Zongqi Xu

Enhanced poly(γ-glutamic acid) fermentation by Bacillus subtilis NX-2 immobilized in an aerobic plant fibrous-bed bioreactor

To enhance poly(γ-glutamic acid) (PGA) production, a novel aerobic plant fibrous-bed bioreactor (APFB) was constructed for immobilized fermentation. Based on the analysis of the kinetics of immobilized-cell fermentation using the APFB and conventional free-cell fermentation, immobilized-cell fermentation exhibited more efficient PGA production. Furthermore, repeated fed-batch cultures for PGA production were conducted to evaluate the stability of the APFB system. Average final PGA concentration and productivity of 71.21±0.83g/L and 1.246±0.008g/L/h were respectively achieved by cells immobilized in bagasse during APFB, which was reused eight times over a period of 457±18h. Analysis of the membrane phospholipids and the key enzyme activities indicated that APFB-adapted cells had better productivity than original cells. Thus, this study demonstrated the significant potential of the APFB culture system in future industrial applications.

Conversion of agroindustrial residues for high poly(γ-glutamic acid) production by Bacillus subtilis NX-2 via solid-state fermentation.

Poly(γ-glutamic acid) (γ-PGA) production by Bacillus subtilis NX-2 was carried out through solid-state fermentation with dry mushroom residues (DMR) and monosodium glutamate production residues (MGPR; a substitute of glutamate) for the first time. Dry shiitake mushroom residue (DSMR) was found to be the most suitable solid substrate among these DMRs; the optimal DSMR-to-MGPR ratio was optimized as 12:8. To increase γ-PGA production, industrial waste glycerol was added as a carbon source supplement to the solid-state medium. As a result, γ-PGA production increased by 34.8%. The batch fermentation obtained an outcome of 115.6 g kg(-1) γ-PGA and 39.5×10(8) colony forming units g(-1) cells. Furthermore, a satisfactory yield of 107.7 g kg(-1) γ-PGA was achieved by compost experiment on a scale of 50 kg in open air, indicating that economically large-scale γ-PGA production was feasible. Therefore, this study provided a novel method to produce γ-PGA from abundant and low-cost agroindustrial residues.

Calcium involved in the poly(γ-glutamic acid)-mediated promotion of Chinese cabbage nitrogen metabolism.

Plant growth can reportedly be promoted by poly(γ-glutamic acid) (γ-PGA). However, the underlying mechanism is unknown. To reveal the mechanism of γ-PGA, we designed an experiment that investigated the effect of γ-PGA on the nitrogen metabolism of Chinese cabbage hydroponic cultured at different calcium (Ca) levels and varied exogenous Ca(2+) inhibitors. The results showed that nitrate reductase (NR), glutamine synthetase (GS), glutamate synthase, and glutamate dehydrogenase activities in leaves and roots were obviously enhanced by γ-PGA at the normal Ca(2+) level (4.0 mM). Meanwhile, γ-PGA increased the content of total nitrogen, soluble protein, and soluble amino acids in leaves. However, the promotional effect of γ-PGA on fresh weight weakened when Ca(2+) was inadequate. Moreover, γ-PGA not only induced the influx of extracellular Ca(2+) and Ca(2+) in organelles into cytoplasm, but also increased the Ca(2+)-ATPase level to modify Ca(2+) homeostasis in plant cells. In addition, exogenous Ca(2+) inhibitors significantly suppressed the γ-PGA-mediated promotion of cytoplasmic free Ca(2+) level, calmodulin (CaM) content, GS and glutamate dehydrogenase activities. In summary, γ-PGA accelerated the nitrogen metabolism of plants through the Ca(2+)/CaM signaling pathway, thereby improving the growth of the plant.

Highly efficient rice straw utilization for poly-(γ-glutamic acid) production by Bacillus subtilis NX-2

Lignocellulosic biomass has been identified as an economic and environmental feedstock for future biotechnological production. Here, for the first time, poly-(γ-glutamic acid) (PGA) production by Bacillus subtilis NX-2 using rice straw is investigated. Based on two-stage hydrolysis and characteristic consumption of xylose and glucose by B. subtilis NX-2, a co-fermentation strategy was designed to better accumulate PGA in a 7.5L fermentor by two feeding methods. The maximum cumulative respective PGA production and PGA productivity were 73.0 ± 0.5 g L(-1) and 0.81 g L(-1) h(-1) by the continuous feeding method, with carbon source cost was saved by 84.2% and 42.5% compared with glucose and cane molasse, respectively. These results suggest that rice straw, a type of abundant, low-cost, non-food lignocellulosic feedstock, may be feasibly and efficiently utilized for industrial-scale production of PGA.

Economic process to co-produce poly(ε-L-lysine) and poly(L-diaminopropionic acid) by a pH and dissolved oxygen control strategy.

This study tended to apply biorefinery of indigenous microbes to the fermentation of target-product generation through a novel control strategy. A novel strategy for co-producing two valuable homopoly(amino acid)s, poly(ε-l-lysine) (ε-PL) and poly(l-diaminopropionic acid) (PDAP), was developed by controlling pH and dissolved oxygen concentrations in Streptomyces albulus PD-1 fermentation. The production of ε-PL and PDAP got 29.4 and 9.6gL(-1), respectively, via fed-batch cultivation in a 5L bioreactor. What is more, the highest production yield (21.8%) of similar production systems was achieved by using this novel strategy. To consider the economic-feasibility, large-scale production in a 1t fermentor was also implemented, which would increase the gross profit of 54,243.5USD from one fed-batch bioprocess. This type of fermentation, which produces multiple commercial products from a unified process is attractive, because it will improve the utilization rate of raw materials, enhance production value and enrich product variety.

Effect of poly(γ-glutamic acid) on microbial community and nitrogen pools of soil

Given the numerous environmental problems caused by chemical fertilizer overuse, agricultural practices are shifting toward the development of environmentally friendly nitrogen (N) fertilizers. In this study, a pot experiment was conducted to investigate the effect of poly(γ-glutamic acid) (γ-PGA), an effective fertilizer synergist, on rapeseed (Brassica napus cv. Qinyou No. 9) productivity, soil N pools, and soil enzymes. Seven treatments were implemented: T1, a check without urea; T2, a check with urea only; T3, urea mixed with glutamic acid; T4, urea mixed with 3 mg of γ-PGA per kilogram of soil; T5, urea mixed with 10 mg of γ-PGA per kilogram of soil; T6, urea coated with γ-PGA (0.9%, m/m); and T7, urea coated with γ-PGA (3.1%, m/m). The results demonstrated that the application of γ-PGA increased the above-ground fresh weight of rapeseed by 7.57–9.26% and the grain yield per pot by 6.46–10.98% compared with T2. The rapeseed grain yield significantly increased from 15.94 ± 0.55 g pot−1 in T2 to 17.69 ± 0.78 g pot−1 in T7. With the increase in pod numbers and seed numbers per pod, the seed yield was significantly improved after γ-PGA application. Culturable soil microbial count and microbial diversity were increased by γ-PGA application. In addition, the effects of application method of γ-PGA have a more important role on the soil microflora than its amount. The reduced loss of N can be attributed to the capacity of γ-PGA to increase the “ turnover pool.” More mineral N in the soil was immobilized by clays, soil microbes, and γ-PGA at the early growth stage, and the immobilized N was then released at the later growth stage for plant growth. Moreover, urease activity also increased with γ-PGA application. These findings indicated that the urea coated with γ-PGA could be a better choice for agricultural applications.

Improvement of poly-γ-glutamic acid biosynthesis in a moving bed biofilm reactor by Bacillus subtilis NX-2.

The production of poly-γ-glutamic acid (γ-PGA) by Bacillus subtilis NX-2 using a moving bed biofilm reactor (MBBR) system was tested for the first time in this study. Polypropylene TL-2 was chosen as a suitable carrier, and γ-PGA concentration of 42.7±0.86g/L and productivity of 0.59±0.06g/(Lh) were obtained in batch fermentation. After application of the strategy of dissolved oxygen (DO)-stat feeding, higher γ-PGA concentration and productivity were achieved than with glucose feedback feeding. Finally, the repeated fed-batch cultures implemented in the MBBR system showed high stability, and the maximal γ-PGA concentration and productivity of 74.2g/L and 1.24g/(Lh) were achieved, respectively. In addition, the promotion of oxygen transfer by an MBBR carrier was well explained by a computational fluid dynamics (CFD) simulation. These results suggest that an MBBR system could be applied to large-scale γ-PGA production.

Development of Jerusalem artichoke resource for efficient one-step fermentation of poly-(γ-glutamic acid) using a novel strain Bacillus amyloliquefaciens NX-2S

This study aimed to develop non-food fermentation for the cost-effective production of poly-(γ-glutamic acid) (γ-PGA) using a novel strain of Bacillus amyloliquefaciens NX-2S. The new isolate assimilated inulin more efficiently than other carbohydrates from Jerusalem artichoke, without hydrolytic treatment. To investigate the effect of inulin on γ-PGA production, the transcript levels of γ-PGA synthetase genes (pgsB, pgsC, pgsA), regulatory genes (comA, degQ, degS), and the glutamic acid biosynthesis gene (glnA) were analyzed; inulin addition upregulated these key genes. Without exogenous glutamate, strain NX-2S could produce 6.85±0.22g/L of γ-PGA during fermentation. Exogenous glutamate greatly enhances the γ-PGA yield (39.4±0.38g/L) and productivity (0.43±0.05g/L/h) in batch fermentation. Our study revealed a potential method of non-food fermentation to produce high-value products.

Systematic unravelling of the biosynthesis of poly (L-diaminopropionic acid) in Streptomyces albulus PD-1.

Poly(L-diaminopropionic acid) (PDAP) is one of the four homopoly(amino acid)s that have been discovered in nature. However, the molecular mechanism of PDAP biosynthesis has yet to be described. In this work, the general layout of the PDAP biosynthetic pathway is characterised in Streptomyces albulus PD-1 by genome mining, gene disruption, heterologous expression and in vitro feeding experiments. As a result, L-diaminopropionic acid (L-DAP), which is the monomer of PDAP, is shown to be jointly synthesised by two protein homologues of cysteine synthetase and ornithine cyclodeaminase. Then, L-DAP is assembled into PDAP by a novel nonribosomal peptide synthetase (NRPS) with classical adenylation and peptidyl carrier protein domains. However, instead of the traditional condensation or thioesterase domain of NRPSs, this NRPS has seven transmembrane domains surrounding three tandem soluble domains at the C-terminus. As far as we know, this novel single-module NRPS structure has only been reported in poly(ε-L-lysine) synthetase. The similar NRPS structure of PDAP synthetase and poly(ε-L-lysine) synthetase may be a common characteristic of homopoly(amino acid)s synthetases. In this case, we may discover and/or design more homopoly(amino acid)s by mining this kind of novel NRPS structure in the future.

The microbe-secreted isopeptide poly-γ-glutamic acid induces stress tolerance in Brassica napus L. seedlings by activating crosstalk between H 2 O 2 and Ca 2+

Poly-γ-glutamic acid (γ-PGA) is a microbe-secreted isopeptide that has been shown to promote growth and enhance stress tolerance in crops. However, its site of action and downstream signaling pathways are still unknown. In this study, we investigated γ-PGA-induced tolerance to salt and cold stresses in Brassica napus L. seedlings. Fluorescent labeling of γ-PGA was used to locate the site of its activity in root protoplasts. The relationship between γ-PGA-induced stress tolerance and two signal molecules, H2O2 and Ca2+, as well as the γ-PGA-elicited signaling pathway at the whole plant level, were explored. Fluorescent labeling showed that γ-PGA did not enter the cytoplasm but instead attached to the surface of root protoplasm. Here, it triggered a burst of H2O2 in roots by enhancing the transcription of RbohD and RbohF, and the elicited H2O2 further activated an influx of Ca2+ into root cells. Ca2+ signaling was transmitted via the stem from roots to leaves, where it elicited a fresh burst of H2O2, thus promoting plant growth and enhancing stress tolerance. On the basis of these observation, we propose that γ-PGA mediates stress tolerance in Brassica napus seedlings by activating an H2O2 burst and subsequent crosstalk between H2O2 and Ca2+ signaling.