Malolactic fermentation in chardonnay wine by immobilised Lactobacillus casei cells

The kinetics of malolactic fermentation in Chardonnay wine by immobilised Lactobacillus casei cells has been studied. Calcium pectate gel and chemically modified chitosan beads were used as supports for immobilisation. Repeated batch fermentations were carried out with different wine samples, some of which were treated with sulfur dioxide (free 19–25 mg/litre and total 80–88 mg/litre), in shake flask at 36, 25 and 20°C without any loss of activity. The degradation of malic acid obtained using immobilised cells was twice as high as that obtained with free cells. At an initial pH 3·2, decrease of malic acid of about 30% was observed at 25°C in one hour using L. casei cells immobilised either in pectate gel or on chitosan. Among the physico-chemical parameters studied, temperature was the main factor affecting metabolism of the organic acids as well as the rate of the malolactic fermentation. Operational stability of calcium pectate gel beads and chemically modified chitosan beads was 6 months after eight fermentations and 2 months after five fermentations, respectively, which proved the possibility of industrial application of the chosen supports in wine making.     

Production of β-glucosidase using immobilised<Emphasis Type="Italic"> Piromyces</Emphasis> sp. KSX1 and<Emphasis Type="Italic"> Orpinomyces</Emphasis> sp. 478P1 in repeat-batch culture

Two anaerobic fungi, one a monocentric strain (Piromyces sp. KSX1) and the other a polycentric strain (Orpinomyces sp. 478P1), were immobilised in calcium alginate beads and cultured in sequential batches where spent medium (containing 0.25% cellobiose) was repeatedly drained and replaced. β-Glucosidase production with KSX1 was maintained for 45 days over six repeated batch cultures yielding a maximum level of 107 mIU/ml. For 478P1, β-glucosidase production was maintained for 30 days over four repeated batches yielding a maximum level of 34 mIU/ml. Although repeat-batch cultures of KSX1 produced more β-glucosidase than strain 478P1, the maximum specific β-glucosidase produced from these immobilised cultures was similar. The immobilised polycentric strain proved to be operationally superior to strain KSX1, as strain 478P1 did not produce any growth in the culture liquor. Electronic Publication     

Studies on an acetate-fermenting strain of Methanosarcina.

An acetate-fermenting strain of Methanosarcina was isolated from an acetate enrichment culture inoculated with anaerobic sludge from a waste treatment digestor. In pure culture, this organism fermented acetate in the absence of added hydrogen at rates comparable in magnitude to those found in digestor systems. This rate was significantly higher than previously obtained for pure cultures of this genus. Mineral components of yeast extract were highly stimulatory for cultures growing on methanol. Comparable stimulation was not observed for cultures growing on acetate. Labeling studies indicated that acetate was converted to methane and CO2 as predicted by previous studies on mixed cultures. Total oxidation or reduction of acetate was not the mechanism of conversion of acetate to methane by the pure culture. The ability of this strain to form colonies or to produce methane from acetate was apparently influenced by the choice of substrate and conditions used for growing the inoculum.     

Artificial immobilization of the two-membered bacterial system Bdellovibrio bacteriovorus 109D--Escherichia coli B in polyacrylamide gel

The interaction of a parasite with a host was studied in the two-membered bacterial system, Bdellovibrio bacteriovorus 109D and Escherichia coli B, immobilised in polyacrylamide gel (PAAG). The parasite localised inside the host cells was found to be more resistant to the toxic action of PAAG components than free B. bacteriovorus. The latter lost its mobility and was inactivated in the matrix of the carrier whereas the intracellular parasite had a normal cycle of development in the periplasm of the infected cells. The dynamics of B. bacteriovorus and E. coli incidence in the liquid phase and in PAAG granules was studied while the immobilised system was incubated. The interaction in the immobilised system could be intensified by growing more bacterial host cells in PAAG particles. The immobilisation was shown to favour the survival of the parasite and the host in the two-membered system.     

Dynamic modelling of aerobic bioprocesses in gel particles with immobilised cells

A dynamic model for aerobic growing cells immobilised into gel beads is developed and its operation is illustrated for the case of gluconic acid production by a strictly aerophilic strain of Gluconobacter oxydans. The model consists of both kinetic and mass transfer equations predicting the time course of bulk and intraparticle concentrations of substrates, products, and biomass. The model includes a product inhibition term. The parameter values are taken from own studies and from the literature. A sensitivity analysis of the model shows that the most significant parameters for the process are the biotransformation rate constant, the specific cell growth rate in the bulk, and the Thiele modulus for glucose. The computer simulation reveals that depending on the parameter values the gel particles might perform as a source or a sink of the product, thus enhancing or retarding the net process. For a specific parameter selection, the biotransformation in the pellets can prevail compared with the bulk in the beginning of the process as long as the direction of the product diffusion flux is from the beads toward the bulk. Since the process in the free culture dominates, the system is more sensitive to parameters associated with the bulk phase (aeration rate, specific microbial growth rate, oxygen uptake rate). The model can be applied for prediction and fast evaluation of the performance of aerobic processes accomplished by immobilised growing cells.     

Induction of Cr(VI) reduction activity in an Anoxybacillus strain under heat stress: a biochemical and proteomic study

A bacterial strain, designated as TSB-6, was isolated from the sediments of a Tantloi (India) hot spring at 65 °C. The strain showed 98% 16S rRNA gene sequence similarity with Anoxybacillus kualawohkensis strain KW12 and was found to grow optimally at 37 °C. However, growing cells, cell suspensions, and cell-free extracts from 65 °C cultures showed higher Cr(VI) reduction activities when assayed at either 37 or 65 °C than those obtained from 37 °C cultures. On fractionation of extracts from cells grown at 65 °C, the chromate reductase activity assayed at 65 °C was found mostly in the soluble fraction. When log-phase cells growing at 37 °C were shifted to 65 °C, the stressed cells produced larger quantities of reactive oxygen species. Consequently, growth of the cells was retarded, but specific Cr(VI) reduction activity increased. 2D gel electrophoresis followed by MALDI-TOF MS/MS identified the proteins whose expression level changed as a result of heat stress. The upregulated set included proteins involved in cellular metabolism of sugar, nucleotide, amino acids, lipids and vitamins, oxidoreductase activity, and protein folding. The downregulated proteins are also involved in cellular metabolism, DNA binding, and environmental signal processing.     

Comparison of the activity of immobilised and freely suspended Streptomyces coelicolor A3(2)

Streptomyces coelicolor was immobilised naturally in porous support materials and its growth, glucose uptake and actinorhodin production were compared with freely suspended culture using defined and complex media. When the defined medium was used, the most pronounced difference between the two cultures was the accumulation of actinorhodin extracellulary in freely suspended and intracellularly in immobilised cultures. In the complex medium, however, actinorhodin was excreted by both cultures. In addition, the complex medium yielded 50 times as much actinorhodin compared to the defined medium. Further increases in product concentration were obtained by repeated batches of immobilised culture, which showed stability for at least 3 months.     

Immunofluorescent Detection of Enterotoxin B in Food and a Culture Medium

Both Staphylococcus aureus strains 243 and S-6 cells producing enterotoxin B and free enterotoxin in food and culture medium were rapidly demonstrated by using the fluorescent-antibody technique. Comparison of cell fluorescence and enterotoxin B production determined by double gel diffusion showed that an estimation of enterotoxin production could be made by observing the degree of cell fluorescence. The fluorescent-antibody technique was used to determine whether cells were producing enterotoxin under varying nutritional and environmental conditions: NaCl concentration, culture aeration, and time and temperature of incubation in Brain Heart Infusion broth and shrimp slurries. At the various NaCl concentrations, the fluorescence of cells was found positively associated with enterotoxin B production only during the first 12 hr of growth. As the NaCl concentration was increased from 0 to 10%, the fluorescence of cells and toxin production decreased. Maximum for cell fluorescence and enterotoxin production was observed at 37 C. Little or no difference in cell fluorescence and enterotoxin production with both strains was found between Brain Heart Infusion broth and shrimp slurry cultures. All results obtained with the fluorescent-antibody technique were verified with double gel diffusion for enterotoxin detection and quantitation.     

Immobilisation of cells in a linear polyacrylamide matrix: Degradation of a mixture of volatile fatty acids byAlcaligenes denitrificans.

Summary Cells of a potent volatile fatty acid degrading strain of the organismAlcaligences denitrificans were immobilised in a linear, prepolymerised, derivatised polyacrylamide gel, cross linked with glyoxal. The beads formed were structurally strong, and immobilised cells had an increased ability compared to an equivalent quantity of free cells, to degrade a mixture of volatile fatty acids at concentrations up to 6 g/l.     

Growth of immobilised plant cells in reticulate polyurethane foam matrices

Summary An inoculum of initially freely suspended cell aggregates ofCapsicum frutescens was immobilised in porous polyurethane foam matrices. Subsequent growth and substrate consumption of these immobilised cells in batch culture were measured and compared with those of suspension cultures. The results showed that the maximum specific growth rate of freely suspended cells was slightly higher than that of immobilised cells but the overall growth patterns and final cell yields were similar.     

Thermally-dried free and immobilized kefir cells as starter culture in hard-type cheese production

In an attempt to seek for suitable dried cultures, thermally-dried kefir was employed as starter in hard-type cheese production and tested in cheeses ripened at 5, 18 and 22 °C. Both free and immobilised on casein kefir cells were used and compared to cheese made without starter culture. Cheese products made with free cells of kefir culture were characterized by longer preservation time, improved aroma, taste, texture characteristics and increased degree of openness. Volatile profiles obtained by GC/MS analysis revealed a 216% increase in total concentration of esters, organic acids, alcohols and carbonyl compounds between cheeses prepared with and without kefir culture.     

Continuous production of mixed lactic starters containing probiotics using immobilized cell technology

The production of a mixed lactic culture containing Lactococcus lactis subsp. lactis biovar. diacetylactis MD and Bifidobacterium longum ATCC 15707 was studied during a 17-day continuous immobilized-cell culture at different temperatures between 32 and 37 degrees C. The two-stage fermentation system was composed of a first reactor (R1) containing cells of the two strains separately immobilized in kappa-carrageenan/locust bean gum gel beads and a second reactor (R2) operated with free cells released from the first reactor. The system allowed continuous production of a concentrated mixed culture with a strain ratio whose composition depended on temperature and fermentation time. A stable mixed culture (with a 22:1 ratio of L. diacetylactis and B. longum) was produced at 35 degrees C in the effluent of R2, whereas the mixed culture was rapidly unbalanced in favor of B. longum at a higher temperature (37 degrees C) or L. diacetylactis at a lower temperature (32 degrees C). Strain redistribution in beads originally immobilizing pure cultures of L. diacetylactis or B. longum was observed. At the end of culture, the strain ratio (7:1 L. diacetylactis/B. longum) in bulk bead samples was similar to that of individual beads. The determination of the spatial distribution of the two strains in gel beads by immunofluorescence and confocal laser-scanning microscopy showed that bead cross-contamination was limited to a 100 microm peripheral layer. Data from this study validate a previous model for population dynamics and cell release in gel beads during mixed immobilized-cell cultures.     

Antigenic variability of Aspergillus fumigatus strains.

The effect of culture media, temperature of incubation, and continuous shaking of cultures, on the reactivity and yield of antigens of Aspergillus fumigatus were evaluated. It was found that AOAC medium was superior to Czapek medium and shake cultures yielded better results than stationary cultures. However, antigens from stationary cultures in AOAC medium incubated at 30 degrees C for 3 weeks were equally as good as antigens obtained from 2-week-old shake cultures. Antigens from 11 selected strains of Aspergillus fumigatus were used to test antibody activity in 33 sera from patients with various forms of aspergillosis and 35 normal controls by the agar gel double-diffusion method. The results showed that the reactivity of individual antigens varied from 42 to 87%, indicating that antigens from more than one strain of Aspergillus fumigatus may be used. The cross-reactivity between strains were studied by two-dimensional immunoelectrophoresis. The use of polyacrylamide gel electrophoresis and crossed immunoelectrophoresis in the quality assurance of Aspergillus antigens is discussed.     

Biosynthesis of the third component of complement in rat liver epithelial cell lines and its stimulation by effector molecules from cultured human mononuclear cells

Summary Rat liver epithelial cell lines, growing in a serum-supplemented medium, synthesize and secrete into the culture medium the third component of complement (C₃). We studied the regulation of C₃ production in this system. We found that human peripheral blood mononuclear leukocytes in culture released one or more soluble factors which stimulated rat liver epithelial cells to produce increased quantitites of C₃. This stimulting effect was strongly enhanced when the mononuclear cell cultures were treated with phytohemagglutinin, a T-lymphocyte mitogen. The factor(s) failed to enhance C₃ biosynthesis by rat dermal fibroblasts, which are known to produce this protein. This reveals a tissue-specific differential response between the fibroblasts and the liver epithelial cells. The physical and chemical characteristics, such as heat sensitivity, 2.8M ammonium sulphate precipitation, and lower activity after digestion by proteases unambiguously indicate that the effector molecules are proteins. When the crude supernatant of mononuclear leukocytes was fractionated by gel filtration, the stimulating factor(s) eluted as two peaks with apparent molecular weight of 25 to 60 and 15 to 20 kdalton, respectively. As to the cellular origin of the C₃-stimulating factor(s), several observations were made: (a) in separate cultures containing either T-cells or monocyte-enriched populations from the same sample of blood mononuclear cells, no activity was detected in the presence or absence of phytohemagglutinin, (b) conditioned media from each of these cultures could not substitute for the corresponding intact cell populations, and (c) the addition of purified T-cells to the monocyte-enriched population in the presence of phytohemagglutinin restored the production of the stimulating activity by the mixed culture. Finally, experiments were carried out to verify whether monokine interleukin 1 affects the hepatic C₃ biosynthesis. It was demonstrated that interleukin 1 enhanced this biosynthesis, but could not completely substitute for conditioned medium from stimulated mononuclear cells.     

Growth of the obligate methylotroph Methylobacillus flagellatum under stationary and nonstationary conditions during continuous cultivation

The growth characteristics of a chemostat culture of the obligate methylotrophic bacterium Methylobacillus flagellatum have been determined. Steady-state cultures growing at a rate of 0.73-0.74 h(-1), equal to the maximal growth rate, were obtained under oxyturbidostat cultivation conditions. The response of a chemostat culture to a pulse increase of methanol concentration was studied. It was shown that slow and rapidly growing cultures of M. flagellatum responded differently to pulse methanol addition. The growth characteristics of slow-growing cultures decreased after methanol addition compared to those of stationary chemostat cultures. The growth characteristics of rapidly growing cultures were practically unchanged with and without pulse methanol addition.     

Timing of initiation of chromosome replication in individual Escherichia coli cells.

The synchrony of initiation of chromosome replication at multiple origins within individual Escherichia coli cells was studied by a novel method. Initiation of replication was inhibited with rifampicin or chloramphenicol and after completion of ongoing rounds of replication the numbers of fully replicated chromosomes in individual cells were measured by flow cytometry. In rapidly growing cultures, with parallel replication of several chromosomes, cells will end up with 2n (n = 1, 2, 3) chromosomes if initiation occurs simultaneously at all origins. A culture with asynchronous initiation may in addition contain cells with irregular numbers (not equal to 2n) of chromosomes. The frequency of cells with irregular numbers of chromosomes is a measure of the degree of asynchrony of initiation. After inhibition of initiation and run-out of replication in rapidly growing B/r A and K-12 cultures, a small fraction of the cells (2-7%) contained 3, 5, 6 or 7 chromosomes. From these measurements it was calculated that initiation at four origins in a single cell occurred within a small fraction, 0.1, of the doubling time (tau). A dnaA(Ts) mutant strain grown at permissive temperature exhibited a very large fraction of cells with irregular numbers of chromosomes after drug treatment demonstrating virtually random timing of initiation. A similar pattern of chromosome number per cell was found after treatment of a recA strain.     

酿酒酵母突变株J-X25胞内合成GSH的研究

以筛选得高产谷胱甘肽(GSH)产生酵母甲硫氨酸缺陷型变株J-X25为试验菌株。对其培养条件进行研究,结果表明：发酵培养基的最适初始pH值为6.0、最佳发酵温度为30℃、最佳装液量为100ml/500ml、接种量10%、摇床转速为220r/min。在酵母细胞培养到对数期,加入过氧化氢刺激细胞发生应激反应和乳酸钠作为表面活性剂改变细胞通透性,GSH总量达到0.253g/L,比不添加两者情况下的GSH产量高出52％。结果表明优化培养条件后,J-X25胞内积累GSH比出发株提高79％。     

可产酸快生小菌的分离鉴定

目的：鉴定在实验过程中分离到的一株生长快速,并且可以将L-山梨糖转化为2-酮基-L-古龙酸的菌株。方法：将快生小菌传代,并进行产酸、抗菌谱、山梨糖脱氢酶活性等分析,通过PCR方法扩增并分析16S rDNA。结果：在传代过程中还分离得到了不产酸菌株;从快生小菌中扩增得到了普通酮古龙酸菌16S rDNA;从产酸菌中能够扩增得到包含酮古龙酸菌和乙酸钙不动杆菌的16S rDNA序列;在不产酸菌中只检测到乙酸钙不动杆菌的16S rDNA序列。结论：产酸的快生小菌可能是普通酮古龙酸菌和乙酸钙不动杆菌形成的融合细胞,这种融合细胞基因组表现为很不稳定,普通酮古龙酸菌基因组容易丢失,且丢失后也失去了产酸能力。     

Combination of extractive solvent addition and immobilization culture for continuous production of scopoletin by tobacco cells

Extractive solvent addition was combined with immobilization cultures of Nicotiana tabacum cells to produce scopoletin. Using various solvents, the partition coefficients of scopoletin between the solvent and water phases and the solvent toxicity to the cell viability were investigated. The effect of the solvent addition on cell growth and scopoletin production was elucidated in the suspension cultures. Coconut oil, one of the natural vegetable oils, was selected as the most suitable extractive solvent. The cells were immobilized in the calcium alginate gel bead coated with a cell-free gel film and then the batch cultures with the addition of various volumes of the coconut oil were performed. The total scopoletin production increased with the solvent volume according to the amount of scopoletin transferred from the medium to the solvent. The maximum productivity obtained in the batch immobilization cultures was about 16 times larger than that in the suspension culture without solvent. A continuous production system, in which the fresh solvent was supplied to the culture system and the solvent containing scopoletin was recovered from it, was constructed. The integrated scopoletin production in the effluent oil attained 2.21 mg/gDCW for 30 days at 100 cm(3)/day without cell leakage.     

丹参冠瘿组织高产株系选择和丹参酮的产生

丹参(Salvia miltiorrhiza)是治疗心血管系统疾病的重要中草药。前文我们已报道了丹参冠瘿组织培养的部分研究结果^[1].利用根癌农杆菌(Agrobacterium tumefaciens)Ti 质粒转化产生的冠瘿组织进行高产株系选择的研究还很少,本文报道丹参冠瘿组织高产株系选择和丹参酮产生的研究结果。