Mechanisms of response to pH shock in microbial fermentation.

The following review highlights pH shock, a novel environmental factor, as a tool for the improvement of fermentation production. The aim of this review is to introduce some recent original studies on the enhancement of microbial fermentation production by pH shock. Another purpose of this review is to improve the understanding of the processes that underlie physiological and genetic differences, which will facilitate future research on the improvement of fermentation production and reveal the associated molecular mechanisms. This understanding will simultaneously promote the application of this strategy to other microbial fermentation systems. Furthermore, improvement of the cellular tolerance of genetically engineered bacteria can also be a new field of research in the future to enhance fermentation production.

Full recycling of citric acid wastewater through anaerobic digestion, air-stripping and pH control.

Anaerobic digestion effluent (ADE) from the anaerobic digestion treatment of citric acid wastewater can be reused as a potential substitute for process water in the citric acid fermentation. However, excessive sodium contained in ADE significantly decreases citric acid production. In this paper, the inhibition mechanism of sodium on citric acid fermentation was investigated. We demonstrated that excessive sodium did not increase oxidative stress for Aspergillus niger, but reduced the pH of the medium significantly over the period 4-24 h, which led to lower activities of glucoamylase and isomaltase secreted by A. niger, with a decrease of available sugar concentration and citric acid production. ADE was pretreated by air-stripping prior to recycle and 18 g/L calcium carbonate was added at the start of fermentation to control the pH of the medium. The inhibition caused by ADE was completely alleviated and citric acid production substantially increased from 118.6 g/L to 141.4 g/L, comparable to the fermentation with deionized water (141.2 g/L). This novel process could decrease wastewater discharges and fresh water consumption in the citric acid industry, with benefit to the environment.

Metabolic analyses of the improved ε-poly-L-lysine productivity using a glucose-glycerol mixed carbon source in chemostat cultures.

The glucose-glycerol mixed carbon source remarkably reduced the batch fermentation time of ε-poly-L-lysine (ε-PL) production, leading to higher productivity of both biomass and ε-PL, which was of great significance in industrial microbial fermentation. Our previous study confirmed the positive influence of fast cell growth on the ε-PL biosynthesis, while the direct influence of mixed carbon source on ε-PL production was still unknown. In this work, chemostat culture was employed to study the capacity of ε-PL biosynthesis in different carbon sources at a same dilution rate of 0.05 h-1. The results indicated that the mixed carbon source could enhance the ε-PL productivity besides the rapid cell growth. Analysis of key enzymes demonstrated that the activities of phosphoenolpyruvate carboxylase, citrate synthase, aspartokinase and ε-PL synthetase were all increased in chemostat culture with the mixed carbon source. In addition, the carbon fluxes were also improved in the mixed carbon source in terms of tricarboxylic acid cycle, anaplerotic and diaminopimelate pathway. Moreover, the mixed carbon source also accelerated the energy metabolism, leading to higher levels of energy charge and NADH/NAD+ ratio. The overall improvements of primary metabolism in chemostat culture with glucose-glycerol combination provided sufficient carbon skeletons and ATP for ε-PL biosynthesis. Therefore, the significantly higher ε-PL productivity in the mixed carbon source was a combined effect of both superior substrate group and rapid cell growth.

Effect of propionic acid on citric acid fermentation in an integrated citric acid-methane fermentation process.

In this study, an integrated citric acid-methane fermentation process was established to solve the problem of wastewater treatment in citric acid production. Citric acid wastewater was treated through anaerobic digestion and then the anaerobic digestion effluent (ADE) was further treated and recycled for the next batch citric acid fermentation. This process could eliminate wastewater discharge and reduce water resource consumption. Propionic acid was found in the ADE and its concentration continually increased in recycling. Effect of propionic acid on citric acid fermentation was investigated, and results indicated that influence of propionic acid on citric acid fermentation was contributed to the undissociated form. Citric acid fermentation was inhibited when the concentration of propionic acid was above 2, 4, and 6 mM in initial pH 4.0, 4.5 and, 5.0, respectively. However, low concentration of propionic acid could promote isomaltase activity which converted more isomaltose to available sugar, thereby increasing citric acid production. High concentration of propionic acid could influence the vitality of cell and prolong the lag phase, causing large amount of glucose still remaining in medium at the end of fermentation and decreasing citric acid production.

Physiological mechanism of the overproduction of ε-poly-L-lysine by acidic pH shock in fed-batch fermentation.

The introduction of an environmental stress of acidic pH shock had successfully solved the common deficiency existed in ε-PL production, viz. the distinct decline of ε-PL productivity in the feeding phase of the fed-batch fermentation. To unravel the underlying mechanism, we comparatively studied the physiological changes of Streptomyces sp. M-Z18 during fed-batch fermentations with the pH shock strategy (PS) and pH non-shock strategy (PNS). Morphology investigation showed that pellet-shape change was negligible throughout both fermentations. In addition, the distribution of pellet size rarely changed in the PS, whereas pellet size and number decreased substantially with time in the PNS. This was consistent with the performances of ε-PL productivity in both strategies, demonstrating that morphology could be used as a predictor of ε-PL productivity during fed-batch fermentation. Furthermore, a second growth phase happened in the PS after pH shock, followed by the re-appearance of live mycelia in the dead core of the pellets. Meanwhile, mycelia respiration and key enzymes in the central metabolic and ε-PL biosynthetic pathways were overall strengthened until the end of the fed-batch fermentation. As a result, the physiological changes induced by the acidic pH shock have synergistically and permanently contributed to the stimulation of ε-PL productivity. However, this second growth phase and re-appearance of live mycelia were absent in the PNS. These results indicated that the introduction of a short-term suppression on mycelia physiological metabolism would guarantee the long-term high ε-PL productivity.

Acidic pH shock induced overproduction of ε-poly-L-lysine in fed-batch fermentation by Streptomyces sp. M-Z18 from agro-industrial by-products.

ε-Poly-L-lysine (ε-PL) is produced by Streptomyces as a secondary metabolite with wide industrial applications, but its production still needs to be further enhanced. Environmental stress is an important approach for the promotion of secondary metabolites production by Streptomyces. In this study, the effect of acidic pH shock on enhancing ε-PL production by Streptomyces sp. M-Z18 was investigated in a 5-L fermenter. Based on the evaluation of acidic pH shock on mycelia metabolic activity and shock parameters optimization, an integrated pH-shock strategy was developed as follows: pre-acid-shock adaption at pH 5.0 to alleviate the damage caused by the followed pH shock, and then acidic pH shock at 3.0 for 12 h (including pH decline from 4.0 to 3.0) to positively regulate mycelia metabolic activity, finally restoring pH to 4.0 to provide optimal condition for ε-PL production. After 192 h of fed-batch fermentation, the maximum ε-PL production and productivity reached 54.70 g/L and 6.84 g/L/day, respectively, which were 52.50 % higher than those of control without pH shock. These results demonstrated that acidic pH shock is an efficient approach for improving ε-PL production. The information obtained should be useful for ε-PL production by other Streptomyces.

Enhancement of ε-poly-lysine production in ε-poly-lysine-tolerant Streptomyces sp. by genome shuffling.

ε-Poly-L-lysine (ε-PL) has been widely used as food additive. However, the self-inhibition of ε-PL on cell growth limits the accumulation of ε-PL in the wild-type strain. Here, we screened ε-PL-tolerant strain of Streptomyces sp. with higher ε-PL productivity by genome shuffling and studied the mechanism for the improvement. The initial mutant library was constructed by diethyl sulfate mutagenesis. After four rounds of protoplast fusion, a shuffled strain F4-22 with 3.11 g/L ε-PL productivity in shake flask, 1.81-fold in comparison with that of parent strain, was obtained. The higher aspartokinase activity was induced in F4-22 whereas no obvious changes have been found in ε-PL synthetic and degrading enzymes which indicated that the upstream reregulation of the precursor lysine synthesis rather than lysine polymerization or ε-PL degradation in shuffled strain accounted for the higher productivity. The activities of key enzymes in the central metabolic pathway were also enhanced in F4-22 which resulted in increased vigor of the strain and in delayed strain lysis during fermentation. These improved properties of shuffled strain led to the success of combining general two-stage fermentation into one-stage one in 5-L bioreactor with 32.7 % more ε-PL production than that of parent strain. The strategy used in this study provided a novel strain breeding approach of producers which suffered from ε-PL-like self-inhibition of the metabolites.

Optimization of the integrated citric acid-methane fermentation process by air stripping and glucoamylase addition.

To solve the problem of extraction wastewater in citric acid industry, an integrated citric acid-methane fermentation process was proposed. In the integrated process, extraction wastewater was treated by mesophilic anaerobic digestion and then reused to make mash for the next batch of citric acid fermentation. In this study, an Aspergillus niger mutant strain exhibiting resistance to high metal ions concentration was used to eliminate the inhibition of 200 mg/L Na(+) and 300 mg/L K(+) in anaerobic digestion effluent (ADE) and citric acid production increased by 25.0 %. Air stripping was used to remove ammonium, alkalinity, and part of metal ions in ADE before making mash. In consequence, citric acid production was significantly improved but still lower by 6.1 % than the control. Results indicated that metal ions in ADE synergistically inhibited the activity of glucoamylase, thus reducing citric acid production. When 130 U/g glucoamylase was added before fermentation, citric acid production was 141.5 g/L, which was even higher than the control (140.4 g/L). This process could completely eliminate extraction wastewater discharge and reduce water resource consumption.

In vivo and in vitro decolorization of synthetic dyes by laccase from solid state fermentation with Trametes sp. SYBC-L4.

Synthetic decolorization of dyes through solid cassava residue substrate fermentation with Trametes sp. SYBC-L4 via in vivo and in vitro processes was investigated in this study. Effects of pH and mediator (1-hydroxybenzotriazole, HBT) concentration on dyes decolorization were evaluated. In vitro, decolorization ratios of dyes differed considerably in pH and increased with the increasing of HBT concentration. Crude laccase (50 U/L) derived from Trametes sp. SYBC-L4 decolorized 67.91 ± 1.25 % Congo red (100 mg/L), 94.58 ± 1.05 % aniline blue (100 mg/L) and 99.02 ± 0.54 % indigo carmine (100 mg/L) with 2.5 mM HBT at pH 4.5 in 36 h of incubation. In vivo, decolorization ratios of dyes were not enhanced by usage of the mediator. After 10 days of fermentation, decolorization ratio of Congo red (1,000 mg/kg), aniline blue (1,000 mg/kg) and indigo carmine (1,000 mg/kg) was 57.82 ± 0.84, 92.53 ± 1.12 and 97.26 ± 1.92 % without the usage of mediator at pH 4.5, respectively. Moreover, there was no obvious difference between the in vivo decolorization of aniline blue and indigo carmine in the pH range of 3.0-9.0. Results showed that Trametes sp. SYBC-L4 had great potential to be used for dyes decolorization via in vivo and in vitro processes. Moreover, in terms of pH range and mediator, in vivo decolorization with Trametes sp. SYBC-L4 was more advantageous since laccase mediator was needless and the applicable range of pH was broader.

Production of citric acid using its extraction wastewater treated by anaerobic digestion and ion exchange in an integrated citric acid-methane fermentation process.

In order to solve the problem of extraction wastewater pollution in citric acid industry, an integrated citric acid-methane fermentation process is proposed in this study. Extraction wastewater was treated by mesophilic anaerobic digestion and then used to make mash for the next batch of citric acid fermentation. The recycling process was done for seven batches. Citric acid production (82.4 g/L on average) decreased by 34.1 % in the recycling batches (2nd-7th) compared with the first batch. And the residual reducing sugar exceeded 40 g/L on average in the recycling batches. Pigment substances, acetic acid, ammonium, and metal ions in anaerobic digestion effluent (ADE) were considered to be the inhibitors, and their effects on the fermentation were studied. Results indicated that ammonium, Na(+) and K(+) in the ADE significantly inhibited citric acid fermentation. Therefore, the ADE was treated by acidic cation exchange resin prior to reuse to make mash for citric acid fermentation. The recycling process was performed for ten batches, and citric acid productions in the recycling batches were 126.6 g/L on average, increasing by 1.7 % compared with the first batch. This process could eliminate extraction wastewater discharge and reduce water resource consumption.

Suitability of anaerobic digestion effluent as process water for corn fuel ethanol fermentation.

A corn fuel ethanol plant integrated with anaerobic digestion treatment of thin stillage increases the net energy balance. Furthermore, the anaerobic digestion effluent (ADE) can be reused as a potential substitute for process water in the ethanol fermentation. In this study, the suitability of ADE as process water for corn ethanol fermentation was investigated by analyzing the potential inhibitory components in the ADE. It was found that ammonium influenced the growth and metabolism of Saccharomyces cerevisiae. Maximum ethanol production was obtained when the concentration of ammonium nitrogen was 200 mg/L, and ammonium could replace urea as the nitrogen source for S. cerevisiae under this concentration. In the ethanol fermentation with a higher concentration of ammonium, more glycerol was produced, thereby resulting in the decrease of ethanol production. In addition, components except ammonium in the ADE caused no inhibition to ethanol production. These results suggest that ADE could be reused as process water for corn ethanol fermentation without negative effect when ammonium concentration is well controlled.

[Characterization of structural change of ascorbate peroxidase from Chinese kale during denaturation by circular dichroism].

Circular dichroism (CD) is a special absorption spectrum. The secondary structure of protein such as α-helix, ß-sheet and ß-turn in the far ultraviolet region (190-250 nm) has a characteristic CD spectrum. In order to understand the activity and structural changes of ascorbate peroxidase from Chinese kale (BaAPX) during denaturation, specific activity and percentage of secondary structure of BaAPX under different time, temperature and concentration were analyzed by CD dynamically. In addition, the percentage of four secondary structures in BaAPX was calculated by CD analysis software Dichroweb. The results show that BaAPX is a full α-type enzyme whose specific activity is positively related to the percentage of α-helix. During denaturation of BaAPX, three kinds of structural changes were proposed: the one-step structural change from initial state (N state) to minimum state of α-helix (R state) under low concentration and low temperature; the one-step structural change from N state to equilibrium state (T state) under high concentration and low temperature; the two-step structural changes from N state through R state to final T state under heat treatment and low temperature renaturation.

Enhancement of ε-poly-L-lysine production coupled with precursor L-lysine feeding in glucose-glycerol co-fermentation by Streptomyces sp. M-Z18.

ε-Poly-L-lysine (ε-PL), one of the only two homo-poly amino acids known in nature, is used as a preservative. In this study, strategies of feeding precursor L-lysine into 5 L laboratory scale fermenters, including optimization of L-lysine concentration and time, was investigated to optimize the production of ε-PL by Streptomyces sp. M-Z18. The optimized strategy was then used in ε-PL fed-batch fermentation in which glucose and glycerol served as mixed carbon sources. In this way, a novel ε-PL production strategy involving precursor L-lysine coupled with glucose-glycerol co-fermentation was developed. Under optimal conditions, ε-PL production reached 37.6 g/l, which was 6.2 % greater than in a previous study in which glucose and glycerol co-fermentation was performed without added L-lysine (35.14 g/l). To the best of our knowledge, this is the first report of the enhancement of ε-PL production through L-lysine feeding to evaluate the use of fermenters. Meanwhile, the role of L-lysine in the promotion of ε-PL production, participating ε-PL synthesis as a whole, was first determined using the L-[U-(13)C] lysine labeling method. It has been suggested that the bottleneck of ε-PL synthesis in Streptomyces sp. M-Z18 is in the biosynthesis of precursor L-lysine. The information obtained in the present work may facilitate strain improvement and efficient large-scale ε-PL production.

Culture medium containing glucose and glycerol as a mixed carbon source improves ε-poly-L-lysine production by Streptomyces sp. M-Z18.

To improve the efficiency of ε-poly-L-lysine (ε-PL) production by Streptomyces sp. M-Z18, batch and fed-batch fermentations with glucose and glycerol (co-fermentations) were performed. The batch fermentations showed that the initial ratio of glucose to glycerol plays an important role in glucose/glycerol co-fermentation. The optimal glucose/glycerol weight ratio was 30/30; this resulted in a maximum ε-PL productivity of 5.26 g/L/d. Glucose and glycerol were consumed synergistically during the co-fermentation process, and the length of time during which the substrate was exhausted was significantly shortened compared with the single carbon source fermentation. Under optimized conditions, fed-batch fermentations with glucose and glycerol as a mixed carbon source achieved maximum ε-PL concentration and productivity values of 35.14 g/L and 4.85 g/L/d, respectively. These values were respectively 1.43- and 1.39-, and 1.17- and 1.16-folds higher than those obtained from fermentations with glucose and glycerol as single carbon sources. The present study is the first to suggest that glucose/glycerol co-fermentation may be an efficient strategy for ε-PL production by Streptomyces sp. M-Z18.

Lactulose biosynthesis by ß-galactosidase from a newly isolated Arthrobacter sp.

Lactulose, a ketose disaccharide, is used in both pharmaceutical and food industries. This study was undertaken to screen and isolate potent ß-galactosidase-producing bacteria and to evaluate their enzymatic production of lactulose. Soil samples from fruit gardens were collected. One isolate designated LAS was identified whose cell extract could convert lactose and fructose into lactulose. The 16S rDNA gene analysis of LAS revealed its phylogenetic relatedness to Arthrobacter sp. The ß-galactosidase produced by LAS was purified 15.7-fold by ammonium sulfate precipitation and subsequent Phenyl-Sepharose hydrophobic chromatography. The optimum pH and temperature for lactulose synthesis by this ß-galactosidase were 6.0 and 20 °C, respectively. The low optimum temperature of this enzyme compared to the currently used ones for lactulose production has the advantage of reducing the nonenzymatic browning in biotransformations. The results indicated that Arthrobacter could be used as a novel bacterial ß-galactosidase source for lactulose production.

Production of ε-poly-L: -lysine using a novel two-stage pH control strategy by Streptomyces sp. M-Z18 from glycerol.

The production of ε-poly-L: -lysine (ε-PL) by Streptomyces sp. M-Z18 from glycerol was investigated in a 5-L jar-fermenter. Batch fermentations by Streptomyces sp. M-Z18 at various pH values ranging from 3.5 to 4.5 were studied. Based on the analysis of the time course of specific cell growth rate and specific ε-PL formation rate, a novel two-stage pH control strategy was developed to improve ε-PL production by shifting the culture pH from 3.5 to 3.8 after 36 h of cultivation. By applying the strategy, the maximal ε-PL concentration and productivity had a significant improvement and reached 9.13 g L(-1) and 4.76 g L(-1) day(-1), respectively, compared with those in one-stage pH control process where the pH value is controlled at 3.5 (7.83 g L(-1) and 3.13 g L(-1) day(-1)). Fed-batch fermentation with two-stage pH control strategy was also applied to produce ε-PL; final ε-PL concentration of 30.11 g L(-1) was obtained, being 3.3-fold greater than that of batch fermentation. To our knowledge, it is the first report on production of ε-PL from glycerol in fermenter scale and achievement of high ε-PL production with two-stage pH control strategy.

Effective ethanol production by reutilizing waste distillage anaerobic digestion effluent in an integrated fermentation process coupled with both ethanol and methane fermentations.

An integrated ethanol-methane fermentation coupled system characterized with full wastewater reutilization was proposed. The waste distillage originated from ethanol distillation was treated with anaerobic digestion and then recycled for medium preparation in the next ethanol fermentation run. This process could enhance wastewater reutilization, save fresh water and reduce energy consumption in the cassava-based ethanol production. The results indicated that, when using anaerobic effluents from the digestion process with only one tank, an ethanol concentration of 10.5% (v/v) compatible with that of conventional one could be achieved, but ethanol fermentation was partially inhibited and operation time gradually prolonged from 48 to 105 h. Using anaerobic effluents from the digestion process with two subsequently connected tanks, ethanol fermentation performance could be largely improved, and the fermentation lag could be completely eliminated. The performance enhancement was due to the concentrations reduction in organic acids, such as acetic and propionic acids in the digestion effluents using two digestion tanks in-series.

Development of Microtiter Plate Culture Method for Rapid Screening of ε-Poly-L-Lysine-Producing Strains.

ε-Poly-L-lysine (ε-PL) produced by Streptomyces albulus possesses a broad spectrum of antimicrobial activity and is widely used as a food preservative. To extensively screen ε-PL-overproducing strain, we developed an integrated high-throughput screening assay using ribosome engineering technology. The production protocol was scaled down to 24- and 48-deep-well microtiter plates (MTPs). The microplate reader assay was used to monitor ε-PL production. A good correlation was observed between the fermentation results obtained in both 24-(48)-deep-well MTPs and conventional Erlenmeyer flasks. Using this protocol, the production of ε-PL in an entire MTP was determined in <5 min without compromising on accuracy. The high-yielding strain selected through this protocol was also tested in Erlenmeyer flasks. The result showed that the ε-PL production of the high-yielding mutants was nearly 45% higher than that of the parent stain. Thus, development of this protocol is expected to accelerate the selection of ε-PL-overproducing strains.

A novel cleaner production process of citric acid by recycling its treated wastewater.

In this study, a novel cleaner production process of citric acid was proposed to completely solve the problem of wastewater management in citric acid industry. In the process, wastewater from citric acid fermentation was used to produce methane through anaerobic digestion and then the anaerobic digestion effluent was further treated with air stripping and electrodialysis before recycled as process water for the later citric acid fermentation. This proposed process was performed for 10 batches and the average citric acid production in recycling batches was 142.4±2.1g/L which was comparable to that with tap water (141.6g/L). Anaerobic digestion was also efficient and stable in operation. The average chemical oxygen demand (COD) removal rate was 95.1±1.2% and methane yield approached to 297.7±19.8mL/g TCODremoved. In conclusion, this novel process minimized the wastewater discharge and achieved the cleaner production in citric acid industry.

Improvement of ε-poly-L-lysine production through seed stage development based on in situ pH monitoring.

Nissin, natamycin, and ε-poly-L-lysine (ε-PL) are three safe, microbial-produced food preservatives used today in the food industry. However, current industrial production of ε-PL is only performed in several countries. In order to realize large-scale ε-PL production by fermentation, the effects of seed stage on cell growth and ε-PL production were investigated by monitoring of pH in situ in a 5-L laboratory-scale fermenter. A significant increase in ε-PL production in fed-batch fermentation by Streptomyces sp. M-Z18 was achieved, at 48.9 g/L, through the optimization of several factors associated with seed stage, including spore pretreatment, inoculum age, and inoculum level. Compared with conventional fermentation approaches using 24-h-old shake-flask seed broth as inoculum, the maximum ε-PL concentration and productivity were enhanced by 32.3 and 36.6 %, respectively. The effect of optimized inoculum conditions on ε-PL production on a large scale was evaluated using a 50-L pilot-scale fermenter, attaining a maximum ε-PL production of 36.22 g/L in fed-batch fermentation, constituting the first report of ε-PL production at pilot scale. These results will be helpful for efficient ε-PL production by Streptomyces at pilot and plant scales.