Removal of skim milk lactose by fermentation using free and immobilized Kluyveromyces marxianus cells

Kluyveromyces marxianus CBS 6164 cells, free or immobilized in Ca-alginate (2%) beads, are able to consume more than 99% of the skim milk lactose in anaerobic conditions. In batches at 30 °C, the lactose consumption after 3.5 h of skim milk fermentation by 30 and 50 g free K. marxianus cells per liter was around 99 and 99.6% respectively, with an approximate conversion of lactose to ethanol and CO₂ of 80%. The immobilized cells, easy to handle and showing a faster and easier separation from the fermented medium compared to the free ones, were used in more than 23 batches (cycles of re-use) without losing their activity.     

Kluyveromyces marxianus: a potential source of diacetyl reductase

Kluyveromyces marxianus had a higher specific activity of diacetyl reductase (EC 1.1.1.5) than all other organisms previously reported. The enzyme was NADH-dependent and irreversibly catalysed the conversion of diacetyl to acetoin with an optimum pH of 7.0. It was stable at 40°C but lost 50% of its activity at 50°C in 30 min. The K _m and V _max values for diacetyl were 1.8 mm and 0.053 mm/min, respectively.The authors are with the Department of Food Science and Technology, Comell University, Geneva, New York 14456, USA     

Production of High-content Galacto-oligosaccharide by Enzyme Catalysis and Fermentation with Kluyveromyces marxianus

Of three β-galactosidases from Aspergillus oryzae, Kluyveromyces lactis and Bacillus sp., used for the production of low-content galacto- oligosaccharides (GOS) from lactose, the latter produced the highest yield of trisaccharides and tetrasaccharides. GOS production was enhanced by mixing β-galactosidase glucose oxidase. The low-content GOS syrups, produced either by β-galactosidase alone or by the mixed enzyme system, were subjected to the fermentation by Kluyveromyces marxianus, whereby glucose, galactose, lactose and other disaccharides were depleted, resulting in up to 97% and 98% on a dry weight basis of high-content GOS with the yields of 31% and 32%, respectively. An erratum to this article can be found at     

Enhanced lovastatin production by solid state fermentation ofMonascus ruber

The purpose of this study was to optimize the solid state cultivation ofMonascus ruber on sterile rice. A single-level-multiple-factor and a single-factor-multiple-level experimental design were employed to determine the optimal medium constituents and to optimize carbon and nitrogen source concentrations for lovastatin production. Simultaneous quantitative analyses of the β-hydroxyacid form and β-hydroxylactone for of lovastatin were performed by the high performance liquid chromatography (HPLC) method with a UV photodiode-array (PDA) detector. The total lovastatin yield (4≈6 mg/g, average of five repeats) was achieved by adding soybean powder, glycerol, sodium nitrate, and acetic acid at optimal composition of the medium increased by almost 2 times the yield observed prior to optimization. The experimental results also indicated that the β-hydroxylactone form of lovastatin (LFL) and the β-hydroxyacid form of lovastatin (AFL) simultaneously existed in solid state cultures ofMonascus ruber, while the latter was the dominant form in the middle-late stage of continued fermentation. These results indicate that optimized culture conditions can be used for industrial production of lovastatin to obtain high yields.     

Fed-batch fermentation for production of Kluyveromyces marxianusFII 510700 cultivated on a lactose-based medium

A strain of Kluyveromyces marxianus was grown in batch culture in lactose-based media at varying initial lactose concentrations (10–60 g L^–1) at 30°C, pH 5.0, dissolved oxygen concentrations greater than 20%. Increasing the concentration of mineral salts three-fold at 40 g L^–1 and 60 g L^–1 initial lactose concentration showed only a small increase in the yield of biomass, from 0.38 g g^–1 to 0.41 g g^–1, indicating that the initial batch cultures were not significantly nutrient- (mineral salts)-limited. A relatively high biomass concentration (105 g L^–1) was obtained in fed-batch culture following extended lactose feeding. An average specific growth rate (0.27 h^–1), biomass yield (0.38 g g^–1) and overall productivity (2.9 g L^–1 h^–1) were obtained for these fed-batch conditions. This fed-batch protocol provides a strategy for achieving relatively high concentrations and productivities of K. marxianus on other lactose-based substrate streams (e.g., whey) from the dairy industry.     

The use of the replicating pDblet plasmid as a cloning vector with enhanced stability in Kluyveromyces marxianus

The replicating plasmid, pDblet, transformed the budding yeast Kluyveromyces marxianus to an efficiency of 10⁴ transformant g^–1 DNA. Transformed cells showed 1% of segregation rate without affecting their growth rate of 0.69 h^–1 and glucose consumption. These results were similar or better than the commonly used pE1 plasmid and suggests that pDblet can be used for cloning genes in K. marxianus.     

Toxin-binding properties of cytochrome P450 in Saccharomyces cerevisiae and Kluyveromyces marxianus

Microsomes from Kluyveromyces marxianus GK1005 examined by carbon monoxide difference spectroscopy showed no evidence of cytochrome P450, in contrast to microsomes isolated from a control strain of Saccharomyces cerevisiae. Benzo[a]pyrene produced a typical Type I-binding spectrum with microsomes of both yeasts, with K _s values of 82 M (S. cerevisiae) and 70 M (K. marxianus). While aflatoxin B₁ generated a typical Type I-binding spectrum with microsomes from S. cerevisiae (K _s of 178 M), the toxin did not produce a recognisable binding spectrum with microsomes from K. marxianus.     

Simultaneous saccharification and fermentation of Kanlow switchgrass by thermotolerant Kluyveromyces marxianus IMB3: the effect of enzyme loading, temperature and higher solid loadings

Switchgrass (Panicum virgatum) was subjected to hydrothermolysis pretreatment and then used to study the effect of enzyme loading and temperature in a simultaneous saccharification and fermentation (SSF) with the thermotolerant yeast strain Kluyveromyces marxianus IMB3 at 8% solid loading. Various loadings of Accellerase 1500 between 0.1 and 1.1 mL g(-1) glucan were tested in SSF at 45 °C (activity of enzyme was 82.2 FPU mL(-1)). The optimum enzyme loading was 0.7 mL g(-1) glucan based on the six different enzyme loadings tested. SSFs were performed at 37, 41 and 45 °C with an enzyme loading of 0.7 mL g(-1) glucan. The highest ethanol concentration of 22.5 g L(-1) was obtained after 168 h with SSF at 45 °C, which was equivalent to 86% yield. Four different batch and fed-batch strategies were evaluated using a total solid loading of 12% (dry basis). About 32 g L(-1) ethanol was produced with the four strategies, which was equivalent to 82% yield.     

Production of pectinases by Aspergillus niger in solid state fermentation at high initial glucose concentrations

Exopectinase (exo-p) and endopectinase (endo-p) production by Aspergillus niger CH4 in solid state culture was studied at initial glucose concentrations of 100, 250, 350 and 450 g/l. The highest activity of exo-p (35 U/g) was produced at 72 and 120 h in the medium containing 100 and 250 g glucose/l, respectively. The maximum endo-p activity (9 U/g) was produced at 72 h in the medium with 250 g glucose/l. The reduction in pectinase production at 350 and 450 g/l initial glucose concentration was due neither to repression of the synthesis of the enzyme nor to the glucose consumption rate of the strain but due to a drastic drop in pH of the medium.S. Solis-Pereyra, E. Favela-Torres, M. Gutiérrez-Rojas, G. Saucedo-Castañeda and G. Viniegra-González are with the Departamento de Biotecnologia, Universidad Autónoma Metropolitana, A.P. 55-535, C.P. 09340, México D.F., México; S. Roussos is with the Laboratoire de Biotechnologie, ORSTOM, B.P. 5045, 34032, Montpellier Cedex, France, and P. Gunasekaran is with the Department of Microbial Technology, School of Biological Sciences, Madurai Kamaraj University, Madurai, 625-021, India.     

Short communication: Ethanol production from cellulose at 45°C using a batch-fed system containing alginate-immobilized Kluyveromyces marxianus IMB3

The thermotolerant yeast, Kluyveromyces marxianus IMB3, was grown in batch culture at 45°C on cellulose-containing media, supplemented with exogenous cellulase activity. At various stages during fermentation, both substrate and enzyme were added in batch mode and fermentation was continued for 220 h. Ethanol production increased to 20 g/l at 200 h, representing 45% of the maximum theoretical yield. In subsequent experiments, the organism was immobilized in calcium alginate beads and these were used in a similar, batch-fed system at 45°C. Again, fermentation was continued for 220 h and ethanol production increased to its maximum, of 28 g/l, within 100 h and this represented in excess of 60% of the maximum theoretical yield.     

嗜热拟青霉固体发酵产木聚糖酶的纯化和性质

研究一株新的嗜热拟青霉J18的固体发酵产木聚糖酶的纯化和性质。固体发酵的粗酶液经硫酸铵沉淀、凝胶过滤层析和离子交换层析得到了一种分子量约为26 kDa的电泳纯木聚糖酶,酶活力回收率为33.5%,纯化了5.27倍。该木聚糖酶具有很好的温度和pH稳定性,在pH7.0~pH 9.0下,60℃处理24 h,酶活力能保存80%以上。该酶水解玉米芯木聚糖生成以木二糖、木三糖和木四糖为主的低聚木糖,薄层层析分析表明不含木糖,适合生产低聚木糖。     

Molecular probes for the detection of Kluyveromyces marxianus chromosomal DNA in electrophoretic karyotypes of intergeneric protoplast fusion products

Random genomic DNA fragments from Kluyveromyces marxianus were cloned in order to identify chromosomal bands in pulsed field electrophoresis patterns of intergeneric hybrid strains which were obtained by protoplast fusion. Molecular hybridization data indicated that the K. marxianus parental strain might be triploid, and it showed strong chromosome length polymorphism. We analyzed the karyotype of two Saccharomyces cerevisiae/K. marxianus hybrid strains (St. 1, St. 46) with our DNA probes and with a Ty1 specific probe. We found indications for recombinational events which lead to the formation of hybrid chromosomal DNA molecules.     

Heat transfer simulation of solid state fermentation in a packed-bed bioreactor

A dynamic two-dimensional mathematical model was developed to simulate the heat transfer in solid state fermentation of rice bran inoculated with Aspergillus niger. Two bioreactors of 47 mm diameter and 300 mm height were used. Forced aeration conditions were simulated and saturated air with 60 ml/g h flow at 30°C and a bed porosity equal to 0.3 were determined as being the optimum operating conditions for the used packed-bed bioreactor. © Rapid Science Ltd. 1998     

Continuous removal of malathion by immobilised biomass of Bacillus species S14 using a packed bed column reactor

Abstract

Biosorption of malathion from aqueous solution was studied using Bacillus sp. S₁₄ immobilised on calcium alginate (3%) using a packed bed column reactor at a temperature of 25 °C and a pH of 7.0. The experiments were conducted to study the effect of important design parameters such as bed height, flow rate and influent malathion concentration. Maximum removal capacity (57%) was found at 4 mL min^-1 flow rate, 6.0 cm bed height and 25 mg L^-1 influent malathion concentration. The Adam-Bohart model, Wolborska model, Thomas model, Yoon-Nelson model were employed to determine characteristic parameters such as saturation concentration, external mass transfer coefficient, Thomas rate constant, the maximum solid phase concentration of the solute, rate constant, and the time required for 50% adsorbate breakthrough time, which are all useful for process design. Experimental data were well fitted with Adam–Bohart model at the lower region of effluent/influent malathion concentration values but at higher region values data fitted well with the Thomas and Yoon-Nelson models.     

Enhanced production of cellulases byPenicillium citrinum in solid state fermentation of cellulosic residue

Penicillium citrinum, using rice husks in a solid state fermentation, produced maximum cellulase yields (37 Units/g) after 12 days with a cellulose utilization of more than 70%. Enzyme yields were three times higher than in shake-flask cultures.     

Fungal solid state fermentation on agro-industrial wastes for acid wastewater decolorization in a continuous flow packed-bed bioreactor

This study was aimed at developing a process of solid state fermentation (SSF) with the fungi Pleurotus ostreatus and Trametes versicolor on apple processing residues for wastewater decolorization. Both fungi were able to colonize apple residues without any addition of nutrients, material support or water. P. ostreatus produced the highest levels of laccases (up to 9 U g⁻¹ of dry matter) and xylanases (up to 80 U g⁻¹ of dry matter). A repeated batch decolorization experiment was set up with apple residues colonized by P. ostreatus, achieving 50% decolorization and 100% detoxification after 24 h, and, adding fresh wastewater every 24 h, a constant decolorization of 50% was measured for at least 1 month. A continuous decolorization experiment was set up by a packed-bed reactor based on colonized apple residues achieving a performance of 100 mg dye L⁻¹ day⁻¹ at a retention time of 50 h.     

Biosorption of Malathion using dry cells of Bacillus species S14 in a packed bed column reactor

Abstract

The ability of dried bacterial strain Bacillus sp. S₁₄ to adsorb Malathion in a packed bed column reactor was studied. The effects of important design parameters such as bed height, flow rate and influent Malathion concentration on Malathion removal from an aqueous solution was studied using a packed bed column reactor. The optimised conditions for maximum Malathion removal were found to be: flow rate: 5 mL min^-1, bed height: 6.0 cm and influent Malathion concentration: 25 mg L^-1. The Adams-Bohart model, Wolborska model, Thomas model, Yoon and Nelson Model were employed to determine characteristic parameters such as N₀ (saturation concentration, mg L^-1), β_o (external mass transfer coefficient, min^-1), k _Th(Thomas rate constant, mL min^-1mg^-1), q₀ (maximum solid phase concentration of the solute, mg L^-1), k_YN (rate constant, min^-1) and τ (time required for 50 % adsorbate breakthrough time, min) which are useful for process design. Data were fitted with Adams-Bohart model at lower region of (C/C₀) values but more accurately fitted with Wolborska and Thomas model.     

Evaluation of Kluyveromyces marxianus FII 510700 grown on a lactose-based medium as a source of a natural bioemulsifier

Mannoprotein with emulsification properties was extracted from the cell walls of Kluyveromyces marxianus grown on a lactose-based medium by autoclaving cells in a citrate buffer at pH 7.The purified product was evaluated for chemical and physical stability to establish its potential use as a natural emulsifier in processed foods. The yield of purified bioemulsifier from this strain of K. marxianus was 4–7% of the original dry cell weight. The purified product, at a concentration of 12 g l^–1, formed emulsions that were stable for 3 months when subjected to a range of pH (3–11) and NaCl concentrations (2–50 g l^–1). The composition of this mannoprotein was 90% carbohydrate (mannan) and 4–6% protein. These values are similar to mannoprotein extracted from cells of Saccharomyces cerevisiae, which is the traditional source. Consequently K. marxianus cultivated on a low-cost lactose-based medium such as whey, a lactose-rich clean waste of the dairy industry, could be developed as a source of bioemulsifier for use in the food industry.     

Modes of lactose uptake in the yeast species Kluyveromyces marxianus

Twelve lactose-assimilating strains of the yeast species Kluyveromyces marxianus and its varieties marxianus, lactis and bulgaricus were studied with respect to transport mechanisms for lactose, glucose and galactose, fermentation of these sugars and the occurrence of extracellular lactose hydrolysis. The strains fell into three groups. Group I (two strains): Fermentation of lactose, glucose and galactose, extracellular lactose hydrolysis, apparent facilitated diffusion of glucose and galactose; Group II (two strains): Lactose not fermented, glucose and galactose fermented and transported by an apparent proton symport, extracellular hydrolysis of lactose present (one strain) or questionable; Group III (eight strains): Lactose, glucose and galactose fermented, lactose transported by an apparent proton symport mechanism, extracellular hydrolysis of lactose and transport modes for glucose and galactose variable.     

金龟子绿僵菌固态发酵产壳聚糖酶

对金龟子绿僵菌（Metarhizium anisopliae）AS3．4606产壳聚糖酶进行了固态发酵条件优化及酶学部分特征的研究。正交实验结果表明,以麸皮为碳源,日本根霉菌丝体粉末为氮源,在起始pH5．0,m（碳）：m（氨）为1：4,m（干重）：m（液体）为1：1．4,27℃下培养120h,壳聚糖酶活性可达35．08U／g（干培养基）。粗酶液的最适反应温度为40℃,最适反应pH为5．0,酶在pH5．0～6．0条件下稳定性最高。该酶不能水解固体甲壳素和纤维素粉末,最适底物为胶体壳聚糖。