Factors influencing the cultivability of lake water bacteria

Counting bacteria in natural water samples by cultivation yields only low recovery efficiencies (ca. 1%), compared to total counts obtained after 4,6-diamidino-2-phenylindol (DAPI) staining. In order to optimize the cultivation of heterotrophic planktonic bacteria from Lake Constance (Germany), selected parameters of the medium composition were modified. The most important factor was the concentration of organic substrate (nutrient broth plus yeast extract), which significantly influenced the "most probable number" obtained in liquid growth medium. Reduced oxygen concentrations (3-12%) lowered the "most probable number". Addition of N-acyl homoserine lactones to the medium increased the cultivability slightly. Low substrate concentrations [0.03-0.06% (w/v)], an incubation atmosphere of 21% oxygen at 16 degrees C for 4 weeks were optimal and increased the cultivability ("most probable number" related to total bacterial counts) to an average cultivability of 18+/-11%, (n=8). The results indicate that cultivabilities of heterotrophic bacteria from lakewater samples can be significantly increased by modifying the cultivation methods.     

Optimization of the extracellular production of a bacterial phytase with Escherichia coli by using different fed-batch fermentation strategies

The extracellular production of Escherichia coli phytase was studied in fed-batch fermentations. Two different feeding strategies were compared: control by keeping the glucose concentration constant, and control by keeping a low constant oxygen level in the medium. For the feeding control based on glucose concentration, a recently developed rapid glucose controlling system was tested for the first time in bacterial cultivations and used to establish the fermentative production of extracellular phytase with E. coli. High activity levels (120 U ml(-1)) at short cultivation times (14 h) were obtained. Even higher activity levels - albeit at longer cultivation times - were reached by applying a feeding control, the main characteristic of which was a constant low oxygen concentration. The optimum oxygen level for the production of phytase was in the range of 5-10% saturation.     

Bacterial oxidation of cholic acid byArthrobacter sp

Some bacteria possess the ability to convert cholic acid (CA) to pharmacologically important chenodeoxycholic acid (CDCA) or to its precursor 3α, 7α-dihydroxy-12-oxo-5β-cholan-24-ic acid (12-oxo-CDCA). The ability of several bacterial strains to convert CA to 12-oxo-CDCA was tested in our work. The highest conversion was reached by one strain ofArthrobacter sp. which converted up to 61% CA to 12-oxo-CDCA in a complex medium containing 4% (W/V) CA under aerobic conditions. 12-Oxo-CDCA was further oxidized to 3,12-dioxo-7ì-hydroxy-5β-cholan-24-ic acid (3,12-dioxo-7-OHCA) that represented up to 10% of total bile acids in the cultivation medium after a 1-d cultivation. The concentration of 12-oxo-CDCA increased rapidly during the first hours of cultivation, maximal yields were achieved between 20 and 24 h of cultivation and they decreased after that due to further bacterial oxidation. Dedicated to Professor C. John on the occasion of his 75th birthday     

A balanced DO-stat and its application to the control of acetic acid excretion by recombinant Escherichia coli

During fed-batch cultivation of a recombinant Escherichia coli AT2471 harboring plasmid pSY130-14 for phenylalanine production, a large amount of acetic acid was excreted by the cells and accumulated in the culture medium. Acetic acid concentration reached 30-35 g/L at the end of a process conducted without special precautions for the reduction of this excretion. Cell growth stopped when acetic acid concentration was about 15 g/L, resulting in poor growth, 16 g/L cell concentration, and poor production - 8 g/L phenylalanine. A novel control strategy, called a balanced DO-stat. was developed to prevent acetic acid excretion. It represents a model-independent two-loop control structure, which is simple, reliable, and convenient for computer application. Using the balanced DO-stat, implemented in a computer control system, acetic acid concentration was kept at zero during the entire cultivation period. As a result, the cell concentration increased to 36 g/L and phenylalanine concentration reached 24 g/L. Aside from the phenylalanine fermentation, the proposed control approach might be applied to cultivation of other bacterial and yeast strains which have similar mechanism of the excretion of fermentative by-products.     

新组合菌系SCB329-SCB933利用L-山梨糖发酵生产维生素C前体——2-酮基-L-古龙酸的研究 Ⅱ.利用L-山梨糖批加技术实现高糖发酵

新组合菌系氧化葡萄糖酸杆菌SCB329-苏芸金芽孢杆菌SCB933能在较长时间内保持高的转化活力且具有极强的抗杂菌污染的特性。在一次投糖分批发酵的基础上,探索在控制溶氧、pH、温度等条件下,分批加入L-山梨糖发酵生产2-酮基-L-古龙酸新工艺。采用新工艺,既充分利用了菌系的优良特性,又避免了高糖浓度可能对菌系造成的不良影响。L-山梨糖最终浓度达到14%（w/v）,产酸120—135g/l,转化率90％左右,发酵周期40—65h。     

Microbial destruction of cyanide and thiocyanate

The role played by a bacterial community composed of Pseudomonas putida, strain 21, Pseudomonas stutzeri, strain 18, and Pseudomonas sp., strain 5, and by physical and chemical factors in the degradation of CN- and SCN- was studied. It was shown that the degradation of CN- is determined both by the action of bacteria and by abiotic physical and chemical factors (pH, O2, temperature, the medium agitation rate, etc.). The contribution of chemical degradation was found to increase drastically at pH below 9.0; when air was blown through the medium (irrespective of the pH value); under active agitation of the medium; and when the medium surface interfacing air was increased. Even at elevated pH values (9.0-9.2), suboptimal for bacterial growth, the microbial degradation could account for at most 20-25 mg/l of CN-, regardless of its initial concentration. When CN- and SCN- were concurrently present in the medium, the former compound was the first to be degraded by microorganisms. The rate of bacterial degradation of SCN- under continuous cultivation in a chain of reactors was found to depend on its concentration, the medium flow rate, agitation rate, and the pattern of carbon source supply and could exceed 1 g/(1 day). CN- and SCN- are utilized by bacteria solely as nitrogen sources. The mechanism of CN- and SCN- degradation by the microbial community is discussed.     

Cyclic AMP and acyl homoserine lactones increase the cultivation efficiency of heterotrophic bacteria from the central Baltic Sea

The effect of signal molecules on the cultivation efficiency of bacteria from the Gotland Deep in the central Baltic Sea was investigated. Numbers of cultivated cells were determined by the most-probable-number (MPN) technique. Artificial brackish water supplemented with different carbon substrates at low concentrations (200 microM each) was employed as the growth medium. Compared to the results of previous studies, this approach yielded significantly higher cultivation efficiencies (up to 11% in fluid media). A further and pronounced increase in cultivation success was accomplished by the addition of cyclic AMP (cAMP), N-butyryl homoserine lactone, or N-oxohexanoyl-DL-homoserine lactone at a low concentration of 10 microM. The most effective inducer was cAMP, which led to cultivation efficiencies of up to 100% of total bacterial counts. From the highest positive dilutions of these latter MPN series, several strains were isolated in pure culture and one strain (G100) was used to study the physiological effect of cAMP. Dot blot hybridization revealed, however, that strain G100 represented only a small fraction of the total bacterial community. This points towards an inherent limitation of the MPN approach, which does not necessarily recover abundant species from highly diverse communities. Bacterial cells of strain G100 that were starved for 6 weeks attained a higher growth rate and a higher biomass yield when resuscitated in the presence of cAMP instead of AMP.     

Intensification of phenol biodegradation by humic substances

Phenol biodegradation was carried out in a batch system by the bacterial strain Cupriavidus metallidurans in the presence of potassium humate that was prepared by alkaline extraction from oxyhumolite. The experiments were focused on the assessment of the humate effect on biodegradation activity of the tested bacterial strain. The achieved results demonstrated that the humate has a positive influence on the biodegradation of phenol and reduces the incubation time necessary for phenol removal. Higher biodegradation rate and more intensive growth were observed during the cultivation in presence of humate in comparison to the cultivation without its addition. Adsorption of the humate on bacterial biomass was observed as well. Subsequently, a phenol biodegradation testing in a continuous-flow system using a biofilm reactor was also carried out. Although the reactor was inoculated by C. metallidurans only, the microbial composition under an aerobic non-aseptic condition during this long-term cultivation changed. The phenol removal efficiency obtained in the biofilm reactor was higher than 92% when phenol concentration in a treated medium was 1200 mg l⁻¹.     

Characteristics of growth and changes in the ultrafine structure of bacteria in the course of continuous cultivation on media containing ethanol

A large number of bacterial strains assimilating chemical ethanol has been isolated using an original technique. Active growth of strains belonging to the genera Pseudomonas and, particularly, Acinetobacter was registered on mineral media containing ethanol. A mathematical model was constructed select a strain of Acinetobacter calcoaceticus K-9 during its continuous cultivation on media containing ethanol. The model makes it possible to determine conditions for producing a present amount of the biomass, the percentage of its yield, and the produc;iveness as a function of the dilution rate, temperature, and the concentration of ethanol and phosphoric acid in the medium. The main characteristics of the growth process in the studied factor space were established. The optimum conditions were calculated for growth of the strain with respect to each of the criteria. Under various conditions of bacterial growth, changes in the morphology and ultra-fine structure of the cells correlated with their physiological activity. The volume of the cells increased with the rate of dilution of the medium: the process can be described by a saturation curve. The presence of mesosomal structures is typical of the cells growing at low flow rates.     

酵母浸出粉在奈瑟脑膜炎双球菌培养中的比较观察

目的探索A群,C群奈瑟脑膜炎双球菌(简称流脑)多糖疫苗生产中奈瑟脑膜炎双球菌培养的最适培养基。方法在培养基配制中用增减酵母浸出粉的方法制备相应的培养基,8h收菌,通过菌体的收获量并参考多糖量来确定较好的培养基配比。结果不同培养基用于A群、C群奈瑟脑膜炎菌培养8h后均有收获,其中2号培养基(酵母浸出粉)培养的菌体的浓度明显高于1号和3号培养基,它们之间有显著性差异(P<0.05)此种培养基能提高奈瑟脑膜炎双球菌的产量。结论添加酵母浸出粉的培养基可作为A群、C群奈瑟脑膜炎双球菌培养的最适培养基。     

Cultivation on cellophane agar as a method for the assay of bacterial enzymes

Production of bacterial enzymes in a liquid medium and on cellophane-agar plates is compared. Using neutral proteinase, urease, α-amylase and thiosulfate sulfurtransferase as examples it was demonstrated that the cultivation on cellophane-agar plates yields higher concentrations of the enzymes and that the enzyme production is detected in a higher number of cultures. The method might be useful for the characterization of bacterial communities.     

一株降血压功能乳酸菌在模拟胃肠环境中抗性的研究

目的研究具有降血压功能乳酸菌DM9057株对pH、胆汁盐、胃蛋白酶及胰蛋白酶的耐受性。方法以MRS为基础培养基,模拟胃肠环境。结果 DM9057在pH2~3、胆汁盐浓度0.1%~0.3%、胃蛋白酶0.592~1.482μg/mL和胰蛋白酶14.24~72.32 U/g的条件下,37℃培养4 h,活菌数均能达到107CFU/mL以上。结论乳酸菌DM9057能顺利通过胃肠道,达到促进健康的作用。     

Study of the reciprocal relationship of the exoantigens ofClostridium perfringens type A in the culture medium and the bacterial cells during cultivation

The times of the active release ofClostridium perfringens antigens—lecithinase, collagenase and alkaline proteinase—were determined. It was found that lecithinase release attained maximum values after 4–9 hours' cultivation of the microorganisms and stopped completely after 24–36 hours. The course of collagenase release was the same. Maximum proteinase release was determined within 36 to 48 hours' cultivation ofClostridium perfringens. Preformed lecithinase disappeared very rapidly from the culture medium. The presence of the components of the exoantigen complex was determined in the bacterial cell cytoplasm by the immunoadsorption method. Their distribution in the fractions isolated by differential high speed centrifugation of homogenates of disintegrated bacterial cells was studied. Their ribosomal fraction, represented by four groups of ribosomes with the maximum sedimentation coefficients 110 and 130S, contains alkaline proteinase. Its activity diminishes during cultivation of the microorganism and proteolytic activity accumulates in the hyaloplasm. The absence of lecithinase and collagenase in the bacterial cells was demonstrated. The plate will be found at the end of the issue.     

The effect of the oxygen transfer rate (OSR) on the formation of cellulases by Trichoderma viride in submersion culture

The formation of cellulases by Trichoderma viride in a medium containing cellulose as a sole source of carbon depends on the oxygen transfer rate (OSR); the OSR, on the other hand, depends on the concentration of cellulose in the medium because the concentration of cellulose strongly affects the viscosity of the medium. In the work presented here, the dependence has been determined for the oxygen transfer rate on geometric relations and viscosity in cellulose-containing media during cultivation in shaken flasks, and the oxygen transfer rate on N(Re') N(G') and N(a) during cultivation in a laboratory fermentor of 3000-mL volume. Two cellulosic materials have been compared with a different effect on viscosity: Microcrystalline beach cellulose and fibrous cellulose. It has been found that, in an applicable range of concentration, microcrystalline cellulose does not affect the oxygen transfer rate (at concentrations up to 3%). Fibrous cellulose increases the OSR during cultivation in shake flasks but decreases it during civilization in fermentors. On the basis of these results, the optimization has been carried out on the cultivation conditions in fermentors.     

Factors Influencing Agnotobiotic Cultures of the Rumen Ciliate, Entodinium simplex

The nutrition of Entodinium simplex was studied, with foliage of bluegrass (Poa pratense), perennial ryegrass (Lolium perenne), and grains of wheat (Triticum vulgare) as substrates in agnotobiotic cultures. Entodinium grew poorly when the substrates were autoclaved; better growth was obtained when the substrates were sterilized with ethylene oxide vapor. The concentration of ethylene oxide and the amount of moisture influenced the sterility and nutritional adequacy of the treated substrate. Autolysates and hydrolysates of mixed rumen protozoa stimulated growth. Protozoal extracts did not replace factors destroyed by autoclaving. Clarified rumen fluid assisted the cultivation of entodinia from small inocula but was detrimental to established cultures. Success of cultures was influenced by the medium used to grow the inoculum as well as by the medium inoculated. The results indicated that the composition of the bacterial population influences the growth of E. simplex.     

Acrylamide and Acrylic Acid Biodegradation by <Emphasis Type="Italic">Alcaligenes faecalis</Emphasis> 2 Planktonic Cells and Biofilms

The ability of the Alcaligenes faecalis 2 strain to utilize acrylamide and acrylic acid upon cultivation with these compounds as the only sources of carbon and energy has been investigated. Complete utilization of the acrylic acid present in the medium at concentrations below 0.113 g/L was observed by cultivation day 5, at a concentration of 0.225 g/L by day 7, and at a concentration of 0.45 g/L by day 17. Complete utilization of the acrylamide present in the medium at concentrations below 0.4 g/L was observed by day 5, at a concentration of 0.9 g/L by day 7, and at a concentration of 1.8 g/L by day 20. Importantly, bacterial growth did not start before complete transformation of acrylamide into acrylic acid. The rate of acrylamide transformation by growing bacteria and a cell suspension in the stationary growth phase amounted to 12.5 mg/L h at a cell concentration of 610 mg/L and 300 mg/L h, at a concentration of 1500 mg/L. A. faecalis 2 cells immobilized on BVV-22 basalt fibers and Carbopon-B-aktiv at concentrations of 3000 and 800 mg dry cells/L, respectively, transformed acrylamide at a rate of 1200 mg/L h.     

Extracellular secretion of levansucrase from Zymomonas mobilis in Escherichia coli

Secretion of levansucrase from Zymomonas mobilis in Escherichiacoli by glycine supplement was investigated. A significant amount of levansucrase (about 25% of total activity) was found in intact whole-cells. Cell fractionation experiments showed that levansucrase was found both in the periplasmic space and in the cytoplasmic fraction of E. coli. None or only trace amounts of levansucrase was detected in the extracellular culture broth at 24 h of cultivation and it accrued with the increasing concentration of glycine in the culture medium and duration of the culture period. Optimal glycine concentration for the maximum secretion of levansucrase was in the range of 0.8-1%, in which approximately 20-50% of levansucrase was released into the extracellular fraction at 24 h of cultivation, although glycine retarded the bacterial growth.     

Cell-free synthesis of silver nanoparticles in spent media of different Aspergillus species

The recovery and valorization of metals and rare earth metals from wastewater are of great importance to prevent environmental pollution and recover valuable resources. Certain bacterial and fungal species are capable of removing metal ions from the environment by facilitating their reduction and precipitation. Even though the phenomenon is well documented, little is known about the mechanism. Therefore, we systematically investigated the influence of nitrogen sources, cultivation time, biomass, and protein concentration on silver reduction capacities of cell-free cultivation media (spent media) of Aspergillus niger, A. terreus, and A. oryzae. The spent medium of A. niger showed the highest silver reduction capacities with up to 15 μmol per milliliter spent medium when ammonium was used as the sole N-source. Silver ion reduction in the spent medium was not driven by enzymes and did not correlate with biomass concentration. Nearly full reduction capacity was reached after 2 days of incubation, long before the cessation of growth and onset of the stationary phase. The size of silver nanoparticles formed in the spent medium of A. niger was influenced by the nitrogen source, with silver nanoparticles formed in nitrate or ammonium-containing medium having an average diameter of 32 and 6 nm, respectively.     

Screening of the common culture conditions affecting crystallinity of bacterial cellulose

By analyzing with X-ray diffraction and FT-IR spectroscopy, the main effective factors among common culture conditions on crystallinity index and I _α fraction of cellulose produced by Gluconacetobacter xylinus were examined with Plackett–Burman design experiment. Varying pH value in the medium by adjusting the composition of citrate buffer or by adding HCl/NaOH solution indicates it is the content of citrate buffer rather than its function of pH buffering that gives the influence on crystallinity. Further experiment reveals that Na⁺ concentration of 0.174 mol/l in medium with citrate buffer added would decrease the crystallinity index significantly. Comparison of carbon sources shows that fructose leads to a higher crystallinity index than glucose, which suggests a relationship between crystallinity and production speed of bacterial cellulose affected by carbon sources. An interesting phenomenon was that a longer period of cultivation would decrease the crystallinity of bacterial cellulose. The reason is assumed to be the dense network of cellulose formed by bacterial cells that restrict the motion of themselves as the incubation period extends. Though the effect of inoculum age is still unclear, the influence on crystallinity of bacterial cellulose caused by variation of some ordinary culture conditions can be drawn out from data of this work.     

Long-Term Continuous Cultivation of Clostridium beijerinckii in a Two-Stage Chemostat with On-Line Solvent Removal

A two-stage continuous cultivation experiment with Clostridium beijerinckii NRRL B592 is described. This strain maintained its ability to produce neutral solvents (acetone, n-butanol, and ethanol) at an overall dilution rate of 0.13 h(sup-1) and achieved an average overall solvent concentration of 9.27 g/liter and an overall solvent productivity of 1.24 g/liter/h for more than 100 overall retention times. The experiment was performed without pH control on a semisynthetic medium containing yeast extract, and product inhibition was the limiting factor. Solid carrier material was present in both stages, and the solvent productivity in both stages was similar. A membrane evaporation module integrated into the recirculation loop of a second-stage bioreactor after 2,166 h increased solvent productivity and improved the yield of solvents by about 40%. The membrane reduced the concentration of solvents, which would otherwise inhibit the fermentation. Additionally, the integrated membrane evaporation dampened metabolic oscillations, which are characteristic of continuous cultivation of clostridia. It was also demonstrated that a moderate concentration buildup (approximately 30% of bioreactor inflow) caused by water flux through the membrane caused no detrimental effects to the bacterial cells. However, much higher water fluxes through the membrane, associated with a much more dramatic increase in the concentration of salts in the medium, did appear to favor cell degeneration.