Microbial mutation breeding has been widely used because it is one of the most efficient and practical breeding strategies in the fermentation industry. However, different mutagenesis methods cause various degrees of DNA damage to individual microorganisms, which lead to diverse characteristics of the mutants. In this study, the effects of four different mutagenesis methods on the mutation breeding of Streptomyces avermitilis for improving avermectin B1a production were investigated with an optimized liquid microtiter plate (MTP) culture system. First, an effective and feasible MTP system for mutant strain screening was evaluated through the optimization of the oxygen transfer rate and rapid titer determination. Then, high energy carbon heavy ion irradiation, diethyl sulfate, ultraviolet- (UV) irradiation combined with lithium chloride, and sodium nitrite were used as the mutagens for mutation breeding, respectively. Results showed that carbon heavy ion irradiation had the advantages of possessing the highest positive mutation rate and mean-production of positive mutant strains in the first generation. Sodium nitrite treatment resulted in mutant strains with better inherited stability than the other three methods. Through the combined treatment of carbon heavy ion irradiation and sodium nitrite treatment, an inheritstable mutant S. avermitilis S-233 with high avermectin B1a production was successfully obtained. The fermentation verification in a 500-liter (L) bioreactor demonstrated that the avermectin B1a produced by mutant S. avermitilis S-233 reached 6818 μg/mL, which was 23.8% higher than that of parent strains.

Aminoglycoside antibiotics affect protein translation fidelity and lead to protein aggregation and an increase in intracellular oxidative stress level as well. The overexpression of the chaperonin GroEL/GroES system promotes short-term tolerance to aminoglycosides in Escherichia coli. Here, we demonstrated that the coexpression of prefoldin or Hsp60 originating from the hyperthermophilic archaeon Pyrococcus furiosus in E. coli cells can rescue cell growth and inhibit protein aggregation induced by streptomycin exposure. The results of our study show that hyperthermophilic chaperones endow E. coli with a higher tolerance to streptomycin than the GroEL/GroES system, and that they exert better effects on the reduction of intracellular protein misfolding, indicating that these chaperones have unique features and functions.

•Trace metal composition was statistically optimized aiming at maximal alcohol titre.•Fermentation at 25 °C overrode bacterial flocculation and enhanced alcohol titre.•Alcohol titre 6.97 g/L exceeded the best reported for P7 in bottle fermentation.Bioconversion of C1 gases to produce chemicals has good application prospects. Here, the combination of trace metals optimization using a statistical method with variable-temperature cultivation was used to enhance alcohol synthesis during CO-rich off-gas fermentation by Clostridium carboxidivorans P7. Based on ATCC medium 1754, the optimum concentration of the trace metals was found to be 5-fold Ni2+, Co2+, SeO42+, and WO42+; 3.48-fold Cu2+; 0.55-fold MoO42+; 0.5-fold Zn2+ and (NH4)2SO4·FeSO4·6H2O; and additional 44.32 μM FeCl3·6H2O. The production of alcohol and organic acid changed to 4.40 g/L and 0.50 g/L from 2.16 g/L and 2.37 g/L, respectively, yielding an increase of alcohol-to-product ratio from 47.7% to 89.8%. By fermenting with the optimized medium and timed control of the incubation temperature (37 °C [0–24 h]–25 °C [24–144 h]), the alcohol titre further increased to 6.97 g/L with 1.67 g/L butanol and 1.33 g/L hexanol, exceeding those previously reported for strain P7.

The extracellular α-amylase from the hyperthermophilic archaeum Pyrococcus furiosus (PFA) is extremely thermostable and of an industrial importance and interest. PFA aggregates and accumulates as insoluble inclusion bodies when expressed as a heterologous protein at a high level in Escherichia coli. In the present study, we investigated the roles of chaperones from P. furiosus in the soluble expression of recombinant PFA in E. coli. The results indicate that co-expression of PFA with the molecular chaperone prefoldin alone significantly increased the soluble expression of PFA. Although, co-expression of other main chaperone components from P. furiosus, such as the small heat shock protein (sHSP) or chaperonin (HSP60), was also able to improve the soluble expression of PFA to a certain extent. Co-expression of chaperonin or sHSP in addition to prefoldin did not further increase the soluble expression of PFA. This finding emphasizes the biotechnological potentials of the molecular chaperone prefoldin from P. furiosus, which may facilitate the production of recombinant PFA.

For strain improvement, robust and scalable high-throughput cultivation systems as well as simple and rapid high-throughput detection methods are crucial. However, most of the screening methods for lactic acid bacteria (LAB) strains were conducted in shake flasks and detected by high-performance liquid chromatography (HPLC), making the screening program laborious, time-consuming and costly. In this study, an integrated strategy for high-throughput screening of high l-lactic acid-productivity strains by Bacillus coagulans in deep-well microtiter plates (MTPs) was developed. The good agreement of fermentation results obtained in the MTPs platform with shake flasks confirmed that 24-well U-bottom MTPs could well alternate shake flasks for cell cultivation as a scale-down tool. The high-throughput pH indicator (bromocresol green) and l-lactate oxidase (LOD) assays were subsequently developed to qualitatively and quantitatively analyze l-lactic acid concentration. Together with the color halos method, the pH indicator assay and LOD assay, the newly developed three-step screening strategy has greatly accelerated the screening process for LAB strains with low cost. As a result, two high l-lactic acid-productivity mutants, IH6 and IIIB5, were successfully screened out, which presented, respectively, 42.75 and 46.10 % higher productivities than that of the parent strain in a 5-L bioreactor.

In the production of L-lactic acid, the residual sugar in fermentation broth would lead to increased sugar content in its commercial products, bringing about lactic acid deterioration because of Maillard reactions and thus shortening their shelf life. In this work, the residual sugar components in L-lactic acid industrial fermentation broth with agricultural biomass-derived sugar were determined by means of high-performance anion-exchange chromatography (HPAEC) coupled with pulsed amperometric detection (PAD). A feasible off-line sample clean-up procedure consisting of sodium oxalate extraction, ethanol extraction and ion-exchange column extraction was developed to remove calcium ions, proteins, anion acids and organic acids before analysis, and their removal efficiencies were 100, 98.74, 97.99 and 99.41%, respectively. Under the optimized analytical conditions, the seventeen sugars, including four monosaccharides, eight disaccharides and five oligosaccharides, have been completely separated in one experimental run on a Dionex Carbopac PA 200 column with sodium hydroxide and sodium acetate as eluents. The method was validated, with good linearity (R2 > 0.99), high precision (RSDs < 5.47%), and sensitive detection limits (0.35–44.61 μg L−1); recoveries were >86.95% for short chain carbohydrates (degree of polymerization, DP < 4), while recoveries ranged from 81.87 to 61.41% for DP4 to DP7. The method was successfully applied for analysis of the residual sugar in lactic acid broth with a salt content as high as 120 g L−1 calcium lactate, using different kinds of starch (corn starch, corn flour and cassava starch) hydrolysates as carbon sources. Isomaltose (47.97–60.43%), sucrose (17.50–29.73%) and trehalose (3.05–7.51%) were the main components of residual sugar in these broths.

An industrial mutant of Streptomyces avermitilis produced avermectin at a high level in industrial complex culture medium. However, almost no avermectin was detected in the cultures of tryptone soya broth (TSB). Its wild-type strain could not synthesize avermectin. To elucidate the regulatory mechanism about avermectin production, proteomic analysis of S. avermitilis was carried out. Results showed that during avermectin biosynthesis, fatty acid metabolism and TCA cycle were repressed. Partial enrichment of glycolytic pathway indicated the critical role of glucose catabolism during avermectin biosynthesis. Some enriched enzymes in amino acid metabolic pathways (glnA, leuC) confirmed the leucine as the possible precursor of avermectin. Highly expressed stress or stress-related proteins indicated a global regulation mechanism at the onset of avermectin production. And highly expressed morphology control proteins revealed an association between hyphal morphology and avermectin production. Further, this study proofed strengthened capability to utilize carbon and nitrogen source in the industrial strain. Some stress or stress-related proteins (eshA, clpC, dnaK and grpE) expressed at low level in the industrial strain cultivated in non-production medium (lower than that in the wild-type strain), but these highly expressed at the onset of avermectin production. More sensible response to environmental stress may be responsible for it.To elucidate the regulatory mechanism about avermectin, proteomic comparison of the responses of S. avermitilis during avermectin production was carried out (show as follows).Highlights► Two strains of Streptomyces avermitilis were used for comparison. ► A global protein abundance analysis using 2DE-based proteomics were carried out. ► Significant protein modulation occurred in the comparison group In-P vs In-NP. ► The comparison group In-NP vs W-NP revealed some information about the mutation.

In this study, a new outer-cycle flat-panel photobioreactor was designed for an anaerobic, photo-fermentation process by Rhodobacter sphaeroides ZX-5. In order to obtain the high hydrogen yield, photo-hydrogen production by fed-batch culture with on-line oxidation–reduction potential (ORP) feedback control was investigated. Meanwhile, the effects of feeding malic acid concentration and pH adjustment on the growth and hydrogen production of R. sphaeroides ZX-5 were studied. In the entire fed-batch culture, biomass (i.e., OD660) rapidly increased up to 1.79 within 18 h, and then OD660 value stayed constant within a range of 1.85–2.18 until the end of the photo-fermentation. The cumulative hydrogen volumes in each phase of fed-batch process were 2339, 1439, 1328, and 510 ml H2/l-culture, respectively. Throughout the entire repeated fed-batch photo-fermentation, the maximum substrate conversion efficiency of 73.03% was observed in the first fed-batch process, obviously higher than that obtained from batch culture process (59.81%). In addition, compared to the batch culture, a much higher maximum hydrogen production rate (102.33 ml H2/l h) was achieved during fed-batch culture. The results demonstrated that photo-hydrogen production using fed-batch operation based on ORP feedback control is a favorable choice of sustainable and feasible strategy to improve phototrophic hydrogen production efficiency.Highlights► The fed-batch culture mode based on ORP level has proved to be capable of attaining high-rate and high-yield phototrophic H2 production. ► The feeding time controlled by computer is conducive to future large-scale photo-hydrogen production. ► A new outer-cycle flat-panel photobioreactor was designed for an anaerobic, photo-fermentation process.

The effects of vitamins (nicotinic acid, vitamin B1 and biotin) on the growth and hydrogen production of Rhodobacter sphaeroides ZX-5 were investigated by batch culture in this study. The results showed that nicotinic acid, as a precursor of NAD+/NADH, plays a crucial role in effectively enhancing the phototrophic hydrogen synthesis during photo-fermentation process. Lack of nicotinic acid in hydrogen production medium resulted in the failure of photo-hydrogen production. In addition, though vitamin B1 and biotin do not have direct impact on photo-hydrogen production, they are still essential and must exist in either growth medium or hydrogen production medium. Without either of them, photo-hydrogen production decreased seriously, regardless of the existence of nicotinic acid.Highlights► Vitamins in medium are important factors making effects on photo-H2 production. ► Nicotinic acid plays a crucial role in enhancing the phototrophic hydrogen synthesis. ► Without either of vitamin B1 and biotin, photo-H2 production decreases seriously.

The mitogen-activated protein kinase Hog1 gene (Kmhog1) was isolated from Kluyveromyces marxianus strain NBRC 1777 by degenerate PCR and genome walking, and then disrupted to construct a mutant strain hog1∆. The mutant was now more sensitive to acetic acid and its growth was nearly completely inhibited by 0.5 M NaCl (97%) and 10 mM H2O2 (93%) as compared with the wild-type cells. However, neither strain grew at 47°C. Kmhog1 may thus be required for adaptation to acetic acid, osmotic, and oxidative stress but is not involved in thermotolerance.

4′′-O-isovalerylspiramycins are the major components of bitespiramycin complex consisting of a group of 4′′-O-acylated spiramycins. The availability of isovaleryl group, usually in vivo derived from leucine, one of the branched-chain amino acids, affects the content of isovaleryispiramycin significantly. In this study, the effect of glucose on the activity of branched-chain α-keto acid dehydrogenase (BCKDH), which catalyzed the rate-limiting as well as the first irreversible reaction oxidative decarboxylation for branched-chain amino acids degradation, and isovaleryispiramycin biosynthesis was investigated. In the initial glucose concentration experiment, when the residual glucose concentration in the medium declined to 2–4 g/L, the BCKDH activity rose rapidly, and glucose deprivation and the summit of BCKDH activity appeared nearly at the same time. After a delay of about 6 h, the maximal isovalerylspiramycin content was observed. However, the shortage of glucose at the later production phase resulted in the marked decrease in BCKDH activity and isovaleryispiramycin content. In the fermentation in a 50 L fermentor, glucose feeding at the late production phase helped to maintain the residual glucose concentration between 0 and 1 g/L, leading to the high level of BCKDH activity and thus isovalerylspiramycin content. These suggested that glucose concentration could be used as a key parameter to regulate BCKDH activity and isovaleryispiramycin biosynthesis in the bitespiramycin production.

Steam-exploded corn stover biomass was used as the substrate for fed-batch separate enzymatic hydrolysis and fermentation (SHF) to investigate the solid concentration ranging from 10% to 30% (w/w) on the lignocellulose enzymatic hydrolysis and fermentation. The treatment of washing the steam-exploded material was also evaluated by experiments. The results showed that cellulose conversion changed little with increasing solid concentration, and fermentation by Saccharomyces cerevisiae revealed a nearly same ethanol yield with the water-washed steam-exploded corn stover. For the washed material at 30% substrate concentration, i.e., 30% water insoluble solids (WIS), enzymatic hydrolysis yielded 103.3 g/l glucose solution and a cellulose conversion of 72.5%, thus a high ethanol level up to 49.5 g/l. With the unwashed steam-exploded corn stover, though a cellulose conversion of 70.9% was obtained in hydrolysis at 30% solid concentration (27.9% WIS), its hydrolysate did not ferment at all, and the hydrolysate of 20% solid loading containing 3.3 g/l acetic acid and 145 mg/l furfural already exerted a strong inhibition on the fermentation and ethanol production.

Biohydrogen has gained attention due to its potential as a sustainable alternative to conventional methods for hydrogen production. In this study, the effect of light intensity as well as cultivation method (standing- and shaking-culture) on the cell growth and hydrogen production of Rhodobacter sphaeroides ZX-5 were investigated in 38-ml anaerobic photobioreactor with RCVBN medium. Thus, a novel shaking and extra-light supplementation (SELS) approach was developed to enhance the phototrophic H2 production by R. sphaeroides ZX-5 using malate as the sole carbon source. The optimum illumination condition for shaking-culture by strain ZX-5 increased to 7000–8000 lux, markedly higher than that for standing-culture (4000–5000 lux). Under shaking and elevated illumination (7000–8000 lux), the culture was effective in promoting photo-H2 production, resulting in a 59% and 56% increase of the maximum and average hydrogen production rate, respectively, in comparison with the culture under standing and 4000–5000 lux conditions. The highest hydrogen-producing rate of 165.9 ml H2/l h was observed under the application of SELS approach. To our knowledge, this record is currently the highest hydrogen production rate of non-immobilized purple non-sulphur (PNS) bacteria. This optimal performance of photo-H2 production using SELS approach is a favorable choice of sustainable and economically feasible strategy to improve phototrophic H2 production efficiency.

Bitespiramycin, a group of 4″-O-acylated spiramycins with 4″-O-isovalerylspiramycins as the major components, is produced by recombinant spiramycin-producing strain Streptomyces spiramyceticus harboring a 4″-O-acyltransferase gene from a carbomycin-producing strain S. mycarofaciens 1748. The effects of leucine feeding on the bitespiramycin fermentation, especially the synthesis of isovalerylspiramycin components, were investigated. The experiment was initially performed in flask culture under the condition of feeding 15.4 mmol/l of leucine at 72 h fermentation, and the culture without leucine feeding was used as control. When 15.4 mmol/l leucine was fed at 72 h, 51.3 ± 0.33% total isovalerylspiramycins was recorded compared to 40.9 ± 0.26% under the control condition after 96 h of fermentation. The improvement of total isovalerylspiramycin content was further achieved in 15 l fermentation when 15.4 mmol/l of leucine was supplemented from 65 to 72 h. These results indicated that isovaleryl group derived from leucine catabolism could act as the precursor of the 4″ side chain of bitespiramycin, which profoundly enhanced the synthesis of isovalerylspiramycins in the bitespiramycin complex.

In the production of L-lactic acid, the residual sugar in fermentation broth would lead to increased sugar content in its commercial products, bringing about lactic acid deterioration because of Maillard reactions and thus shortening their shelf life. In this work, the residual sugar components in L-lactic acid industrial fermentation broth with agricultural biomass-derived sugar were determined by means of high-performance anion-exchange chromatography (HPAEC) coupled with pulsed amperometric detection (PAD). A feasible off-line sample clean-up procedure consisting of sodium oxalate extraction, ethanol extraction and ion-exchange column extraction was developed to remove calcium ions, proteins, anion acids and organic acids before analysis, and their removal efficiencies were 100, 98.74, 97.99 and 99.41%, respectively. Under the optimized analytical conditions, the seventeen sugars, including four monosaccharides, eight disaccharides and five oligosaccharides, have been completely separated in one experimental run on a Dionex Carbopac PA 200 column with sodium hydroxide and sodium acetate as eluents. The method was validated, with good linearity (R2 > 0.99), high precision (RSDs < 5.47%), and sensitive detection limits (0.35–44.61 μg L−1); recoveries were >86.95% for short chain carbohydrates (degree of polymerization, DP < 4), while recoveries ranged from 81.87 to 61.41% for DP4 to DP7. The method was successfully applied for analysis of the residual sugar in lactic acid broth with a salt content as high as 120 g L−1 calcium lactate, using different kinds of starch (corn starch, corn flour and cassava starch) hydrolysates as carbon sources. Isomaltose (47.97–60.43%), sucrose (17.50–29.73%) and trehalose (3.05–7.51%) were the main components of residual sugar in these broths.