Improved elastase production by Bacillus sp. EL31410—further optimization and kinetics studies of culture medium for batch fermentation

An efficient culture medium producing a bacterial elastase with high yields was developed further following preliminary studies by means of response surface method. Central composite design (CCD) and response surface method-ology were applied to optimize the medium constituents. A central composite design was used to explain the combined effect of three medium constituents, viz, glucose, K2HPO4, MgSO47H2O. The strain produced more elastase in the completely optimized medium, as compared with the partially optimized medium. The fitted model of the second model, as per RSM, showed that glucose was 7.4 g/100 ml, casein 1.13 g/100 ml, corn steep flour 0.616 g/100 ml, K2HPO4 0.206 g/100 ml and MgSO47H2O 0.034 g/100 ml. The fermentation kinetics of these two culture media in the flask experiments were analyzed. It was found that the highest elastase productivity occurred at 54 hours. Higher glucose concentration had inhibitory effect on elastase production. At the same time, we observed that the glucose consumption rate was slow in the completely optimized medium, which can explain the lag period of the highest elastase production. Some metal ions and surfactant additives also affected elastase production and cell growth.

Establishment of Kinetics Models for Batch Fermentation Process of β-mannase with Bacillus licheniformis HDYM-04

In order to improve the yield of β-mannase and to investigate the rules of fermentation production, a high-yield β-mannase producing strain, Bacillus licheniformis HDYM-04, was used to investigate the kinetics models based on the optimal fermentation conditions： HDYM-04 strain was fermented at 37℃ for 30 h with agitation speed at 300 r/min and aeration rate at 3 L/min in a 5 L fermenter, the initial addition amount of konjac flour was 2%（w/v）, the initial pH of medium was 8.0, and the inoculum concentration was 6.7%（v/v）. Three batch fermentation kinetic models were established （cell growth kinetic model, substrate consumption kinetic model, product formation kinetic model） bases on Logistic and Luedeking-Piret equations. To be specific, cell growth kinetic model was dX/dt =0.431X （1- X/ 15.522 ）, substrate consumption kinetic model was -ds/dt =1.11 dX/dt ＋0.000 2 dP/dt ＋0.000 8X, and product formation kinetic model was dP/dt=133.1 dX ＋222.87X. The correlation coefficients R^2 of the three equations were 0.990 21, 0.989 08 and 0.988 12, respectively, which indicated a good correlation between experimental values and models. Therefore, the three equations could be used to describe the processes of cell growth, enzyme synthesis and substrate consumption during batch fermentation using B. licheniformis strain HDYM-04. The establishment of batch fermentation kinetic models （cell growth kinetic model, substrate depletion kinetic model, product formation kinetic model） could lay the theoretical foundation and provide practical reference for the applica- tion of HDYM-04 in fermentation industry.

Principles and Case Studies of Fed Batch Fermentation and Continuous Fermentation

Modern fermentation processes include a variety of fermentation methods,such as fed batch fermentation and continuous fermentation.This paper will focus on the principles and case studies of these two methods.Fed batch fermentation originates from fractionation fermentation with closed culture and adjustment of the pH value of the carbon source,to which the process of feeding the carbon source to the cell culture in a controlled manner has been added.This type of fermentation is more commonly used compared to the other.Continuous fermentation is also a closed fermentation system,which can operate without restrictions by continuous or intermittent addition of fresh nutrient media to the fermenter;however,it is susceptible to contamination by stray bacteria and metabolic inconvenience.

Mass Transfer, Gas Holdup, and Kinetic Models of Batch and Continuous Fermentation in a Novel Rectangular Dynamic Membrane Airlift Bioreactor

Compared with conventional cylinder airlift bioreactors(CCABs)that produce coarse bubbles,a novel rectangular dynamic membrane airlift bioreactor(RDMAB)developed in our lab produces fine bubbles to enhance the volumetric oxygen mass transfer coefficient(k_(L)a)and gas holdup,as well as improve the bioprocess in a bioreactor.In this study,we compared mass transfer,gas holdup,and batch and con-tinuous fermentation for RNA production in CCAB and RDMAB.In addition,unstructured kinetic models for microbial growth,substrate utilization,and RNA formation were established.In batch fermentation,biomass,RNA yield,and substrate utilization in the RDMAB were higher than those in the CCAB,which indicates that dynamic membrane aeration produced a high k_(L)a by fine bubbles;a higher k_(L)a is more bene-ficial to aerobic fermentation.The starting time of continuous fermentation in the RDMAB was 20 h ear-lier than that in the CCAB,which greatly improved the biological process.During continuous fermentation,maintaining the same dissolved oxygen level and a constant dilution rate,the biomass accumulation and RNA concentration in the RDMAB were 9.71% and 11.15% higher than those in the CCAB,respectively.Finally,the dilution rate of RDMAB was 16.7% higher than that of CCAB during con-tinuous fermentation while maintaining the same air aeration.In summary,RDMAB is more suitable for continuous fermentation processes.Developing new aeration and structural geometry in airlift bioreac-tors to enhance k_(L)a and gas holdup is becoming increasingly important to improve bioprocesses in a bioreactor.

Repeated batch fermentation with water recycling and cell separation for microbial lipid production

Large waste water disposal was the major problem in microbial lipid fermentation because of low yield of lipid. In this study, the repeated batch fermentation was investigated for reducing waste water generated in the lipid fermentation of an oleaginous yeast Trichosporon cutaneum CX1 strain. The waste fermentation broth was recycled in the next batch operation after the cells were separated using two different methods, centrifugation and flocculation. Two different sugar substrates, glucose and inulin, were applied to the proposed operation. The result showed that at least 70% of the waste water was reduced, while lipid production maintained satisfactory in the initial four cycles. Furthermore, it is suggested that T. cutaneum CX1 cells might produce certain naturally occurring inulin hydrolyzing enzyme（s） for obtaining fructose and glucose from inulin directly. Our method provided a practical option for reducing the waste water generated from microbial lipid fermentation.

MULTISTAGE DYNAMIC SYSTEM OF MICROBIAL BATCH FERMENTATION AND ITS PARAMETER IDENTIFICATION

Based on 1,3-propanediol production from batch fermentation of glycerol by Klebsiella pneurnoniae, a multistage dynamic system and its parameter identification are discussed in this paper. The batch fermentation process is divided into three stages exhibiting different dynamic behaviors and characteristics, from which a corresponding nonlinear multistage dynamic system is built. We then propose a parameter identification optimization model whose objective function is the average relative error. The model is solved by particle swarm optimization weighted by inertia, and the result shows that the relative error of our proposed model is 2-10%smaller than those of existing models.

Batch and fed-batch production of butyric acid by Clostridium butyricum ZJUCB

The production of butyric acid by Clostridium butyricum ZJUCB at various pH values was investigated. In order to study the effect of pH on cell growth, butyric acid biosynthesis and reducing sugar consumption, different cultivation pH values ranging from 6.0 to 7.5 were evaluated in 5-L bioreactor. In controlled pH batch fermentation, the optimum pH for cell growth and butyric acid production was 6.5 with a cell yield of 3.65 g/L and butyric acid yield of 12.25 g/L. Based on these results, this study then compared batch and fed-batch fermentation of butyric acid production at pH 6.5. Maximum value (16.74 g/L) of butyric acid concentration was obtained in fed-batch fermentation compared to 12.25 g/L in batch fermentation. It was concluded that culti- vation under fed-batch fermentation mode could enhance butyric acid production significantly (P<0.01) by C. butyricum ZJUCB.

Effect of temperature on batch elastase production by Bacillus sp. EL31410

The production of elastase by Bacillus sp. EL31410 at various temperatures was investigated. In order to study the effect of temperature on elastase fermentation, different cultivation temperatures, ranging from 39 ℃ to 28 ℃, were evaluated in shake flask. The result indicated that 37 ℃ was best for cell growth at earlier stage; while maximum elastase activity was obtained when the cells were cultivated at 30 ℃. This result was verified by batch fermentation in 5-L bio-reactor under 37 ℃ and 30 ℃ temperature, respectively. The specific cell growth rate at 37 ℃ was higher than that at 30 ℃ during earlier stage of cultivation. The maximum value [5.5 U/(h-g DCW)] of elastase formation rate occurred at 24 h at 30 ℃ compared to 4.6 U/(h-g DCW) at 30 h at 37 ℃. Based on these results, two-stage temperature shift strategy and oscillatory temperature cultivation mode were evaluated in the next study. When compared to single temperature of 37 ℃ or 30 ℃, both two-stage temperature shift strategy and oscillatory temperature strategy improved biomass but did not yield the same result as expected for elastase production. The maximum biomass (both 8.6 g/L) was achieved at 30 h at 37 ℃, but at 42 h using two-stage temperature cultivation strategy. The highest elastase production (652 U/ml) was observed at 30 ℃ in batch process. It was concluded that cultivation at constant temperature of 30 ℃ was appropriate for elastase production by Bacillus sp. EL31410.

Learning control of fermentation process with an improved DHP algorithm

Control of the fed-batch ethanol fermentation processes to produce maximum product ethanol is one of the key issues in the bioreactor system.However,ethanol fermentation processes exhibit complex behavior and nonlinear dynamics with respect to the cell mass,substrate,feed-rate,etc.An improved dual heuristic programming algorithm based on the least squares temporal difference with gradient correction(LSTDC) algorithm(LSTDC-DHP) is proposed to solve the learning control problem of a fed-batch ethanol fermentation process.As a new algorithm of adaptive critic designs,LSTDC-DHP is used to realize online learning control of chemical dynamical plants,where LSTDC is commonly employed to approximate the value functions.Application of the LSTDC-DHP algorithm to ethanol fermentation process can realize efficient online learning control in continuous spaces.Simulation results demonstrate the effectiveness of LSTDC-DHP,and show that LSTDC-DHP can obtain the near-optimal feed rate trajectory faster than other-based algorithms.

Effects of urea ammonia pretreatment on the batch anaerobic fermentation efficiency of corn stovers

In order to enhance the biogas production and provide nitrogen sources for the growth of microorganisms,experiments on urea ammonia pretreatment of corn stovers were implemented at(35±1)°C to investigate the effects of urea ammonia pretreatment on the batch anaerobic fermentation efficiency of corn stovers.This study assessed the effects of urea ammonia contents(2%,4%,and 6%)and moisture contents(30%,50%,70%and 90%)on the physical structures of lignocelluloses and the efficiency of biogas production from anaerobic fermentation of corn stovers.The results indicated that the methane production reached 230.31 mL/g VS(volatile solids)at pretreatment with 4%urea ammonia and 70%moisture contents for the batch anaerobic fermentation,which was 26.6%higher than that of the untreated group.The degradation rates of cellulose and hemicellulose were 66.34%and 75.47%after the anaerobic fermentation,respectively,which were about 22.6%and 20.9%higher than that of the untreated group,respectively.Thus,it was concluded that urea ammonia pretreatment can improve the efficiency of biogas production from anaerobic fermentation of corn stovers.

Development of a new cleaner production process for cassava ethanol

A new cleaner production process for cassava ethanol has been developed, in which the thin stillage by-product was treated initially by anaerobic digestion, and the digestate further processed by hydrogen-form cation exchange resin before being recycled as process water to make mash for the next ethanol fermentation batch.Thus wastewater was eliminated and freshwater and energy consumption was significantly reduced. To evaluate the new process, ten consecutive batches of ethanol fermentation and anaerobic digestion at lab scale were carried out. Average ethanol production in the recycling batches was 11.43%(v/v) which was similar to the first batch, where deionized(DI) water was used as process water. The chemical oxygen demand(COD) removal rate reached 98% and the methane yield was 322 ml per gram of COD removed, suggesting an efficient and stable operation of the anaerobic digestion. In conclusion, the application of the new process can contribute to sustainable development of the cassava ethanol industry.

Effect of different gas releasing methods on anaerobic fermentative hydrogen production in batch cultures

Decreasing hydrogen partial pressure can not only increase the activity of the hydrogen enzyme but also decrease the products inhibition, so it is an appropriate method to enhance the fermentative hydrogen production from anaerobic mixed culture. The effect ofbiogas release method on anaerobic fermentative hydrogen production in batch culture system was compared, i.e., Owen method with intermediately release, continuous releasing method, and continuous releasing ＋ CO2 absorbing. The experi- mental results showed that, at 35℃, initial pH 7.0 and glucose concentration of 10 g.L-1, the hydrogen produc- tion was only 28 mL when releasing gas by Owen method, while it increased two times when releasing the biogas continuously. The cumulative hydrogen production could reach 155 mL when carbon dioxide in the gas stream was continuously absorbed by 1 mol.L-1 NaOH. The results showed that acetate was dominated, accounting for 43% in the dissolved fermentation products in Owen method, whereas the butyrate predominated and reached 47%-53% of the total liquid end products when releasing gas continuously. It is concluded that the homoacetogenesis could be suppressed when absorbing CO2 in the gas phase in fermentative hydrogen production system.