport was upregulated at elevated temperature. Graphical abstract.

Metabolic engineering of Corynebacterium glutamicum for hyperproduction of polymer-grade L- and D-lactic acid.

Strain development is critical for microbial production of bio-based chemicals. The stereo-complex form of polylactic acid, a complex of poly-L- and poly-D-lactic acid, is a promising polymer candidate due to its high thermotolerance. Here, we developed Corynebacterium glutamicum strains producing high amounts of L- and D-lactic acid through intensive metabolic engineering. Chromosomal overexpression of genes encoding the glycolytic enzymes, glucokinase, glyceraldehyde-3-phosphate dehydrogenase, phosphofructokinase, triosephosphate isomerase, and enolase, increased L- and D-lactic acid concentration by 146% and 56%, respectively. Chromosomal integration of two genes involved in the Entner-Doudoroff pathway (6-phosphogluconate dehydratase and 2-dehydro-3-deoxyphosphogluconate aldolase), together with a gene encoding glucose-6-phosphate dehydrogenase from Zymomonas mobilis, to bypass the carbon flow from glucose, further increased L- and D-lactic acid concentration by 11% and 44%, respectively. Finally, additional chromosomal overexpression of a gene encoding NADH dehydrogenase to modulate the redox balance resulted in the production of 212 g/L L-lactic acid with a 97.9% yield and 264 g/L D-lactic acid with a 95.0% yield. The optical purity of both L- and D-lactic acid was 99.9%. Because the constructed metabolically engineered strains were devoid of plasmids and antibiotic resistance genes and were cultivated in mineral salts medium, these strains could contribute to the cost-effective production of the stereo-complex form of polylactic acid in practical scale.

Metabolic engineering of Corynebacterium glutamicum for production of sunscreen shinorine.

Ultraviolet-absorbing chemicals are useful in cosmetics and skin care to prevent UV-induced skin damage. We demonstrate here that heterologous production of shinorine, which shows broad absorption maxima in the UV-A and UV-B region. A shinorine producing Corynebacterium glutamicum strain was constructed by expressing four genes from Actinosynnema mirum DSM 43827, which are responsible for the biosynthesis of shinorine from sedoheptulose-7-phosphate in the pentose phosphate pathway. Deletion of transaldolase encoding gene improved shinorine production by 5.2-fold. Among the other genes in pentose phosphate pathway, overexpression of 6-phosphogluconate dehydrogenase encoding gene further increased shinorine production by 60% (19.1 mg/L). The genetic engineering of the pentose phosphate pathway in C. glutamicum improved shinorine production by 8.3-fold in total, and could be applied to produce the other chemicals derived from sedoheptulose-7-phosphate.

Design of Wall-Destructive but Membrane-Compatible Solvents.

We report an extremely biocompatible solvent for plant cell walls based on a polar liquid zwitterion that dissolves cellulose, the most recalcitrant component of the plant cell walls. The polar liquid zwitterion does not affect the viability and activity of Escherichia coli, even at high concentrations. We demonstrate conversion of cell walls to ethanol via a starch-like process, namely successive dissolution, hydrolysis and fermentation in the same reaction pot.

Sucrose purification and repeated ethanol production from sugars remaining in sweet sorghum juice subjected to a membrane separation process.

The juice from sweet sorghum cultivar SIL-05 (harvested at physiological maturity) was extracted, and the component sucrose and reducing sugars (such as glucose and fructose) were subjected to a membrane separation process to purify the sucrose for subsequent sugar refining and to obtain a feedstock for repeated bioethanol production. Nanofiltration (NF) of an ultrafiltration (UF) permeate using an NTR-7450 membrane (Nitto Denko Corporation, Osaka, Japan) concentrated the juice and produced a sucrose-rich fraction (143.2 g L-1 sucrose, 8.5 g L-1 glucose, and 4.5 g L-1 fructose). In addition, the above NF permeate was concentrated using an ESNA3 NF membrane to provide concentrated permeated sugars (227.9 g L-1) and capture various amino acids in the juice, enabling subsequent ethanol fermentation without the addition of an exogenous nitrogen source. Sequential batch fermentation using the ESNA3 membrane concentrate provided an ethanol titer and theoretical ethanol yield of 102.5-109.5 g L-1 and 84.4-89.6%, respectively, throughout the five-cycle batch fermentation by Saccharomyces cerevisiae BY4741. Our results demonstrate that a membrane process using UF and two types of NF membranes has the potential to allow sucrose purification and repeated bioethanol production.

Engineering cell factories for producing building block chemicals for bio-polymer synthesis.

Synthetic polymers are widely used in daily life. Due to increasing environmental concerns related to global warming and the depletion of oil reserves, the development of microbial-based fermentation processes for the production of polymer building block chemicals from renewable resources is desirable to replace current petroleum-based methods. To this end, strains that efficiently produce the target chemicals at high yields and productivity are needed. Recent advances in metabolic engineering have enabled the biosynthesis of polymer compounds at high yield and productivities by governing the carbon flux towards the target chemicals. Using these methods, microbial strains have been engineered to produce monomer chemicals for replacing traditional petroleum-derived aliphatic polymers. These developments also raise the possibility of microbial production of aromatic chemicals for synthesizing high-performance polymers with desirable properties, such as ultraviolet absorbance, high thermal resistance, and mechanical strength. In the present review, we summarize recent progress in metabolic engineering approaches to optimize microbial strains for producing building blocks to synthesize aliphatic and high-performance aromatic polymers.

FudC, a protein primarily responsible for furfural detoxification in Corynebacterium glutamicum.

Lignocellulosic hydrolysates contain compounds that inhibit microbial growth and fermentation, thereby decreasing the productivity of biofuel and biochemical production. In particular, the heterocyclic aldehyde furfural is one of the most toxic compounds found in these hydrolysates. We previously demonstrated that Corynebacterium glutamicum converts furfural into the less toxic compounds furfuryl alcohol and 2-furoic acid. To date, however, the genes involved in these oxidation and reduction reactions have not been identified in the C. glutamicum genome. Here, we show that Cgl0331 (designated FudC) is mainly responsible for the reduction of furfural into furfuryl alcohol in C. glutamicum. Deletion of the gene encoding FudC markedly diminished the in vivo reduction of furfural to furfuryl alcohol. Purified His-tagged FudC protein from Escherichia coli was also shown to convert furfural into furfuryl alcohol in an in vitro reaction utilizing NADPH, but not NADH, as a cofactor. Kinetic measurements demonstrated that FudC has a high affinity for furfural but has a narrow substrate range for other aldehydes compared to the protein responsible for furfural reduction in E. coli.

Glucose consumption rate critically depends on redox state in Corynebacterium glutamicum under oxygen deprivation.

Rapid sugar consumption is important for the microbial production of chemicals and fuels. Here, we show that overexpression of the NADH dehydrogenase gene (ndh) increased glucose consumption rate in Corynebacterium glutamicum under oxygen-deprived conditions through investigating the relationship between the glucose consumption rate and intracellular NADH/NAD(+) ratio in various mutant strains. The NADH/NAD(+) ratio was strongly repressed under oxygen deprivation when glucose consumption was accelerated by the addition of pyruvate or sodium hydrogen carbonate. Overexpression of the ndh gene in the wild-type strain under oxygen deprivation decreased the NADH/NAD(+) ratio from 0.32 to 0.13, whereas the glucose consumption rate increased by 27%. Similarly, in phosphoenolpyruvate carboxylase gene (ppc)- or malate dehydrogenase gene (mdh)-deficient strains, overexpression of the ndh gene decreased the NADH/NAD(+) ratio from 1.66 to 0.37 and 2.20 to 0.57, respectively, whereas the glucose consumption rate increased by 57 and 330%, respectively. However, in a lactate dehydrogenase gene (L-ldhA)-deficient strain, although the NADH/NAD(+) ratio decreased from 5.62 to 1.13, the glucose consumption rate was not markedly altered. In a tailored D-lactate-producing strain, which lacked ppc and L-ldhA genes, but expressed D-ldhA from Lactobacillus delbrueckii, overexpression of the ndh gene decreased the NADH/NAD(+) ratio from 1.77 to 0.56, and increased the glucose consumption rate by 50%. Overall, the glucose consumption rate was found to be inversely proportional to the NADH/NAD(+) ratio in C. glutamicum cultured under oxygen deprivation. These findings could provide an option to increase the productivity of chemicals and fuels under oxygen deprivation.

Overexpression of the phosphofructokinase encoding gene is crucial for achieving high production of D-lactate in Corynebacterium glutamicum under oxygen deprivation.

We previously reported on the impacts of the overexpression of individual genes of the glycolytic pathway encoding glucokinase (GLK), glyceraldehyde phosphate dehydrogenase (GAPDH), phosphofructokinase (PFK), triosephosphate isomerase (TPI), and bisphosphate aldolase (FBA) on D-lactate productivity in Corynebacterium glutamicum under oxygen-deprived conditions. Searching for synergies, in the current study, we simultaneously overexpressed the five glycolytic genes in a stepwise fashion to evaluate the effect of the cumulative overexpression of glycolytic genes on D-lactate production. Interestingly, the final D-lactate concentration markedly differed depending on whether or not the PFK encoding gene was overexpressed when combined with overexpressing other glycolytic genes. The simultaneous overexpression of the GLK, GAPDH, TPI, and FBA encoding genes led to the highest initial D-lactate concentration at 10 h. However, this particular recombinant strain dramatically slowed producing D-lactate when a concentration of 1300 mM was reached, typically after 32 h. In contrast, the strain overexpressing the PFK encoding gene together with the GLK, GAPDH, TPI, and FBA encoding genes showed 12.7 % lower initial D-lactate concentration at 10 h than that observed with the strain overexpressing the genes coding for GLK, GAPDH, TPI, and FBA. However, this recombinant strain continued to produce D-lactate after 32 h, reaching 2169 mM after a mineral salts medium bioprocess incubation period of 80 h. These results suggest that overexpression of the PFK encoding gene is essential for achieving high production of D-lactate. Our findings provide interesting options to explore for using C. glutamicum for cost-efficient production of D-lactate at the industrial scale.

Recent advances in the metabolic engineering of Corynebacterium glutamicum for the production of lactate and succinate from renewable resources.

Recent increasing attention to environmental issues and the shortage of oil resources have spurred political and industrial interest in the development of environmental friendly and cost-effective processes for the production of bio-based chemicals from renewable resources. Thus, microbial production of commercially important chemicals is viewed as a desirable way to replace current petrochemical production. Corynebacterium glutamicum, a Gram-positive soil bacterium, is one of the most important industrial microorganisms as a platform for the production of various amino acids. Recent research has explored the use of C. glutamicum as a potential cell factory for producing organic acids such as lactate and succinate, both of which are commercially important bulk chemicals. Here, we summarize current understanding in this field and recent metabolic engineering efforts to develop C. glutamicum strains that efficiently produce L- and D-lactate, and succinate from renewable resources.

Two-step production of D-lactate from mixed sugars by growing and resting cells of metabolically engineered Lactobacillus plantarum.

To develop cost-effective systems for D-lactate production, here, the effect of high-cell density cultivation of metabolically engineered Lactobacillus plantarum on D-lactate production was evaluated. A xylose-assimilating strain of L. plantarum was anaerobically cultured with mixed sugars (glucose and xylose) as substrates. Compared to undiluted nutrient-rich de Man, Rogosa, and Sharpe (MRS) medium, D-lactate production by cultivating in 10-fold diluted MRS (0.1 MRS) medium or normal saline solution was 89.7 and 81.3 %, respectively. Notably, the xylose consumption rate was comparable in the three cultures, whereas the glucose consumption rate decreased by 18.3 and 26.1 % in 0.1 MRS medium and normal saline solution, respectively, resulting in a reduction of the D-lactate production rate. The D-lactate productivity in high-cell density cultivation was proportional to the initial cell concentrations. The use of a two-step cultivation process involving growing and resting cells in a single bioreactor revealed that the ratio of the glucose and xylose consumption rates (based on grams consumed) in resting cell conditions was 1.88, whereas that in growing conditions was 2.58. Cultivation of L. plantarum in growing conditions for 24 h produced 73.2 g/l D-lactate with the yield of 0.90 g/g, whereas cells cultivation under resting cell conditions in a saline solution for 24 h produced 68.7 g/l D-lactate with the yield of 0.93 g/g. In total, 141.9 g/l D-lactate was produced after 48 h cultivation, a value that represents the highest reported concentration of D-lactate produced from mixed sugars to date. Our findings contribute to the cost-effective, large-scale production of D-lactate.

Detoxification of furfural in Corynebacterium glutamicum under aerobic and anaerobic conditions.

The toxic fermentation inhibitors in lignocellulosic hydrolysates raise serious problems for the microbial production of fuels and chemicals. Furfural is considered to be one of the most toxic compounds among these inhibitors. Here, we describe the detoxification of furfural in Corynebacterium glutamicum ATCC13032 under both aerobic and anaerobic conditions. Under aerobic culture conditions, furfuryl alcohol and 2-furoic acid were produced as detoxification products of furfural. The ratio of the products varied depending on the initial furfural concentration. Neither furfuryl alcohol nor 2-furoic acid showed any toxic effect on cell growth, and both compounds were determined to be the end products of furfural degradation. Interestingly, unlike under aerobic conditions, most of the furfural was converted to furfuryl alcohol under anaerobic conditions, without affecting the glucose consumption rate. Both the NADH/NAD(+) and NADPH/NADP(+) ratio decreased in the accordance with furfural concentration under both aerobic and anaerobic conditions. These results indicate the presence of a single or multiple endogenous enzymes with broad and high affinity for furfural and co-factors in C. glutamicum ATCC13032.

Reactions upstream of glycerate-1,3-bisphosphate drive Corynebacterium glutamicum (D)-lactate productivity under oxygen deprivation.

We previously demonstrated the simplicity of oxygen-deprived Corynebacterium glutamicum to produce D-lactate, a primary building block of next-generation biodegradable plastics, at very high optical purity by introducing heterologous D-ldhA gene from Lactobacillus delbrueckii. Here, we independently evaluated the effects of overexpressing each of genes encoding the ten glycolytic enzymes on D-lactate production in C. glutamicum. We consequently show that while the reactions catalyzed by glucokinase (GLK), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), phosphofructokinase (PFK), triosephosphate isomerase (TPI), and bisphosphate aldolase had positive effects on D-lactate productivity by increasing 98, 39, 15, 13, and 10 %, respectively, in 10 h reactions in minimal salts medium, the reaction catalyzed by pyruvate kinase had large negative effect by decreasing 70 %. The other glycolytic enzymes did not affect D-lactate productivity when each of encoding genes was overexpressed. It is noteworthy that all reactions associated with positive effects are located upstream of glycerate-1,3-bisphosphate in the glycolytic pathway. The D-lactate yield also increased by especially overexpressing TPI encoding gene up to 94.5 %. Interestingly, overexpression of PFK encoding gene reduced the yield of succinate, one of the main by-products of D-lactate production, by 52 %, whereas overexpression of GAPDH encoding gene increased succinate yield by 26 %. Overexpression of GLK encoding gene markedly increased the yield of dihydroxyacetone and glycerol by 10- and 5.8-fold in exchange with decreasing the D-lactate yield. The effect of overexpressing glycolytic genes was also evaluated in 80 h long-term reactions. The variety of effects of overexpressing each of genes encoding the ten glycolytic enzymes on D-lactate production is discussed.

Utilization of lactic acid bacterial genes in Synechocystis sp. PCC 6803 in the production of lactic acid.

Metabolic pathway engineering of cyanobacteria for the production of industrially important chemicals from atmospheric CO2 has generated interest recently. Here, we engineered Synechocystis sp. PCC 6803 to produce lactic acid using a lactate dehydrogenase (ldh) gene from various lactic acid-producing bacteria, Lactococcus lactis (ldhB and ldhX), Lactobacillus plantarum (ldhL and ldh), and Lactobacillus rhamnosus (ldhL). The lactic acid was secreted outside the cell using a transporter (lldp) gene from L. plantarum. Expression of each ldh in Synechocystis sp. PCC6803 was ascertained by reverse-transcriptase polymerase chain reaction. Five transformants led to the production of L-lactic acid. Co-expression of lldp with ldhB from L. plantarum or ldhL from L. rhamnosus led to the secretion of lactic acid into the medium at concentration of 0.17 ± 0.02 or 0.14 ± 0.02 mM after 18 d of cultivation.

Enhanced production of itaconic acid from enzymatic hydrolysate of lignocellulosic biomass by recombinant Corynebacteriumglutamicum.

Itaconic acid (IA) is a value-added chemical currently produced by Aspergillus terreus from edible glucose and starch but not from inedible lignocellulosic biomass owing to the high sensitivity to fermentation inhibitors present in the hydrolysate of lignocellulosic biomass. To produce IA from lignocellulosic biomass, a gram-positive bacterium, Corynebacterium glutamicum, with a high tolerance to fermentation inhibitors was metabolically engineered to express a fusion protein composed of cis-aconitate decarboxylase from A. terreus responsible for IA formation from cis-aconitate and a maltose-binding protein (malE) from Escherichia coli. The codon-optimized cadA_malE gene was expressed in C. glutamicum ATCC 13032, and the resulting recombinant strain produced IA from glucose. IA concentration increased 4.7-fold by the deletion of the ldh gene encoding lactate dehydrogenase. With the Δldh strain HKC2029, an 18-fold higher IA production was observed from enzymatic hydrolysate of kraft pulp as a model lignocellulosic biomass than from glucose (6.15 and 0.34 g/L, respectively). The enzymatic hydrolysate of kraft pulp contained various potential fermentation inhibitors involved in furan aldehydes, benzaldehydes, benzoic acids, cinnamic acid derivatives, and aliphatic acid. Whereas cinnamic acid derivatives severely inhibited IA production, furan aldehydes, benzoic acids, and aliphatic acid improved IA production at low concentrations. The present study suggests that lignocellulosic hydrolysate contains various potential fermentation inhibitors; however, some of them can serve as enhancers for microbial fermentation likely due to the changing of redox balance in the cell.

Enhanced methane production from cellulose using a two-stage process involving a bioelectrochemical system and a fixed film reactor.

BACKGROUND: It is desirable to improve the anaerobic digestion processes of recalcitrant materials, such as cellulose. Enhancement of methane (CH4) production from organic molecules was previously accomplished through coupling a bioelectrochemical system (BES); however, scaling-up BES-based production is difficult. Here, we developed a two-stage process consisting of a BES using low-cost and low-reactive carbon sheets as the cathode and anode, and a fixed film reactor (FFR) containing conductive material, i.e., carbon fiber textiles (CFTs) (:BES → FFR). By controlling the cathodic current at 2.7 µA/cm2 without abiotic H2 production, the three-electrode BES system was operated to mimic a microbial electrolysis cell. RESULTS: The thermophilic BES (inlet pH: 6.1) and FFR (inlet pH: 7.5) were operated using hydraulic retention times (HRTs) of 2.5 and 4.2 days, respectively, corresponding to a cellulose load of 3555.6 mg-carbon (C)/(L day). The BES → FFR process achieved a higher CH4 yield (37.5%) with 52.8 vol% CH4 in the product gas compared to the non-bioelectrochemical system (NBES) → FFR process, which showed a CH4 yield of 22.1% with 46.8 vol% CH4. The CH4 production rate (67.5 mM/day) obtained with the BER → FFR process was much higher than that obtained using electrochemical methanogenesis (0.27 mM/day). Application of the electrochemical system or CFTs improved the yields of CH4 with the NBES → FFR or BES → non-fixed film reactor process, respectively. Meta 16S rRNA sequencing revealed that putative cellulolytic bacteria (identified as Clostridium species) were present in the BES and NBES, and followed (BES→ and NBES→) FFR. Notably, H2-consuming methanogens, Methanobacterium sp. and Methanosarcina sp., showed increased relative abundances in the suspended fraction and attached fraction of (BES→) FFR, respectively, compared to that of (NBES→) FFR, although these methanogens were observed at trace levels in the BES and NBES. CONCLUSIONS: These results indicate that bioelectrochemical preprocessing at a low current effectively induces interspecies H2 transfer in the FFR with conductive material. Sufficient electrochemical preprocessing was observed using a relatively short HRT. This type of two-stage process, BES → FFR, is useful for stabilization and improvement of the biogas (CH4) production from cellulosic material, and our results imply that the two-stage system developed here may be useful with other recalcitrant materials.