Lipase catalysed hydrolysis of ricebran oil by free and immobilised enzyme system in batch stirred reactor

A change of the reaction rate was observed for the lipasecatalysed hydrolysis of ricebran oil in a batch stirred tank reactor using immobilized lipase enzyme as compared to free enzyme. The reactor rate was observed to be controlled mainly by factors like temperature, pH, initial enzyme concentration, initial substrate concentration and initial products concentration.     

初始pH值对产氢产乙酸/耗氢产乙酸两段耦合工艺定向生产乙酸的影响

以葡萄糖为底物,以经加热预处理并活化过的厌氧污泥为种泥,研究了初始pH值对产氢产乙酸/耗氢产乙酸两段耦合工艺厌氧发酵定向生产乙酸的影响。实验考察了7个初始pH值(5、6、7、8、9、10、11)条件下的底物降解、产物产生和发酵过程pH值的变化。结果表明:产氢产乙酸段初始pH值的变化不仅影响本阶段产酸,而且影响耗氢产乙酸段产酸。初始pH=5时主要进行乙醇型发酵;pH=6和7时主要进行丁酸型发酵;pH=8时混合酸型发酵类型逐渐占优势,pH=8~11时均以乙酸为主要产物,耦合系统生产乙酸最优初始pH值为10。在初始pH=8~11范围内,产氢产乙酸段初期的乙醇浓度一般较高,但到后期因乙醇被微生物进一步代谢转化成乙酸而使其含量下降。     

The production of ethanol by immobilized yeast cells

Saccharomyces cerevisiae cells were immobilized in calcium alginate beads for use in the continuous production of ethanol. Yeasts were grown in medium supplemented with ethanol to selectively screen for a culture which showed the greatest tolerance to ethanol inhibition. Yeast beads were produced from a yeast slurry containing 1.5% alginate (w/v) which was added as drops to 0.05M CaCl₂ solution. To determine their optimum fermentation parameters, ethanol production using glucose as a substrate was monitored in batch systems at varying physiological conditions (temperature, pH, ethanol concentration), cell densities, and gel concentration. The data obtained were compared to optimum free cell ethanol fermentation parameters. The immobilized yeast cells examined in a packed-bed reactor system operated under optimized parameters derived from batch-immobilized yeast cell experiments. Ethanol production rates, as well as residual sugar concentration were monitored at different feedstock flow rates.     

The effect of pH and temperature manipulation on metabolite composition during acidogenesis in a hybrid anaerobic digester

Summary A synthetic medium containing 9 g/l sucrose was hydrolyzed in a novel hybrid reactor. A minimum hydraulic retention time (HRT) of 9.9 h, with a gas production rate of 1.07 m³/m³·d, was obtained without continuous neutralization. A viable anaerobic cell count of 10⁹ organisms/ml was obtained in the reactor fluid. The results showed that both pH and temperature significantly influenced the type and concentration of the various metabolites formed. These include ethanol, formic, acetic, propionic and butyric acids as primary metabolites and caproic acid as secondary metabolite. From the results obtained, it is suggested that to obtain the energetically most favourable products, a substrate pH of 6.5 and a temperature of 35°C must be used in anaerobic acidogenic digesters.     

Ethanol production from xylose by Fusarium oxysporum and the optimization of culture conditions

Bioethanol is the most commonly used renewable biofuel as an alternative to fossil fuels. Many microbial strains can convert lignocellulosics into bioethanol. However, very few natural strains with a high capability of fermenting pentose sugars and simultaneously utilizing various sugars have been reported. In this study, fermentation of sugar by Fusarium oxysporum G was performed for the production of ethanol to improve the performance of the fermentation process. The influences of pH, substrate concentration, temperature, and rotation speed on ethanol fermentation are investigated. The three significant factors (pH, substrate concentration, and temperature) are further optimized by quadratic orthogonal rotation regression combination design and response surface methodology (RSM). The optimum conditions are pH 4, 40?g/L of xylose, 32?°C, and 110?rpm obtained through single factor experiment design. Finally, it is found that the maximum ethanol production (10.0?g/L) can be achieved after 7 d of fermentation under conditions of pH 3.87, 45.2?g/L of xylose, and 30.4?°C. Glucose is utilized preferentially for the glucose–xylose mixture during the initial fermentation stage, but glucose and xylose are synchronously consumed without preference in the second period. These findings are significant for the potential industrial application of this strain for bioethanol production.     

Evaluation of the feasibility of alcohols serving as external carbon sources for biological phosphorus removal induced by the oxic/extended‐idle regime

Recently, a novel operational regime (i.e., the oxic/extended‐idle [OEI] regime) has been reported to successfully achieve enhanced biological phosphorus removal (EBPR) when employing glucose and volatile fatty acids as the sole substrate. In the OEI regime, polyphosphate accumulating organisms (PAOs) could get a selective advantage over other populations during the extended‐idle period where polyphosphate released but polyhydroxyalkanoates and glycogen transformations were negligible/low, thus energy requirements for maintenance purposes in the period could be covered by polyphosphate release. This study further evaluated the feasibility of alcohols as external carbon sources for EBPR induced by the OEI regime, as the available substrate in the raw wastewater is often deficient. First, phosphorus removal in the OEI process was compared, respectively, with methanol and ethanol as the sole substrate. The results showed that the ethanol‐reactor achieved 90.8 ± 2.3% of phosphorus removal, which was approximate twofold than the methanol‐reactor. Further studies displayed that the cells in the ethanol‐reactor contained more PAOs, and had higher activities of exopolyphosphatase and polyphosphate kinase than those in the methanol‐reactor. Also, the aerobic transformations of polyhydroxyalkanoates and glycogen in the ethanol‐reactor were, respectively, higher and lower than those in the methanol‐reactor, which were consistent with the reactors performances. Then, the feasibility of using ethanol as external substrate to enhance EBPR in the OEI process was confirmed for a municipal wastewater. Finally, EBPR performance and metabolic transformation values between the OEI and the anaerobic/oxic (A/O) regimes with ethanol as the sole substrate were compared. The results showed that EBPR in the ethanol‐OEI reactor was higher than that in the ethanol‐A/O reactor. All the above results proved that ethanol was a favorable external substrate to the OEI regime for EBPR enhancement. Biotechnol. Bioeng. 2013; 110: 827–837. © 2012 Wiley Periodicals, Inc.     

Optimization of the production of aroma compounds by Kluyveromyces marxianus in solid-state fermentation using factorial design and response surface methodology

Studies were carried out for the production of aroma compounds in solid-state fermentation using factorial design and response surface methodology (RSM) experiments. Five agro-industrial residues were evaluated as substrate for cultivating a strain of Kluyveromyces marxianus. The results proved the feasibility of using cassava bagasse and giant palm bran (Opuntia ficus indica) as substrates to produce fruity aroma compounds by the yeast culture. In order to test the influence of the process parameters on the culture to produce volatile compounds, two statistical experimental designs were performed. The parameters studied were initial substrate pH, addition of glucose, cultivation temperature, initial substrate moisture and inoculum size. Using a 2(5) factorial design, addition of glucose and initial pH of the substrate was found statistically significant for aroma compounds production on palm bran. Although this experimental design showed that addition of glucose did not have a significant role with cassava bagasse, 2(2) factorial design revealed that glucose addition was significant at higher concentrations. Head-space analysis of the culture by gas chromatography showed the production of nine and eleven compounds from palm bran and cassava bagasse, respectively, which included alcohols, esters and aldehyde. In both the cases, two compounds remained unidentified and ethyl acetate, ethanol and acetaldehyde were the major compounds produced. Esters produced were responsible for the fruity aroma in both the cases. With palm bran, ethanol was the compound produced in highest concentration, and with cassava bagasse (both supplemented with 10% glucose), ethyl acetate was produced at highest concentration, accumulating 418 and 1395μmoll(-1) head-spaceg(-1) substrate in 72h, respectively.     

Conversion of corticosteroid hormones by immobilized cells of Flavobacterium dehydrogenans,Curvularia lunata and Arthrobacter simplex on ceramic carriers

Different possibilities for converting pregnenolone triacetate to prednisolone using immobilized preparations of Flavobacterium dehydrogenans, Curvularia lunata and Arthrobacter simpelex in a fixed-bed loop reactor were investigated. The effects of the carrier, substrate concentration, pH and temperature on the rate of the substrate conversion were studied also. The biotransformations were performed with a continuous or semicontinuous substrate supply. A convenient pathway for the formation of prednisolone is proposed on the basis of the results obtained. Received: 8 July 1996 / Accepted: 5 August 1996     

High ethanol tolerance of the thermophilic anaerobic ethanol producer Thermoanaerobacter BG1L1

The low ethanol tolerance of thermophilic anaerobic bacteria, generally less than 2% (v/v) ethanol, is one of the main limiting factors for their potential use for second generation fuel ethanol production. In this work, the tolerance of thermophilic anaerobic bacterium Thermoanaerobacter BG1L1 to exogenously added ethanol was studied in a continuous immobilized reactor system at a growth temperature of 70°C. Ethanol tolerance was evaluated based on inhibition of fermentative performance e.g. inhibition of substrate conversion. At the highest ethanol concentration tested (8.3% v/v), the strain was able to convert 42% of the xylose initially present, indicating that this ethanol concentration is not the upper limit tolerated by the strain. Long-term strain adaptation to high ethanol concentrations (6–8.3%) resulted in an improvement of xylose conversion by 25% at an ethanol concentration of 5% v/v, which is the concentration required in practice for economically efficient product recovery. For all ethanol concentrations tested, relatively high and stable ethanol yields (0.40–0.42 g/g) were seen. The strain demonstrated a remarkable ethanol tolerance, which is the second highest displayed by thermophilic anaerobic bacteria known to the authors. This appears to be the first study of the ethanol tolerance of these microorganisms in a continuous immobilized reactor system.     

Ethanol fermentation by immobilized cells in a trickle bed reactor

A modified discontinuous packed bed reactor with CO₂ ventilation ports, resembling a trickle bed reactor was employed to overcome gas holdup and bed compaction problems which are commonly encountered in cell immobilized packed bed reactors for ethanol fermentation. The reactor consisting of yeast cells entrapped in alginate matrix was operated by varying the substrate concentration, bed volume and inlet flow rates. The number of recirculation cycles (passes) and total stages were dependent upon the liquid flow rate, though the total contact time for complete conversion remains the same for a particular initial substrate level. The total contact time was 1.5, 3 and 4.5 h for initial substrate concentrations of 0.555, 0.933 and 1.3 kmol/m³ respectively. The number of cycles and in turn stages increased with the increase in initial sugar level. A graphical method for predicting the number of stages required for complete conversion was proposed based on material balance equation and evaluated for the operating variables of the present study. The reactor was operated continuously for 30 days producing 1.05– 1.15 kmol/m³.     

Cell growth and death rates as factors in the long-term performance, modeling, and design of immobilized cell reactors

The viable fraction of immobilized cells in a bioreactor may be critical in predicting long-term or steady-state reactor performance. The assumption of near 100% viable cells in a bioreactor may not be valid for portions of immobilized cell reactors (ICRs) characterized by conditions resulting in appreciable death rates. A mathematical model of an adsorbed cell type ICR is presented in which a steady-state viable cell fraction is predicted, based on the assumptions of no cell accumulation in the reactor and a random loss of cells from the reactor. Data on cell death rates, cell growth rates, and productivity rates as functions of temperature, substrate, and ethanol concentration for the lactose utilizing yeast K. fragillis were incorporated into this model. The steady-state reactor viable cell fraction as predicted by this model is a strong function of both temperature and ethanol concentration. For example, a stable 20% viable fraction of the immobilized cells is predicted in ICR locations experiencing continuous conditions of either 30 g/L ethanol at 40 degrees C, or 95 g/L ethanol at 25 degrees C. Steady-state ICR "plug flow" concentration profiles and column productivities are predicted at three operating temperatures, 20, 30, and 40 degrees C using two different models for ethanol inhibition of productivity. These profiles suggest that the reactor operating temperature should be low if higher outlet ethanol concentrations are desired. Three reactor design strategies are presented to maximize the viable cell fraction and improve long-term ethanol productivity in ICR's: (1) reducing outlet ethanol concentrations, (2) rotating segments of an ICR between high and low ethanol environments, and (3) simultaneous removal of the ethanol produced from the reactor as it is formed.     

Cellulase production by trichoderma harzianum in static and mixed solid-state fermentation reactors under nonaseptic conditions

Cellulase production from lignocellulosic materials was studied in solid-state cultivation by both static and mixed techniques under nonaseptic conditions. The effects of fermentation conditions, such as moisture content, pH, temperature, and aeration, on cellulase production by Trichoderma harzianum using a mixture of wheat straw (80%) and bran (20%) were investigated. With a moisture content of 74% and a pH of 5.8., 18 IU filter paper activity and 198 IU endoglucanase activity/g initial substrate content were obtained in 66 h. The extension from static column cultivation to stirred tank reactor of 65 L capacity gave similar yields of cellulase.     

Yeast cells entrapped in low-gelling temperature agarose for the continuous production of ethanol

Summary Continuous ethanol production byS. uvarum immobilized in a low-gelling temperature agarose namely SeaPlaque agarose was studied in a packed bed reactor at 30°C using sugarcane molasses containing 13.5% fermentable sugars as feed. The productivity at 95% conversion was 23 g/l.h (on reactor volume basis). The bioreactor was run continuously at a fixed dilution rate and it retained 60% of its initial activity upto 80 days.     

Optimization of medium constituents and fermentation conditions for the production of ethanol from palmyra jaggery using response surface methodology

The quantitative effects of sugar concentration, nitrogen concentration, EDTA, temperature, pH and time of fermentation on ethanol production were optimized using a Box-Wilson central composite design (CCD) experiment. It was found that palmyra jaggery (sugar syrup from the palmyra palm) is a suitable substrate for the production of high concentrations of ethanol using Saccharomyces cerevisiae NCIM 3090 by submerged fermentation. A maximum ethanol concentration of 129.4 g/l was obtained after optimizing media components and conditions of fermentation. The optimum values were a temperature of 26.2 °C, pH of 8.4, time of fermentation of 4.2 days with 398.5 g of substrate/l, 3.1 g of urea/l and 0.51 g of EDTA/l. Thus by using the CCD, it is possible to determine the accurate values of the fermentation parameters where maximum production of ethanol occurs.     

一株新型同型产乙酸菌Clostridium sp.的自养和异养生长特性

【背景】同型产乙酸菌是一类利用乙酰辅酶A途径固定CO_2合成自身细胞物质并生成乙酸、乙醇等代谢产物的厌氧菌群,其分布广泛、种类繁多且代谢多样。深入研究同型产乙酸菌菌株的代谢能力及特性,对探索该种群的生理生化特性及其环境作用至关重要。【目的】研究一株同型产乙酸菌Clostridium sp. BXX的最适培养条件及其自养与异养生长特性。【方法】设置BXX菌株培养温度10-55°C、初始pH 6.0-9.0、NaCl浓度0-2.0%、不同氮源,测定菌体细胞含量和产物生成浓度,确定菌株最适培养条件。研究BXX菌株分别以H_2/CO_2、合成气、CO、葡萄糖、1,2-丙二醇、甲酸钠、乙二醇甲醚、甘油、丙酮酸和乳酸为底物时的底物消耗、产物生成、菌体细胞含量和pH等,探究其自养和异养生长特性。【结果】BXX菌株的最适培养温度为30°C,初始pH为7.0,NaCl浓度为1.0%,氮源为酵母粉。BXX菌株能以H2/CO2、合成气、葡萄糖、1,2-丙二醇、甲酸钠、乙二醇甲醚和甘油为底物生长,不能以CO、丙酮酸或乳酸为底物生长。【结论】BXX菌株既能自养生长产乙酸,又能异养生长产乙醇。BXX菌株是乙酸发酵的优良菌种资源,有较好的工业应用潜力。     

Optimization of Operating Temperature for Continuous Immobilized Glucose Isomerase Reactor with Pseudo Linear Kinetics

In this work, the optimal operating temperature for the enzymatic isomerization of glucose to fructose using a continuous immobilized glucose isomerase packed bed reactor is studied. This optimization problem describing the performance of such reactor is based on reversible pseudo linear kinetics and is expressed in terms of a recycle ratio. The thermal deactivation of the enzyme as well as the substrate protection during the reactor operation is considered. The formulation of the problem is expressed in terms of maximization of the productivity of fructose. This constrained nonlinear optimization problem is solved using the disjoint policy of the calculus of variations. Accordingly, this method of solution transforms the nonlinear optimization problem into a system of two coupled nonlinear ordinary differential equations (ODEs) of the initial value type, one equation for the operating temperature profile and the other one for the enzyme activity. The ODE for the operating temperature profile is dependent on the recycle ratio, operating time period, and the reactor residence time as well as the kinetics of the reaction and enzyme deactivation. The optimal initial operating temperature is selected by solving the ODEs system by maximizing the fructose productivity. This results into an unconstrained one‐dimensional optimization problem with simple bounds on the operating temperature. Depending on the limits of the recycle ratio, which represents either a plug flow or a mixed flow reactor, it is found that the optimal temperature of operation is characterized by an increasing temperature profile. For higher residence time and low operating periods the residual enzyme activity in the mixed flow reactor is higher than that for the plug flow reactor, which in turn allows the mixed flow reactor to operate at lower temperature than that of the plug flow reactor. At long operating times and short residence time, the operating temperature profiles are almost the same for both reactors. This could be attributed to the effect of substrate protection on the enzyme stability, which is almost the same for both reactors. Improvement in the fructose productivity for both types of reactors is achieved when compared to the constant optimum temperature of operation. The improvement in the fructose productivity for the plug flow reactor is significant in comparison with the mixed flow reactor.     

Effect of initial substrate concentration on the rate of nitrification in a batch experiment

The intrinsic rate of nitrification was observed in a batch reactor by eliminating external and internal diffusional resistances. The former were minimized by means of intense agitation, and the latter by mechanical rupture of the activated sludge flocs using high mixer rotational speeds. The optimum temperature and pH for the intrinsic nitrification rate were found to be 30–35°C and 8.0, respectively. Initial ammonium concentration was found to have a strong effect on the value of the kinetic parameters of the Michaelis–Menten rate expression at low ammonium levels. However, at high initial concentrations both parameters attained a constant maximum value that is independent of the initial substrate level.     

Effect of pH on the production of lipolyzed butter oil by a calf pregastric esterase immobilized in a hollow-fiber reactor: I. uniresponse kinetics

A calf pregastric esterase immobilized in a hollow-fiber reactor was employed to hydrolyze milkfat, thereby producing a lipolyzed butteroil. The reaction kinetics can be modeled by a two-parameter model of the general Michaelis-Menten form based on a ping-pong bi-bi mechanism; the rate of enzyme deactivation can be modeled as a first-order reaction. The initial concentration of accessible glyceride bonds, [G](O), was estimated by complete saponification of the substrate butteroil as 2400 mM. An extra sum of squares test indicated that not only the parameters of the kinetic generalized Michaelis-Menten model, but also the deactivation-rate constant varied significantly with pH. The optimum pH, for lypolysis is near 6.0 at a temperature of 40 degrees C because at this pH the rate of deactivation of the esterase is minimized.     

Preparation of L-aspartic acid by means of immobilized Alcaligenes metalcaligenes cells

Two types of biocatalysts based on immobilized cells of Alcaligenes metalcaligenes exhibiting aspartate ammonia-lyase activity (EC 4.3.1.1) were developed for the enzymic preparation of L-aspartic acid from ammonium fumarate. The first type of the biocatalyst consists in individual covalently crosslinked and permeabilized cells(I), while the second type is represented by cell aggregates (II). For the above preparation, biocatalyst I can be used only discontinuously in a mixed reactor. After termination of the reaction between individual cycles of its use, the biocatalyst is returned to the reactor in the form of a highly concentrated cell suspension or paste. Biocatalyst II can be used discontinuously or continuously in a fixed-bed column of the catalyst. The effects of pH, substrate concentration and temperature on the reaction velocity and effectivity of enzymic conversion was investigated. Optimal parameters of the reaction are as follows: pH 8.5, initial substrate concentration, 1.35 mol/L, temperature for discontinuous process, 37 degrees C, and temperature for continuous process, 25 degrees C. Under these conditions the enzymic conversion of substrate to product is quantitative. Under optimal toring conditions, the specific activity of both catalysts does not change within a period of one year. The operational half-life of the biocatalyst II during continuous use in a fixed-bed column of the catalyst under standard reaction conditions depends on the quality of the substrate. The discontinuous preparation of L-asparatic acid with the aid of biocatalyst I and continuous preparation of this product with the aid of biocatalyst II have been verified under pilot-plant conditions.     

Efficient production of methionine from 2-amino-4-methylthiobutanenitrile by recombinant Escherichia coli harboring nitrilase

Methionine as an essential amino acid has been attracting more attention for its important applications in food and feed additives. In this study, for efficient production of methionine from 2-amino-4-methylthiobutanenitrile, a codon-optimized nitrilase gene was newly synthesized and expressed, and the catalytic conditions for methionine production were studied. The optimal temperature and pH for methionine synthesis were 40 °C and 7.5, respectively. The recombinant nitrilase was thermo-stable with half-life of 5.52 h at 40 °C. The substrate loading was optimized in given amount of catalyst and fixed substrate/catalyst ratio mode to achieve higher productivity. Methionine was produced in 100 % conversion within 120 min with a substrate loading of 300 mM. The production of methionine with the immobilized resting cells in packed-bed reactor was investigated. The immobilized nitrilase exhibited good operation stability and retained over 80 % of the initial activity after operating for 100 h. After separation, the purity and the total yield of methionine reached 99.1 and 97 %, respectively. This recombinant nitrilase could be a potential candidate for application in production of methionine.