Weifeng Cao

β‐poly(l‐malic acid) production by fed‐batch culture of Aureobasidium pullulans ipe‐1 with mixed sugars

β‐poly(l‐malic acid) (PMLA) is a biopolyester, which has attracted growing attention due to its potential applications in medicine and other industries. In this study, the biosynthetic pathway of PMLA and the fermentation strategies with mixed sugars were both investigated to enhance PMLA production by Aureobasidium pullulans ipe‐1. Metabolic intermediates and inhibitors were used to study the biosynthetic pathway of PMLA. It showed that exogenous addition of l‐malic acid, succinic acid, TFA, and avidin had negligible effect on PMLA production, while pyruvic acid and biotin were the inhibitors, indicating that PMLA biosynthesis was probably related to phosphoenolpyruvate via oxaloacetate catalyzed by phosphoenolpyruvate carboxylase. Sucrose was suitable for achieving the highest PMLA concentration, while fructose generated a higher yield of PMLA (PMLA produced per biomass). Furthermore, the fed‐batch culture using fed solution with different sugar mixture for PMLA production was implemented. During the fed‐batch culture with mixed solution, fructose could increase PMLA production. Compared with the batch culture, the feeding with mixed sugar (sucrose and glucose) increased PMLA concentration by 23.9%, up to 63.2 g/L, and the final volume of the broth was increased by 25%. These results provide a good reference for process development and optimization of PMLA production.

High molecular weight beta-poly(L-malic acid) produced by A. pullulans with Ca2+ added repeated batch culture

β-Poly(malic acid) (PMLA) has attracted increasing attentions because of its potential application in medicine and other industries. In this study, the variation of PMLA molecular weight (Mw) in the batch culture and the strategies to enhance PMLA Mw were studied. Adding exogenous Ca(2+) (0.1g/L CaCl2) to the medium caused a significant increase in both PMLA concentration and Mw (11.38% and 26.3%, respectively) when Na2CO3 was used as the neutralizer. The Mw of PMLA during the process of batch culture, which associated with the specific PMLA production per unit cell mass (Yp/x) before glucose was depleted, increased from 12.522 KDa to its maximum 18.693 KDa and then kept decreasing until the end of the culture. Compared with the results in batch culture, Mw increased by 84.4% (up to 19.51 kDa) with a productivity of 1.1 gh(-1)L(-1) when the cells were maintained in exponential growth phase during Ca(2+) added repeated batch culture. The present work provides an efficient approach to obtain superior quality PMLA product with high Mw.

α, ω-Dodecanedioic acid production by Candida viswanathii ipe-1 with co-utilization of wheat straw hydrolysates and n-dodecane

Candida viswanathii ipe-1 was used to produce α, ω-dodecanedioic acid (DC12), which showed capability to ferment xylose and glucose simultaneously, while arabinose utilization was less efficient. A low concentration of furfural enhanced cell growth, and the addition of 4.0g/L sodium acetate largely increased DC12 production. It indicated that detoxification of the wheat straw hydrolysates was not necessary for the biosynthesis of DC12. Based on the promising features of our strain, an efficient process was developed to produce DC12 from co-utilization of wheat straw hydrolysates and n-dodecane. Using this process, 129.7g/L DC12 with a corresponding productivity of 1.13g·L-1·h-1 could be produced, which was increased by 40.0% compared with a sole carbon of glucose. The improved DC12 yield by the co-utilization of wheat straw hydrolysates and n-dodecane using C. viswanathii ipe-1 demonstrates the great potential of using biomass as a feedstock in the production of DC12.

Role of oxygen supply in α, ω-dodecanedioic acid biosynthesis from n-dodecane by Candida viswanathii ipe-1: Effect of stirring speed and aeration

α, ω‐Dodecanedioic acid (DC12) usually serves as a monomer of polyamides or some special nylons. During the biosynthesis, oxygenation cascaded in conversion of hydrophobic n‐dodecane to DC12, while the oxidation of n‐dodecane took place in the intracellular space. Therefore, it was important to investigate the role of oxygen supply on the cell growth and DC12 biosynthesis. It was found that stirring speed and aeration influenced the dissolved oxygen (DO) concentration which in turn affected cell growth as well as DC12 biosynthesis. However, the effect of culture redox potential (Orp) level on DC12 biosynthesis was more significant than that of DO level. For DC12 biosynthesis, the first step was to form the emulsion droplets through the interaction of n‐dodecane and the cell. When the stirring speed was enhanced, slits in the surface layer of the emulsion droplets would be increased. Thus, the substances transportation by water through the slits would be intensified, leading to an enhanced DC12 production. Compared with the batch culture at a lower stirring speed (400 rpm) without culture redox potential (Orp) control, the DC12 concentration was increased by 5 times up to 201.3 g/L with Orp controlled above 0 mV at a higher stirring speed (800 rpm).

Toward understanding the key enzymes involved in β-poly (L-malic acid) biosynthesis by Aureobasidium pullulans ipe-1

β‐poly (L‐malic acid) (PMLA) is a biopolyester which has attracted industrial interest for its potential application in medicine and other industries. A high dissolved oxygen concentration (DO) was beneficial for PMLA production, while the mechanisms of DO in PMLA biosynthesis by Aureobasidium pullulans are still poorly understood. In this work, the amount of PMLA was first compared when A. pullulans ipe‐1 were cultured under a high DO level (70% saturation) and a low DO level (10% saturation). Meanwhile, the key enzymes involved in different pathways of the precursor L‐malic acid biosynthesis were studied. The results revealed that the activities of glucose‐6‐phosphate dehydrogenase (G6PDH) and phosphoenolpyruvate carboxylase (PEPC) were positively correlated with cell growth and PMLA production, while the activities of phosphofructokinases (PFK), pyruvic carboxylase (PC) and citrate synthetase (CS) did no show such correlations. It indicated that the Pentose Phosphate Pathway (PPP) may play a vital role in cell growth and PMLA biosynthesis. Moreover, the precursor L‐malic acid for PMLA biosynthesis was mainly biosynthesized through phosphoenolpyruvic acid (PEP) via oxaloacetate catalyzed by PEPC. It was also found that low concentration of sodium fluoride (NaF) might impel carbon flux flow to the oxaloacetate through PEP, but inhibit the flux to the oxaloacetate via pyruvic acid.

Exploring the potential of lactic acid production from lignocellulosic hydrolysates with various ratios of hexose versus pentose by Bacillus coagulans IPE22

The aim of this study was to investigate the feasibility of utilizing different lignocellulosic hydrolysates with various hexose versus pentose (H:P) ratios to produce lactic acid (LA) from Bacillus coagulans IPE22 by fermentations with single and mixed sugar. In single sugar utilization, glucose tended to promote LA production, and xylose preferred to enhance cell growth. In mixed sugar utilization, glucose and pentose were consumed simultaneously when glucose concentration was lower than 20 g/L, and almost the same concentration of LA (50 g/L) was obtained regardless of the differences of H:P values. Finally, LA production from corn cob hydrolysates (CCH) contained 60 g/L mixed sugar verified the mechanisms found in the fermentations with simulated sugar mixture. Comparing with single glucose utilization, CCH utilization was faster and the yield of LA was not significantly affected. Therefore, the great potential of producing LA with lignocellulosic materials by B. coagulans was proved.

One step open fermentation for lactic acid production from inedible starchy biomass by thermophilic Bacillus coagulans IPE22

The aim of this study was to establish a simplified operational process for lactic acid (LA) production by Bacillus coagulans IPE22 from inedible starchy biomass with open fermentation method. First, 29.47 mU/mg specific amylase activity was detected in direct batch fermentation from soluble starch, but the activity of the produced amylase was too low for effective production of LA. Then seven batches from 72 g/L soluble starch were conducted without sterilization. It was found that one step simultaneous liquefaction, saccharification and fermentation (SLSF) with the addition of mesothermal α-amylase and glucoamylase was the optimal mode with LA concentration, yield and productivity of 68.72 g/L, 0.99 g/g and 1.72 g/L h respectively. Finally, inedible starchy biomass, cassava and sorghum flours, were proved to be alternatives to refined soluble starch. For the first time, one step open SLSF of inedible starchy biomass was reported for LA production by B. coagulans.

Improving α, ω-dodecanedioic acid productivity from n-dodecane and hydrolysate of Candida cells by membrane integrated repeated batch fermentation

The aim of the present study is to develop an effective production process for α, ω-dodecanedioic acid (DC12) biosynthesis using n-dodecane and hydrolysate of Candida cells as substrates by membrane integrated repeated batch fermentation. Cells and n-dodecane were simultaneously recycled during the filtration of fermentation broth (FB) with a 150 kDa ceramic membrane under a cross-flow velocity of 4 m/s and a trans-membrane pressure of 0.2 MPa, and it was also revealed that the cells in the broth could alleviate the membrane fouling during the FB filtration. Moreover, the hydrolysate of the collected cells could be successfully used as a nitrogen source to replace 50% yeast extract for decreasing the DC12 production cost. With repeated-batch culture in a membrane bioreactor, the maximal DC12 productivity could be enhanced by 57.8% compared with the batch culture, meanwhile n-dodecane and cells could be recovered and used for the next fermentation cycle.

Effectively converting carbon dioxide into succinic acid under mild pressure with Actinobacillus succinogenes by an integrated fermentation and membrane separation process

The aim of the present study is to develop an effective bioprocess for converting CO2 into succinic acid (SA) with Actinobacillus succinogenes by an integrated fermentation and membrane separation process. CO2 could be effectively converted into SA using NaOH as the neutralizer under the completely closed exhaust pipe case with self-circulation of CO2 in the bioreactor. Meanwhile, the optimal CO2 partial pressure was 0.4 bar. In addition, a 300 kDa ultrafiltration (UF) membrane was preferred for constructing the membrane bioreactor. Moreover, a high conductivity was toxic to the cells during SA biosynthesis. After removing the high concentration salts by in-situ membrane filtration, the SA productivity and CO2 fixation rate increased by 39.2% compared with the batch culture, reaching 1.39 g·L-1·h-1 and 0.52 g·L-1·h-1 respectively. Furthermore, nanofiltration (NF) was suitable for purifying the SA and recovering the residual substrates in the UF permeate for the next fermentation.

Succinic acid biosynthesis from cane molasses under low pH by Actinobacillus succinogenes immobilized in luffa sponge matrices

Succinic acid (SA) production by Actinobacillus succinogenes 130Z using cane molasses as a low cost carbon source was developed. With molasses pretreated by 150 kDa membrane, the highest SA concentration (45.6 g/L), productivity (1.27 g/L·h) and yield (0.76 g SA/g sugars) were obtained under an optimal pH 6.4, which were increased by 1.04 folds compared to those with model sugar mixture due to the effect of vitamins in molasses. Meanwhile, the ratio of sugars in the cane molasses had little effect on SA production. To further enhance SA productivity, the cells were immobilized in luffa sponge matrices (LSM), and repeated batch cultures were carried out for 5 cycles, demonstrating a stable and reliable long-term performance. Compared with the batch culture, the SA productivity enhanced by 49.6% in the LSM system with repeated batch culture. These results suggest that the cell immobilization approach is promising for industrial applications.