Hongji Zhu

Conversion of spent mushroom substrate to biofertilizer using a stress-tolerant phosphate-solubilizing Pichia farinose FL7.

To develop high-efficient biofertilizer, an environmental stress-tolerant phosphate-solubilizing microorganism (PSM) was isolated from agricultural wastes compost, and then applied to spent mushroom substrate (SMS). The isolate FL7 was identified as Pichia farinose with resistance against multiple environmental stresses, including 5-45°C temperature, 3-10 pH range, 0-23% (w/v) NaCl and 0-6M ammonium ion. Under the optimized cultivation condition, 852.8 mg/l total organic acids can be produced and pH can be reduced to 3.8 after 60 h, meanwhile, the soluble phosphate content reached 816.16 mg/l. The P. farinose was used to convert SMS to a phosphate biofertilizer through a semi-solid fermentation (SSF) process. After fermentation of 10 days, cell density can be increased to 5.6 × 10(8)CFU/g in biomass and pH in this medium can be decreased to 4.0. SMS biofertilizer produced by P. farinose significantly improved the growth of soybean in pot experiments, demonstrating a tremendous potential in agricultural application.

Combined alkali and acid pretreatment of spent mushroom substrate for reducing sugar and biofertilizer production.

Spent mushroom substrate (SMS) was pretreated with alkaline reagents including potassium hydroxide, lime and ammonia to enhance enzymatic saccharification. Under the best pretreatment conditions (1M KOH, 80 °C, 90 min; 1M lime, 80 °C, 120 min; 10 M ammonia, 70 °C, 120 min), the total reducing sugar (TRS) yield reached 258.6, 204.2 and 251.2 mg/g raw SMS, which were respectively 6.15, 4.86, and 5.98 times of untreated SMS. The effects of pretreatment by above alkaline reagents and sulfuric acid on the composition and structure of SMS were evaluated to provide comparative performance data. A new process, combined alkali and acid (CAA) pretreatment followed by enzymatic hydrolysis, was innovatively proposed to improve the cost-effectiveness and avoid environmental problems. The SMS residue after CAA pretreatment-enzymatic hydrolysis process was converted to biofertilizer with Pichia farinose FL7 and a cell density of 3.0×10(8) cfu/g in biomass was attained.

Production of spent mushroom substrate hydrolysates useful for cultivation of Lactococcus lactis by dilute sulfuric acid, cellulase and xylanase treatment

Spent mushroom substrate (SMS) was treated with dilute sulfuric acid followed by cellulase and xylanase treatment to produce hydrolysates that could be used as the basis for media for the production of value added products. A L9 (3(4)) orthogonal experiment was performed to optimize the acid treatment process. Pretreatment with 6% (w/w) dilute sulfuric acid at 120°C for 120 min provided the highest reducing sugar yield of 267.57 g/kg SMS. No furfural was detected in the hydrolysates. Exposure to 20PFU of cellulase and 200 XU of xylanase per gram of pretreated SMS at 40°C resulted in the release of 79.85 g/kg or reducing sugars per kg acid pretreated SMS. The dilute sulfuric acid could be recycled to process fresh SMS four times. SMS hydrolysates neutralized with ammonium hydroxide, sodium hydroxide, or calcium hydroxide could be used as the carbon source for cultivation of Lactococcus lactis subsp. lactis W28 and a cell density of 2.9×10(11)CFU/mL could be obtained. The results provide a foundation for the development of value-added products based on SMS.

Improving xylose utilization of defatted rice bran for nisin production by overexpression of a xylose transcriptional regulator in Lactococcus lactis

Present investigation explores the potential of defatted rice bran (DRB) serving as sole carbon source and partial nitrogen source to support Lactococcus lactis growth and nisin production. To retain the nutrients in DRB, especially protein fractions, thermal pretreatment followed by enzymatic hydrolysis without washing step was applied for saccharification. A maximum of 45.64g reducing sugar mainly containing 30.26g glucose and 5.66g xylose from 100g DRB was attained in hydrolysates of DRB (HD). A novel strategy of xylR (xylose transcriptional regulator) overexpression followed by evolutionary engineering was proposed, which significantly increased the capacity of L. lactis to metabolize xylose. Subsequently, RT-PCR results indicated that xylR overexpression stimulated expression of xylose assimilation genes synergistically with exposure to xylose. In HD medium, the highest nisin titer of the engineered strain FEXR was 3824.53IU/mL, which was 1.37 times of that in sucrose medium by the original strain F44.

Preparation, characterization and antioxidant activity of polysaccharide from spent Lentinus edodes substrate

This study explored the potential of spent Lentinus edodes substrate, a by-product of mushroom industries causing environmental pollution, serving as materials to produce antioxidant polysaccharide. The extraction process of spent Lentinus edodes substrate polysaccharide (SLSP) was optimized and the effects of drying methods on chemical composition, morphological property and antioxidant activity were investigated. Results showed that freeze-dried SLSP (SLSP-F) exhibited the best quality in terms of the polysaccharide yield (13.00%) and antioxidant activity. The EC50 values of SLSP-F on DPPH, ABTS and superoxide anion radicals was 0.051mg/mL, 0.379mg/mL, 0.719mg/mL, respectively, which was significantly lower than that of freeze-dried Lentinus edodes polysaccharide (LP-F). After purification by Sephadex G-150, the purified SLSP-F (PSP) has a molecular weight of 16.77kDa. Compared with LP-F, PSP has more reducing sugars and uronic acids in chemical composition and higher contents of xylose, glucose and galactose in monosaccharide composition. FT-IR and NMR spectroscopy analysis revealed that PSP has both α and β glycosidic bonds and massive acetyl groups, which is different from LP-F mainly composed of 1, 3 linked α-D-Manp residue with some acetyl groups. The findings provided a reliable approach for the development of antioxidant polysaccharide from spent Lentinus edodes substrate.

Improving nitrogen source utilization from defatted soybean meal for nisin production by enhancing proteolytic function of Lactococcus lactis F44

Nisin, one kind of natural antimicrobial peptide, is produced by certain Lactococcus lactis strains, which generally require expensive high-quality nitrogen sources due to limited ability of amino acids biosynthesis. Here we use defatted soybean meal (DSM) as sole nitrogen source to support L. lactis growth and nisin production. DSM medium composition and fermentation conditions were optimized using the methods of Plackett-Burman design and central composite design. The highest nisin production of 3879.58 IU/ml was obtained in DSM medium, which was 21.3% higher than that of commercial medium. To further increase the utilization ability of nitrogen sources, we enhanced the proteolytic function in L. lactis through rationally expressing the related enzymes, which were selected according to the compositions of amino acids and molecular weight of peptides in DSM medium. Significantly, an artificial proteolytic system consisting of a heterologous protease (NprB), an oligopeptides transporter subunit (OppA) and two peptidases (PepF and PepM) was introduced into L.lactis. The constructed strain BAFM was capable of achieving efficient biomass accumulation and nisin yield with 30% decreased amount of DSM hydrolysates, which further reduced the cost of nisin production. The strategy described here offers opportunities for low-cost L. lactis fermentation and large-scale nisin production in industry.

Contribution of YthA, a PspC Family Transcriptional Regulator of Lactococcus lactis F44 Acid Tolerance and Nisin Yield: a Transcriptomic Approach

ABSTRACT To overcome the adverse impacts of environmental stresses during growth, different adaptive regulation mechanisms can be activated in Lactococcus lactis. In this study, the transcription levels of eight transcriptional regulators of L. lactis subsp. lactis F44 under acid stress were analyzed using quantitative reverse transcription-PCR. Eight gene-overexpressing strains were then constructed to examine their influences on acid-resistant capability. Overexpressing ythA, a PspC family transcriptional regulator, increased the survival rate by 3.2-fold compared to the control at the lethal pH 3.0 acid shock. Moreover, the nisin yield was increased by 45.50%. The ythA-overexpressing strain FythA appeared to have higher intracellular pH stability and nisin-resistant ability. Subsequently, transcriptome analysis revealed that the vast majority of genes associated with amino acid biosynthesis, including arginine, serine, phenylalanine, and tyrosine, were predominantly upregulated in FythA. Arginine biosynthesis (argG and argH), arginine deiminase pathway, and polar amino acid transport (ysfE and ysfF) were proposed to be the main regulation mechanisms of YthA. Furthermore, the transcription of genes associated with pyrimidine and exopolysaccharide biosynthesis were upregulated. The transcriptional levels of nisIPRKFEG genes were substantially higher in FythA, which directly contributed to the yield and resistance of nisin. Three potential DNA-binding sequences were predicted by computer analysis using the upstream regions of genes with prominent changes. This study showed that YthA could increase acid resistance and nisin yield and revealed a putative regulation mechanism of YthA. IMPORTANCE Nisin, produced by Lactococcus lactis subsp. lactis, is widely used as a safe food preservative. Acid stress becomes the primary restrictive factor of cell growth and nisin yield. In this research, we found that the transcriptional regulator YthA was conducive to enhancing the acid resistance of L. lactis F44. Overexpressing ythA could significantly improve the survival rate and nisin yield. The stability of intracellular pH and nisin resistance were also increased. Transcriptome analysis showed that nisin immunity and the biosynthesis of some amino acids, pyrimidine, and exopolysaccharides were enhanced in the engineered strain. This study elucidates the regulation mechanism of YthA and provides a novel strategy for constructing robust industrial L. lactis strains.

Two-stage carbon distribution and cofactor generation for improving l-threonine production of Escherichia coli: LIU et al.

L‐Threonine, a kind of essential amino acid, has numerous applications in food, pharmaceutical, and aquaculture industries. Fermentative l‐threonine production from glucose has been achieved in Escherichia coli. However, there are still several limiting factors hindering further improvement of l‐threonine productivity, such as the conflict between cell growth and production, byproduct accumulation, and insufficient availability of cofactors (adenosine triphosphate, NADH, and NADPH). Here, a metabolic modification strategy of two‐stage carbon distribution and cofactor generation was proposed to address the above challenges in E. coli THRD, an l‐threonine producing strain. The glycolytic fluxes towards tricarboxylic acid cycle were increased in growth stage through heterologous expression of pyruvate carboxylase, phosphoenolpyruvate carboxykinase, and citrate synthase, leading to improved glucose utilization and growth performance. In the production stage, the carbon flux was redirected into l‐threonine synthetic pathway via a synthetic genetic circuit. Meanwhile, to sustain the transaminase reaction for l‐threonine production, we developed an l‐glutamate and NADPH generation system through overexpression of glutamate dehydrogenase, formate dehydrogenase, and pyridine nucleotide transhydrogenase. This strategy not only exhibited 2.02‐ and 1.21‐fold increase in l‐threonine production in shake flask and bioreactor fermentation, respectively, but had potential to be applied in the production of many other desired oxaloacetate derivatives, especially those involving cofactor reactions.