Lactic acid production using free cells of bacteria and filamentous fungi and cells immobilized in polyvinyl alcohol cryogel: A comparative analysis of the characteristics of biocatalysts and processes

The results from studies and a comparative analysis of process characteristics are presented for lactic acid (LA) production from glucose, performed under batch conditions of the long-duration use of different biocatalyst samples comprising cells of Lactobacillus сasei bacteria and Rhizopus oryzae filamentous fungi immobilized in polyvinyl alcohol (PVA) cryogel or applied as concentrated suspensions. It is established that for LA production, the bacteria and fungi must be used in the form of PVA-cryogel-immobilized cells because their half-life in this form is considerably longer than that of concentrated cell suspensions. After 200 h of the batch use of the same immobilized cell samples, the amounts of accumulated LA were similar for both fungal (920 ± 5 g) and bacterial (895 ± 5 g) biocatalysts. The fungal biocatalyst, however, was characterized by a twice higher rate of substrate conversion to product (0.92 g LA per 1 g glucose) than the bacterial biocatalyst. The half-life of the immobilized fungal biocatalyst was 80 days (96 working cycles), ten times longer than that of the bacterial biocatalyst. A comparison of our data and the literature data demonstrated the promise of using fungal cells immobilized in PVA cryogel to produce LA: the process based on their use is superior to all known processes in its main indicators, i.e., the rate of LA conversion to glucose and the maximum accumulated concentration of the product.     

Lipase‐catalyzed synthesis of butyl esters by direct esterification in solvent‐free system

BACKGROUND: Reactions performed under solvent‐free conditions give processes that are environmentally friendly, since most solvents are polluting agents. In this work, the performance of Candida rugosa lipae (CRL) immobilized on styrene‐divinylbenzene (STY‐DVB) or controlled pore silica (CPS), and the commercial lipase Novozym 435, was evaluated for the synthesis of butyl esters in solvent–free systems (SFS). A 2² full factorial design was used to study the influence of the organic acid chain length and the biocatalyst concentration on the esterification performance. RESULTS: When CRL on STY‐DVB was used, the ester formation was influenced by both variables and their interaction. The reaction conversion was higher (63%) using 10% of immobilized system and lauric acid, corresponding to a productivity of 3.62 g L^?1 h^?1 For CRL on CPS, only the effect of biocatalyst concentration was significant, and the highest yield was attained using 20% of immobilized system and caprilic acid. In the case of Novozym 435, the highest yield (49%) was obtained using butyric acid as acyl donor at 15% of immobilized lipase. CONCLUSION: The results allowed better understanding of the influence of important parameters in this environmentally friendly process, which also has the process advantage of a higher volumetric productivity when compared with a solvent system. Copyright © 2007 Society of Chemical Industry     

L(+)‐Lactic acid production using poly(vinyl alcohol)‐cryogel‐entrapped Rhizopus oryzae fungal cells

A new immobilized biocatalyst based on Rhizopus oryzae fungal cells entrapped in poly(vinyl alcohol)‐cryogel was evaluated in both the batch and semi‐batch processes of L (+)‐lactic acid (LA) production, when glucose, acid hydrolysates of starch or gelatinized potato starch were used as the main substrates. Under the batch conditions, the immobilized biocatalyst developed produced LA with yields of 94% and 78% from glucose and acid starch hydrolysates, respectively. Semi‐batch conditions enabled product yields of 52% and 45% to be obtained with the corresponding substrates. The highest process productivity (up to 173 g L^?1) was reached under semi‐batch conditions. Potato starch (5–70 g L^?1) was also transformed into lactic acid by immobilized R. oryzae. It was shown that long‐term operation of the immobilized biocatalyst (for at least 480 h) produced a low decrease in metabolic activity. Copyright © 2006 Society of Chemical Industry     

Characteristics of cellulase modified with amphiphilic copolymer in organic solvent

Characteristics of modified cellulases in organic solvents were studied. Cellulases modified with amphiphilic copolymer of polyoxyalkylene (POA)-derivative and maleic acid anhydride (MAA). Amino groups of the cellulase molecule were easily coupled with the MAA functional group of the copolymer. At the maximum degree of modification (DM) of 55%, the activity of modified cellulase retained more than 80% of the unmodified native cellulase activity. The modified cellulase using AKM-1511 with DM greater than 40% was found to be more than 90% soluble in aqueous solution of acetone and ethanol, leaving the native cellulase and impurities in the fermentation broth with the residue. Modified cellulase showed excellent stability against water-insoluble solvent. Moreover, cellulase modified with hydrophobic copolymer, which consists of ethylene oxide (EO) and propylene oxide (PO), could be dissolved in these solvents.     

金黄节杆菌CYC705腈水解酶催化亚氨基二乙酸合成的研究

将金黄节杆菌CYC705(Arthrobacter aurescens CYC705) 腈水解酶用于生物催化合成亚氨基二乙酸(IDA),从生物催化剂的形式、生物催化反应过程优化和反应体系放大三个方面进行了考察。在氨基载体固定化酶、环氧基载体固定化酶、海藻酸钠固定化细胞、壳聚糖固定化细胞和游离全细胞几种生物催化剂形式中,壳聚糖固定化细胞催化效率最高、稳定性最好。通过反应体系、反应温度、金属离子、底物浓度、固定化细胞投量等因素的优化,确定了最佳的生物催化反应条件：以50 mmol/L pH=6.6的磷酸氢二钠-柠檬酸缓冲液作为反应体系,底物亚氨基二乙腈(IDAN)的浓度为200 mmol/L,添加CoCl2至终浓度为1 mmol/L,反应温度37 ˚C,固定化细胞投量为0.25 g每5 mL反应体积。在此条件下,反应2h可将IDAN完全转化为IDA。进一步将反应体系放大10倍,催化200 mmol/L的IDAN完全转化为IDA仅需1h。     

Biocatalytic production of extracellular exopolysaccharide dextran synthesized by cells of <Emphasis Type="Italic">Leuconostoc mesenteroides</Emphasis>

Results are presented from studies and a comparative analysis of the production of the commercially important product dextran from sucrose using fed-batch cultivated cells of the Leuconostoc mesenteroides subsp. dextranicum B-5481 bacterium either immobilized in a polyvinyl alcohol (PVA) cryogel or in the form of a suspension. It is shown that under identical process conditions, the concentration of dextran is 1.2 times higher when using immobilized cells instead of free cells. The high productivity of dextran formation (4.2 g/(L h)) under the conditions of fed-batch cultivation of the immobilized cells and the ability of these cells to function without losing their metabolic activity for at least five operating cycles are demonstrated. The productivity of the developed biocatalyst is 5 times higher than that of Weissella confusa cells immobilized in a calcium alginate gel and 34 times higher than that of Leuconostoc mesenteroides KIBGE HA1 cells immobilized in a polyacrylamide gel. The molecular weight of the dextran samples produced by the immobilized L. mesenteroides B-5481 cells is half that of the polymer produced by the free cells, expanding the range of possible applications of the polysaccharide with no additional hydrolysis.     

Highly concentrated populations of <Emphasis Type="Italic">Aureobasidium pullulans</Emphasis> cells in biocatalytic pullulan production processes

Results are presented from studies and a comparative analysis of highly concentrated populations of free and immobilized Aureobasidium pullulans Y-4137 cells in the biocatalytic processes of pullulan production in glucose-containing media. The possibility of effectively using the developed biocatalyst in the form of immobilized cells is demonstrated. The process characteristics are determined for pullulan production from hydrolysates of various sources of renewable feedstocks (Jerusalem artichoke tubers, aspen wood, Chlorella vulgaris microalgal biomass, and potato pulp) under the action of the catalyst. It is established that A. pullulans cells immobilized in a polyvinyl alcohol (PVA) cryogel consume glucose 1.5 times faster and accumulate a 1.7 times higher concentration of the target polysaccharide in the medium than free cells. The immobilized cells can function for at least 15 operating cycles with a slight (no more than 10%) reduction in their metabolic activity. Analysis of the obtained data confirms that cell immobilization in a PVA gel for the production of pullulan allows us to shorten the duration of operating cycles in similar processes by a factor of 1.4 while reaching a comparable yield of the target product.     

Enzymatic Hydrolysis of Carboxymethylcellulose and Filter Paper by Immobilized Cellulases on Lignophenols

The cost of cellulase is a major factor limiting the enzymatic hydrolysis of cellulosic biomass. Thus, immobilization of cellulase would be an important advancement. Lignophenol is a lignin-based functional phenolic polymer synthesized from a lignocellulosic material and a phenol derivative at ambient temperature. Cellulase derived from Trichoderma reesei is easily immobilized by softwood and hardwood lignocresol simply by mixing to produce a water-insoluble lignophenol-cellulase complex. Enzymatic hydrolysis performances of cellulases immobilized on lignocresols are approximately 80–90% and 30–50% relative to that of free cellulase in the hydrolysis of carboxymethylcellulose (CMC) and filter paper, respectively. Cellulase was active enough even after adsorption on lignocresols. Limited physical contact between solid substrates and immobilized cellulase due to the presence of lignocresol seems to lead to lower enzymatic activity for solid substrates. Hardwood lignocresol-immobilized cellulase exhibits slightly higher activity than softwood lignocresol-immobilized cellulase when the same amount of cellulase is used per gram lignocresol. Although cellulase activity gradually decreases with recycling, sufficient enzymatic activity, at least for hydrolyzing soluble substrates, remains that it can be reused.     

Preparation of clay–poly(glycidyl methacrylate) composite support for immobilization of cellulase

A composite material was synthesized by grafting of glycidyl methacrylate onto clay using surface initiation atom transfer radical polymerization (SI-ATRP) technique. Epoxy group of the grafted p(GMA) chains was reacted with hexamethylenediamine (HMDA). The composite material was characterized using scanning electron microscopy (SEM) and FTIR. Cellulase from Trichoderma reesei was immobilized on the aminated composite particles via adsorption and covalent binding methods. The amounts of adsorbed enzyme on the aminated composite particles were 43.4 mg/g. The recovered activities of the adsorbed and covalently immobilized cellulase were found to be 87.7% and 73.2% for the substrate, carboxymethyl cellulose (CMC, 1.0 g/L). The pH stabilities and thermo-stabilities, repeated use and storage stabilities of both immobilized cellulase preparations were evaluated. The immobilized cellulase preparations have better stabilities and higher retained activities with respect to pH, temperature and storage than those of the free enzyme. Operational stability of the covalently immobilized cellulase was tested in a continuous flow system, and the activity loss was about 4% at the end of 48 h operation period.     

纤维素酶固定化载体的研究进展

重点介绍了可溶性载体、不溶性载体以及可溶-不可溶性载体固定化纤维素酶的研究进展,3种载体都能不同程度地提高纤维素酶的稳定性与重复使用性。可溶性载体能提高纤维素酶的操作稳定性,有利于水解不溶性的纤维素,但回收不方便。不溶性载体固定化纤维素酶,回收方便,操作稳定性提高,但即使是提高了比表面积和减少了酶与底物的传质阻力的不溶性磁性纳米材料与膜材料固定化纤维素酶,也大多停留在水解可溶的羧甲基纤维素(CMC)阶段,不能高效率地水解不溶的纤维素底物。可溶-不可溶性载体固定纤维素酶,既能方便回收,又能水解不溶性的纤维素底物,但存在难固定,沉淀-溶解过程酶活损失大的缺点,期待开发新的固定方法与新的可溶-不可溶性载体。     

A novel method to prepare chitosan powder and its application in cellulase immobilization

In this study, chitosan microspheres and sponges with uniform spherical and porous morphologies were prepared by coiling the stretched chains of chitosan with addition of salt and choosing different kinds of organic solvents as evaporation solvents. Cellulase was immobilized to the support by a covalent method. The enzyme exhibited a considerable affinity to the support, and the protein loading of 145.5 mg g^?1 support was fairly high. The immobilized cellulase had a higher K_m than free cellulase and had better stability with respect to pH, thermal stability, reuses and storage stability than free cellulase. Copyright © 2005 Society of Chemical Industry     

Carbonaceous–siliceous composite materials as immobilization support for lipase from Alcaligenes sp.: Application to the synthesis of antioxidants

Two carbonaceous–siliceous composite materials, produced by hydrothermal and carbonization processes, were evaluated as immobilization support for lipase from Alcaligenes sp. These materials exhibited similar chemical characteristics but their carbon content and porous characteristics were different, which explain the catalytic behavior and stability of the biocatalysts immobilized on them. Higher activity and immobilization selectivity was achieved with the microporous material that had higher carbon content. The lipase immobilized on the mesoporous material had a higher thermal stability at 55 °C, pH 7.0 or at 40 °C in tert-butanol, simulating the reaction conditions required for organic synthesis. Both biocatalysts were tested in the synthesis of palmitoyl ascorbate and they were compared with the commercial biocatalyst QLC. The synthesis conversions with the lipase immobilized in mesoporous materials and with the biocatalyst QLC were similar (50%), but only the former could be reused. These are promising biocatalysts for industrial applications.     

Biocatalysts based on immobilized cells of microorganisms in the production of bioethanol and biobutanol

In this work, we discuss the processes for the production of bioethanol and biobutanol, which are promising alternative fuels, using biocatalysts based on cells of various microorganisms immobilized in poly(vinyl alcohol) cryogel. Biocatalysts based on immobilized cells reliably allow ethanol production from a variety of industrial and agricultural wastes (wheat straw, beet and sugarcane bagasse, parchment, corn cobs, soybean processing waste) with a high degree of conversion of consumed substrates to the target product. Ethanol concentrations are appreciably higher in media with biocatalysts than in free cells of the same microorganisms. It is found that immobilized cells of filamentous fungi can convert a wider range of the sugars contained in processed media to ethanol than commonly used yeasts. It is shown that the immobilization of the genus Clostridium cells that produce butanol enables us to reliably change the ratio of solvents that accumulate in the medium during acetone-butanol-ethanol fermentation in the direction of a greater amount of butanol, thereby improving the process’s characteristics relative to present-day technologies based on free bacterial cells.     

Characterization of Candida rugosa Lipase Immobilized on Poly(N‐methylolacrylamide) and Its Application in Butyl Butyrate Synthesis

Candida rugosa lipase was immobilized on poly(N‐methylolacrylamide) by physical adsorption. The biocatalyst performance (immobilized lipase) was evaluated in both aqueous (hydrolysis) and organic (butyl butyrate synthesis) media. In the first case, a comparative study between free and immobilized derivatives was provided in terms of pH, temperature and thermal stability following the olive oil hydrolysis, establishing new optimum values. In the second case, the influence of temperature, biocatalyst concentration and acid/alcohol molar ratio was simultaneously studied according to a 2³ full experimental design. The highest molar conversion (96 %), volumetric productivity (1.73 g L^–1 h^–1) and specific esterification activity (1.00 μM mg^–1 min^–1) were obtained when working at the lowest level of temperature and butyric acid in excess. Under these conditions, repeated batch use of the immobilized enzyme was performed and half‐life time (t_1/2) was found to be 145 h.     

Steroid bioconversion: Towards green processes

There is an increasing trend towards reducing the use of organic solvents in industry due to environmental constraints and the adoption of green chemistry guidelines. To overcome the low volumetric productivity of aqueous bioconversion systems involving sparingly water soluble hydrophobic compounds, processes are being developed and designed to incorporate green solvent such as supercritical fluids, ionic liquids and natural oils, and liquid polymers, among others as an alternative to organic solvents. Moreover, processes are developed and redesigned to use/reuse chemicals and reagents derived from waste or renewable feed stocks in order to diminish E-factors.In this work, the use of green solvents as key components in the bioconversion media for a multi-step microbial bioconversion was assessed in a suspended whole cell system, combined with the use of by-products as raw materials, ultimately used as carbon source for cell growth and as sterol substrate for bioconversion. The model system is the selective cleavage of the side-chain of β-sitosterol performed by free resting cells of Mycobacterium sp. NRRL B-3805, a well-established industrial multi-enzymatic process involving the use of nine catabolic enzymes in a 14-step metabolic pathway.Bioconversion yields in silicone media were higher than the ones obtained in polyethylene glycol (PEG), polypropylene glycol (PPG) and ionic liquids, as well as in dioctyl phthalate (DOP), an organic solvent that has previously been shown to allow high conversion yields. Total conversion of 12 mM substrate in silicone media was consistently obtained at the end of 120-h bioconversion runs. Similar bioconversion profiles were attained during a 50-fold scale-up, maintaining constant the power consumption per unit of volume.     

重吸附法回收利用纤维素酶的工艺优化

以玉米芯为底物,分别考察了Tween 80质量分数、加酶量和底物质量分数对纤维素酶解过程中纤维素酶回收利用的影响规律,结果表明,酶回收率随Tween 80质量分数和加酶量的增加呈先增大再减小的趋势。此外,酶回收率随底物质量分数的增加而逐渐增加,确定重吸附法回收纤维素酶的最优工艺条件:Tween 80质量分数为0.23%;加酶量(以葡聚糖计)为40 FPU/g;底物质量分数为3.6%,在最优条件下,纤维素酶回收率可达83.0%。该研究为木质纤维素生物炼制过程中降低纤维素酶成本提供了有效的解决方法。     

Catalytic Behaviour of Lipase Free and Immobilized in Biphasic Membrane Reactor with Different Low Water-Soluble Substrates

The catalytic activity and reaction rate of lipase have been studied using the biocatalyst free in an organic/aqueous emulsion and immobilized in a biphasic organic/aqueous membrane reactor. The first reaction system was realized in a stirred tank reactor. The other was obtained by immobilizing the enzyme in the sponge layer of an asymmetric capillary membrane and recirculating the two phases along the two separate circuits of the membrane module. The performance of the reactors has been studied using two different low water-soluble substrates: triglycerides present in commercial olive oil and (R,S)-cyanomethyl-[2-(4-isobutylphenyl)propionate] (CNE). The effects of substrate viscosity and flow dynamics conditions, such as organic phase flow rate, on the biphasic membrane reactor performance have been evaluated on the basis of observed reaction rate and catalytic activity of free and immobilized lipase for both substrates. It has been observed that free lipase showed higher catalytic activity with olive oil, while immobilized lipase showed higher catalytic activity with CNE which has a lower viscosity than olive oil. The increase of organic phase flow rate negatively affected the reactor performance, with a minor effect when using CNE rather than olive oil. The influence of temperature on the biocatalyst performance with the two substrates has also been investigated. The optimal temperature value of lipase was different for the two substrates: 28°C with CNE and 40°C with olive oil. © 1997 SCI.     

Mathematical Simulating the Biokatalytic Transformation of Methyl Phenyl Sulfide into (<Emphasis Type="Italic">R</Emphasis>)-Sulfoxide

A mathematical model is proposed for describing the biotransformation of methyl phenyl sulfide to (R)-methyl phenyl sulfoxide by immobilized Gordonia terrae IEGM 136 cells. Kinetic patterns of the biotransformation of methyl phenyl sulfide are determined using experimental data on the initial concentration of sulfide and the amount of biocatalyst. The experimental data are compared to simulations of sulfide biotransformation scaling in a laboratory bioreactor. A mathematical model is developed for describing the biotransformation of methyl phenyl sulfide with repeated use of the biocatalyst. The resulting data can be used for optimizing the biotransformation of a wide range of organic aryl alkyl sulfides to optically active sulfoxides.     

利用纤维原料在串联式生物反应器中协同酶解发酵乳酸

采用廉价的纤维素原料代替粮食发酵生产乳酸,具有重要的社会意义和经济价值。在串联式生物反应器中,将纤维原料酶水解与固定化乳酸杆菌发酵相耦联,纤维素的酶解产物葡萄糖被有效转化成乳酸,纤维素对乳酸的转化率和乳酸产率分别为70.3%和0.713g?(L?h)?1。在酶解体系中添加纤维二糖酶可以提高酶解得率。将酶解罐、固定化纤二糖酶柱和固定化细胞柱相串联,可有效消除纤维二糖积累所造成的反馈抑制作用,纤维素对乳酸的转化率和乳酸产率分别提高到90.6%和0.986g?(L?h)?1。串联式生物反应器性能稳定,在重复分批发酵工艺中,连续10批纤维素对乳酸的转化率平均为89.6%。采用分批添料工艺,纤维底物的终浓度可增加到200g?L?1,发酵终点的乳酸浓度达105.2g?L?1,乳酸产率为1.315g?(L?h)?1。对等量底物而言,反应时间明显缩短,同时纤维素酶的利用率也得到了有效提高。     

Wood blocks as a carrier for Saccharomyces Cerevisiae used in the production of ethanol and fructose

Saccharomyces cerevisiae ATCC 39859 was immobilized onto small cubes of wood in order to produce very enriched fructose syrup from synthetic glucose-fructose mixtures, through the selective fermentation of glucose. The kinetics of growth and ethanol production rates were studied. Several tests to assess the influence of substrate and product concentration on the production rates were carried out and appropriate rate equations were proposed as a design basis for continuous immobilized reactors. The ethanol production rate and cell growth rate were found to be inhibited linearly by both substrate and product concentrations. A maximum ethanol productivity of 21.9 g 1⁻¹ h⁻¹ was attained from a feed containing 10% (by weight) glucose and 10% (by weight) fructose. The ethanol concentration was 29.6 g 1⁻¹, the glucose conversion was 78% and a fructose yield of 99% was obtained. This resulted in a final fructose:glucose ratio of 2.7. At lower ethanol productivity levels the fructose:glucose ratio increased, as did the ethanol concentration in the effluent. The ethanol productivities obtained in this study were 33%–132% higher than those obtained in a previous study using the same system, under similar conditions, with the cells immobilized in alginate beads.