基于吐温-80下2株功能菌协同降解多环芳烃的特性研究

【目的】研究恶臭假单胞菌B6-2和克雷伯氏菌CW-D3T构建的混合功能菌对多环芳烃的协同修复效能,并探究非离子表面活性剂吐温-80对混菌降解多环芳烃的影响,以期为芳烃化合物的生物修复提供技术参考和理论依据。【方法】通过生长曲线及平板菌落计数法反映混菌生长情况及比例,从而评估混菌降解体系的可行性;通过高效液相色谱法探究各体系以及不同吐温-80浓度下混培体系对多环芳烃的降解效能;最后通过烷烃吸附法测定细胞表面疏水性,以探究吐温-80对混合功能菌降解多环芳烃的影响机制。【结果】等比例混合的2株菌共培养生长状态优于纯培体系,对混合多环芳烃(菲、荧蒽、芘)的降解率分别为33.4%、30.1%、28.6%(7 d),相较于菌CW-D3T,分别提高了1.31倍、1.46倍、1.42倍。混培体系中加入500 mg/L的吐温-80对菲、荧蒽、芘的降解率分别为47.7%、43.2%、38.8%(7 d),相较于对照组各提高了1.55倍、1.38倍、1.31倍,而更高浓度的吐温-80无明显促进作用或轻微抑制。添加吐温-80使菌CW-D3T和混菌的表面疏水性提高,而菌B6-2表面疏水性降低。结合细菌生长量分析...     

The role of iron in the bacterial degradation of organic matter derived from Phaeocystis antarctica

In high-nutrient low-chlorophyll areas, bacterial degradation of organic matter may be iron-limited. The response of heterotrophic bacteria to Fe addition may be directly controlled by Fe availability and/or indirectly controlled through the effect of enhanced phytoplankton productivity and the subsequent supply of organic matter suitable for bacteria. In the present study, the role of Fe on bacterial carbon degradation was investigated through regrowth experiments by monitoring bacterial response to organic substrates derived from Phaeocystis antarctica cultures set up in <1 nM Fe (LFe) and in Fe-amended (HFe) Antarctic seawater. Results showed an impact of Fe addition on the morphotype dominance (colonies vs. single cells) of P. antarctica and on the quality of Phaeocystis-derived organic matter. Fe addition leaded to a decrease of C/N ratio of Phaeocystis material. The bacterial community composition was modified as observed from denaturing gradient gel electrophoresis (DGGE) profiles in LFe as compared to HFe bioassays. The percentage of active bacteria as well as their specific metabolic activities (ectoenzymatic hydrolysis, growth rates and bacterial growth efficiency) were enhanced in HFe bioassays. As a consequence, the lability of Phaeocystis-derived organic matter was altered, i.e., after seven days more than 90% was degraded in HFe and only 9% (dissolved) and 55% (total) organic carbon were degraded in LFe bioassays. By inducing increased bacterial degradation and preventing the accumulation of dissolved organic carbon, the positive effect of Fe supply on the carbon biological pump may partly be counteracted.     

Modification of the technical properties of Lactobacillus johnsonii NCC 533 by supplementing the growth medium with unsaturated fatty acids

The aim of this study was to investigate the influence of supplementing growth medium with unsaturated fatty acids on the technical properties of the probiotic strain Lactobacillus johnsonii NCC 533, such as heat and acid tolerance, and inhibition of Salmonella enterica serovar Typhimurium infection. Our results showed that the membrane composition and morphology of L. johnsonii NCC 533 were significantly changed by supplementing a minimal Lactobacillus medium with oleic, linoleic, and linolenic acids. The ratio of saturated to unsaturated plus cyclic fatty acids in the bacterial membrane decreased by almost 2-fold when minimal medium was supplemented with unsaturated fatty acids (10 μg/ml). The subsequent acid and heat tolerance of L. johnsonii decreased by 6- and 20-fold when the strain was grown in the presence of linoleic and linolenic acids, respectively, compared with growth in oleic acid (all at 10 μg/ml). Following acid exposure, significantly higher (P < 0.05) oleic acid content was detected in the membrane when growth medium was supplemented with linoleic or linolenic acid, indicating that saturation of the membrane fatty acids occurred during acid stress. Cell integrity was determined in real time during stressed conditions using a fluorescent viability kit in combination with flow cytometric analysis. Following heat shock (at 62.5°C for 5 min), L. johnsonii was unable to form colonies; however, 60% of the bacteria showed no cell integrity loss, which could indicate that the elevated heat inactivated vital processes within the cell, rendering it incapable of replication. Furthermore, L. johnsonii grown in fatty acid-enriched minimal medium had different adhesion properties and caused a 2-fold decrease in S. enterica serovar Typhimurium UK1-lux invasion of HT-29 epithelial cells compared with bacteria grown in minimal medium alone. This could be related to changes in the hydrophobicity and fluidity of the membrane. Our study shows that technical properties underlying probiotic survivability can be affected by nutrient composition of the growth medium.     

Simultaneous activated carbon adsorption within a membrane bioreactor for an enhanced micropollutant removal

Significant adsorption of sulfamethoxazole and carbamazepine to powdered activated carbon (PAC) was confirmed by a series of adsorption tests. In contrast, adsorption of these micropollutants to the sludge was negligible. The removal of these compounds in membrane bioreactor (MBR) was dependent on their hydrophobicity and loading as well as the PAC dosage. Sulfamethoxazole exhibited better removal rate during operation under no or low (0.1 g/L) PAC dosage. When the PAC concentration in MBR was raised to 1.0 g/L, a sustainable and significantly improved performance in the removal of both compounds was observed - the removal efficiencies of sulfamethoxazole and carbamazepine increased to 82 ± 11% and 92 ± 15% from the levels of 64 ± 7%, and negligible removal, respectively. The higher removal efficiency of carbamazepine at high (1.0 g/L) PAC dosage could be attributed to the fact that carbamazepine is relatively more hydrophobic than sulfmethoxazole, which subsequently resulted in its higher adsorption affinity toward PAC.     

Differences in membrane proteins,cyclic AMP levels,and glucose transport between batch and chemostat cultures ofEscherichia coli

Escherichia coli bacteria were grown in minimal-salt medium with glucose as carbon source under either batch or chemostat culture conditions. The physiological state of chemostatgrown bacteria is significantly different from those grown in batch cultures. Differences were measured in outer membrane protein composition,K _m for glucose uptake, and intracellular cyclic AMP levels. Since chemostat growth conditions more closely resemble conditions bacteria encounter in nature, these physiological differences may be significant for bacterial adaptation and survival.     

Pelodictyon phaeoclathratiforme sp. nov., a new brown-colored member of the Chlorobiaceae forming net-like colonies

A new strain of the green sulfur bacteria was isolated from the monimolimnion of Buchensee (near Radolfzell, Lake Constance region, FRG). Single cells were rod-shaped, nonmotile and contained gas vacuoles. Typical net-like colonies were formed by ternary fission of the cells. As photosynthetic pigments bacteriochlorophylls a, e, isorenieratene and -isorenieratene were present. Sulfide, sulfur and thiosulfate were used as electron donors during anaerobic phototrophic growth. Besides carbon dioxide, acetate and propionate could serve as carbon sources under mixotrophic conditions in the light. Like all other members of the green sulfur bacteria, the new bacterium is strictly anaerobic and obligately phototrophic. The possession of gas vacuoles and the formation of net-like colonies and the guanine plus cytosine content of the DNA (47.9 mol% G+C) are typical characteristics of the genus Pelodictyon. Because of its photosynthetic pigments which differ from those of Pelodictyon clathratiforme, strain BU 1 represents a new species, P. Phaeoclathratiforme sp. nov.     

Synthesis of the unusual lipid bis(monoacylglycero)phosphate in environmental bacteria

The bacterial membrane is constantly remodelled in response to environmental conditions and the external supply of precursor molecules. Some bacteria are able to acquire exogenous lyso-phospholipids and convert them to the corresponding phospholipids. Here, we report that some soil-dwelling bacteria have alternative options to metabolize lyso-phosphatidylglycerol (L-PG). We find that the plant-pathogen Agrobacterium tumefaciens takes up this mono-acylated phospholipid and converts it to two distinct isoforms of the non-canonical lipid bis(monoacylglycero)phosphate (BMP). Chromatographic separation and quadrupole-time-of-flight MS/MS analysis revealed the presence of two possible BMP stereo configurations acylated at either of the free hydroxyl groups of the glycerol head group. BMP accumulated in the inner membrane and did not visibly alter cell morphology and growth behaviour. The plant-associated bacterium Sinorhizobium meliloti was also able to convert externally provided L-PG to BMP. Other bacteria like Pseudomonas fluorescens and Escherichia coli metabolized L-PG after cell disruption, suggesting that BMP production in the natural habitat relies both on dedicated uptake systems and on head-group acylation enzymes. Overall, our study adds two previously overlooked phospholipids to the repertoire of bacterial membrane lipids and provides evidence for the remarkable condition-responsive adaptation of bacterial membranes.     

Characterization of the cyanobacteria and associated bacterial community from an ephemeral wetland in New Zealand

New Zealand ephemeral wetlands are ecologically important, containing up to 12% of threatened native plant species and frequently exhibiting conspicuous cyanobacterial growth. In such environments, cyanobacteria and associated heterotrophs can influence primary production and nutrient cycling. Wetland communities, including bacteria, can be altered by increased nitrate and phosphate due to agricultural practices. We have characterized cyanobacteria from the Wairepo Kettleholes Conservation Area and their associated bacteria. Use of 16S rRNA amplicon sequencing identified several operational taxonomic units (OTUs) representing filamentous heterocystous and non‐heterocystous cyanobacterial taxa. One Nostoc OTU that formed macroscopic colonies dominated the cyanobacterial community. A diverse bacterial community was associated with the Nostoc colonies, including a core microbiome of 39 OTUs. Identity of the core microbiome associated with macroscopic Nostoc colonies was not changed by the addition of nutrients. One OTU was highly represented in all Nostoc colonies (27.6%–42.6% of reads) and phylogenetic analyses identified this OTU as belonging to the genus Sphingomonas. Scanning electron microscopy showed the absence of heterotrophic bacteria within the Nostoc colony but revealed a diverse community associated with the colonies on the external surface.     

Electrophoretic mobility and hydrophobicity as a measured to predict the initial steps of bacterial adhesion

The relationship between physiochemical surface parameters and adhesion of bacterial cells to negatively charged polystyrene was studied. Cell surface hydrophobicity and electrokinetic potential were determined by contact angle measurement and electrophoresis, respectively. Both parameters influence cell adhesion. The effect of the electrokinetic potential increases with decreasing hydrophobicity. Cell surface characteristics determining adhesion are influenced by growth conditions. At high growth rates, bacterial cells tend to become more hydrophobic. This fact can be of ecological significance for controlling the spread of bacteria throughout the environment.     

THE USE OF MICROBIAL MEMBRANES TO ACHIEVE ANAEROBIOSIS

Several years ago, it was observed that sterile microbial membrane preparations stimulated recovery of certain radiation-injured bacteria. Later it was noted that these same preparations reduce dissolved oxygen to water in a variety of environments, including bacteriological media. This reduction of oxygen is an enzymatic process and is influenced by parameters such as temperature, pH, and the availability of specific oxidizable substrates. Oxygenreducing membrane preparations can be made from several different bacterial species. When added to liquid or solid bacteriological media, membrane preparations rapidly produce and maintain anaerobic conditions favorable for the growth of a wide variety of oxygen-sensitive microorganisms. When used with a specifically designed disposable dish, membrane preparations allow the development of colonies of many anaerobic microorganisms on the surface of agar without the use of anaerobic hoods or other devices. In addition to providing conditions suitable for the growth of anaerobes, membrane preparations stimulate recovery of heat and cold injured bacteria of several different genera including facultative organisms. These results are reminiscent of the early observations regarding the recovery of radiation-injured bacteria. In addition to their usefulness in microbiology, oxygen-reducing membrane preparations have the potential for protecting a wide variety of oxygen-sensitive organic compounds.     

Isolation and metagenomic characterization of bacteria associated with calcium carbonate and struvite precipitation in a pure moving bed biofilm reactor-membrane bioreactor

A bench-scale pure moving bed bioreactor-membrane bioreactor (MBBR-MBR) used for the treatment of urban wastewater was analyzed for the identification of bacterial strains with the potential capacity for calcium carbonate and struvite biomineral formation. Isolation of mineral-forming strains on calcium carbonate and struvite media revealed six major colonies with a carbonate or struvite precipitation capacity in the biofouling on the membrane surface and showed that heterotrophic bacteria with the ability to precipitate calcium carbonate and struvite constituted ~7.5% of the total platable bacteria. These belonged to the genera Lysinibacillus, Trichococcus, Comamomas and Bacillus. Pyrosequencing analysis of the microbial communities in the suspended cells and membrane biofouling showed a high degree of similarity in all the samples collected with respect to bacterial assemblage. The study of operational taxonomic units (OTUs) identified through pyrosequencing suggested that ~21% of the total bacterial community identified in the biofouling could potentially form calcium carbonate or struvite crystals in the pure MBBR-MBR system used for the treatment of urban wastewater.     

Electrophoretic mobility and hydrophobicity as a measured to predict the initial steps of bacterial adhesion.

The relationship between physiochemical surface parameters and adhesion of bacterial cells to negatively charged polystyrene was studied. Cell surface hydrophobicity and electrokinetic potential were determined by contact angle measurement and electrophoresis, respectively. Both parameters influence cell adhesion. The effect of the electrokinetic potential increases with decreasing hydrophobicity. Cell surface characteristics determining adhesion are influenced by growth conditions. At high growth rates, bacterial cells tend to become more hydrophobic. This fact can be of ecological significance for controlling the spread of bacteria throughout the environment.     

Consequences of protist-stimulated bacterial production for estimating protist growth efficiencies

The trophic link between bacteria and bacterivorous protists is a complex interaction that involves feedback of inorganic nutrients and growth substrates that are immeadiately available for prey growth. These interactions were examined in the laboratory and in incubations of concentrated natural assemblages of bacterioplankton. Growth dynamics of estuarine and marine bacterivorous protists were determined in laboratory culture using Vibrio natriegens as prey and were compared to growth of protists on bacterioplankton assemblages concentrated by tangential flow filtration from four northwest Florida Estuaries. Biomass transfers from bacteria to protists were monitored by tracing elemental carbon and nitrogen in particulate fractions of protist added and grazer free controls. Gross growth efficiencies of the protists on naturally occurring bacteria were within the range determined in lab estimates of growth efficiency on cultured bacteria (50%). However, bacterial response to protist excretion products was different in the lab and field incubations, and bacterial growth contributed to the biomass available to protists in the field incubations. As determined by radioisotope-labeled substrate incorporation, a time lag in bacterial reponse to protist excretion products was observed for laboratory batch cultures, allowing accurate estimation of growth efficiency. In incubations with concentrated natural bacterial assemblages, bacterial growth response coincided with protist growth and excretion. The additional bacterial production on protist excretion products reached a maximum of 2–3-fold higher than protist-free controls. In addition, ammonium concentrations increased with protist grazing and growth in lab cultures, but ammonium excreted by protists in concentrates did not accumulate. The C:N values for the bacterial concentrates suggests that these bacteria were nitrogen limited. It is speculated that dissolved organic carbon, concentrated by tangential flow filtration (> 100,000 MW membrane) with the bacterioplankton, was utilized by bacteria when nitrogen was supplied as ammonium and amino acids from protist excretion. Thus, estimates of protist growth efficiency on naturally occurring bacterioplankton, corrected for protist-stimulated bacterial production, were in the range of 13–21%.     

Enrichment and identification of polycyclic aromatic compound-degrading bacteria enriched from sediment samples

The degradation of polycyclic aromatic compounds (PACs) has been widely studied. Knowledge of the degradation of PACs by microbial populations can be utilized in the remediation of contaminated sites. To isolate and identify PAC-degrading bacteria for potential use in future bioremediation programmes, we established a series of PAC enrichments under the same experimental conditions from a single sediment sample taken from a highly polluted estuarine site. Enrichment cultures were established using the pollutants: anthracene, phenanthrene and dibenzothiophene as a sole carbon source. The shift in microbial community structure on each of these carbon sources was monitored by analysis of a time series of samples from each culture using 16S rRNA polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE). Significantly, our findings demonstrate that shifts in the constituent species within each degradative community are directly attributable to enrichment with different PACs. Subsequently, we characterized the microorganisms comprising the degradative communities within each enrichment using 16S rRNA sequence data. Our findings demonstrate that the ability to degrade PACs is present in five divisions of the Proteobacteria and Actinobacteria. By determining the precise identity of the PAC-degrading bacterial species isolated from a single sediment sample, and by comparing our findings with previously published research, we demonstrate how bacteria with similar PAC degrading capabilities and 16S rRNA signatures are found in similarly polluted environments in geographically very distant locations, e.g., China, Italy, Japan and Hawaii. Such a finding suggests that geographical barriers do not limit the distribution of key PAC-degrading bacteria; this finding is in accordance with the Baas-Becking hypothesis “everything is everywhere; the environment selects” and may have significant consequences for the global distribution of PAC-degrading bacteria and their use in bioremediation.     

Identification and production of a rhamnolipidic biosurfactant by a Pseudomonas species

 A glycolipid-producing bacterium, Pseudomonas aeruginosa GL1, was isolated from the soil contaminated with polycyclic aromatic hydrocarbons (PAH) from a manufactured gas plant. The glycolipid produced was characterized in detail by chromatographic procedures as a mixture of four rhamnolipids, consisting of different associations of rhamnose and hydroxy fatty acids: the main component was monorhamnosyl di-3-hydroxydecanoic acid. The rhamnolipid composition presented marked analogies with a defined part of P. aeruginosa outer membrane lipopolysaccharides (lipopolysaccharide band A). Rhamnolipid production was stimulated under conditions of nitrogen limitation. Glycerol yielded higher productions than did hydrophobic carbon sources. Cell hydrophobicity decreased during growth on glycerol and on n-hexadecane whereas glycolipid production increased. P. aeruginosa GL1 was found to be unable to grow on a variety of 2, 3 and 4 cycle PAH. However, it was shown to persist after at least 12 subcultures in a bacterial population growing on a mixture of pure PAH, suggesting a physiological role for rhamnolipid as a means to enhance PAH availability in a mutualistic PAH-degrading bacterial community. Received: 4 July 1995/Received revision: 7 September 1995/Accepted: 13 September 1995     

Enhanced production of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) copolymer and antimicrobial yellow pigmentation from Cupriavidus sp. USMAHM13 with antibiofilm capability

Antibiofilm polymers have the ability to inhibit bacterial biofilm formation, which is known to occur ubiquitously in the environment and pose risks of infection. In this study, production of P(3HB-co-4HB) copolymer and antimicrobial yellow pigment from Cupriavidus sp. USMAHM13 are enhanced through medium optimization. Before the improvement of yellow pigment production, screening for the best additional supplement was performed resulting in high-yield yellow pigmentation using yeast extract with optimum concentration of 2?g/L. Effects of different concentrations of 1,4-butanediol, ammonium acetate, and yeast extract were studied using central composite design. Under optimal conditions, 53?wt% of polyhydroxyalkanoate (PHA) content, 0.35?g/L of pigment concentration, and 5.87?g/L of residual biomass were achieved at 0.56?wt% C of 1,4-butanediol, 1.14?g/L of ammonium acetate, and 2?g/L of yeast extract. Antibiofilm tests revealed that the yellow pigment coated on P(3HB-co-4HB) copolymer had significant effect on the inhibition of bacteria proliferation and colonization from 6?hr onward reaching 100% inhibition by 12?hr, hence effectively inhibiting the biofilm formation.     

Diurnal variations in the auto- and heterotrophic activity of cyanobacterial phycospheres (Gloeotrichia echinulata) and the identity of attached bacteria

1. We assessed the role of cyanobacterial–bacterial consortia (Gloeotrichia echinulata phycospheres) for net changes in inorganic carbon, primary production (PP) and secondary production in Lake Erken (Sweden). 2. At the time of sampling, large colonies of G. echinulata formed a massive bloom with abundances ranging from 102 colonies L^?1 in the pelagic zone to 5000 colonies L^?1 in shallow bays. These colonies and their surrounding phycospheres contributed between 17 and 92% of total PP, and phycosphere‐associated bacteria contributed between 8.5 and 82% of total bacterial secondary production. PP followed a diurnal cycle, whereas bacterial production showed no such pattern. Over a 24 h period, carbon dioxide measurements showed that the phycospheres were net autotrophic in the top layer of the water column, whereas they were net heterotrophic below 2 m depth. 3. Sequencing and phylogenetic analysis of 16S rRNA genes of attached bacteria revealed a diverse bacterial community that included populations affiliated with Proteobacteria, Bacteriodetes, Acidobacteria, Fusobacteria, Firmicutes, Verrucomicrobia, and other Cyanobacteria. 4. Compared with their planktonic counterparts, bacteria associated with cyanobacterial phycospheres had lower affinity for arginine, used as a model compound to assess uptake of organic compounds. 5. Extrapolation of our data to the water column of lake Erken suggests that microorganisms that were not associated with cyanobacteria dominated CO₂ production at the ecosystem scale during our experiments, as CO₂ fixation balanced CO₂ production in the cyanobacterial phycospheres.     

Microbial activity and phosphorus dynamics in eutrophic lake sediments enriched with Microcystis colonies

1. Sediments from hypereutrophic Lake Vallentunasjön were enriched with Microcystis colonies from the lake water, thereby simulating the conditions after the autumn sedimentation. Release of phosphorus to the overlying lake water was followed during 2–3 weeks in the laboratory. X-ray microanalysis of individual Microcystis and bacterial cells, and chemical phosphorus fractionation, were used to assess the phosphorus pool size in different fractions of the sediment. 2. Benthic Microcystis colonies, most of these having survived within the sediment for 1 year or more, were less susceptible to decomposition, and the specific growth rate of bacteria in their mucilage was lower than for other sediment bacteria. 3. Pelagic Microcystis colonies from late August were resistant to decomposition, when placed on the sediments. When Microcystis colonies from a declining pelagic population in October were added to the sediments, however, a substantial fraction of these colonies was decomposed. The specific growth rate of mucilage bacteria was five times higher than for other sediment bacteria. 4. Release of molybdate-reactive phosphorus to the overlying lake water was larger from sediment cores enriched with Microcystis colonies than from control cores. Chemical phosphorus fractionation showed a decrease in organic-bound phosphorus (residual P). 5. X-ray microanalysis showed that the phosphorus bound in Microcystis cells decreased by -0.300 mg g^?1 DW in the October experiment, due both to a decrease in biomass (i.e. mineralization) and to a decrease in phosphorus content in the remaining cells. Heterotrophic bacteria increased their cellular concentration of phosphorus. The net release of phosphorus from the Microcystis and bacterial pools corresponded to 74% of the decrease of organic-bound phosphorus in the chemical phosphorus fractionation, and to 65% of the decrease of total phosphorus in the upper 0–1 cm of the sediment. 6. Benthic bacteria and cyanobacteria may thus contribute significantly to changes in phosphorus content and turnover of the sediment by changes in their biomass, turnover rate and cellular phosphorus content.     

LapF and Its Regulation by Fis Affect the Cell Surface Hydrophobicity of Pseudomonas putida

The ability of bacteria to regulate cell surface hydrophobicity is important for the adaptation to different environmental conditions. The hydrophobicity of cell surface can be determined by several factors, including outer membrane and surface proteins. In this study, we report that an adhesin LapF influences cell surface hydrophobicity of Pseudomonas putida. Cells lacking LapF are less hydrophobic than wild-type cells in stationary growth phase. Moreover, the overexpression of the global regulator Fis decreases surface hydrophobicity by repressing the expression of lapF. Flow cytometry analysis revealed that bacteria producing LapF are more viable when confronted with methanol (a hydrophilic compound) but are more susceptible to 1-octanol (a hydrophobic compound). Thus, these results revealed that LapF is the hydrophobicity factor for the cell surface of P. putida.     

Enrichment culture and isolation of slow-growing bacteria

 Enrichment containing large numbers of slow-growing bacteria was developed by repeated batch culture under high biomass concentrations (more than 10 000 mg biomass/l). The characteristics of slow-growing bacterial populations were elucidated by application of colony-forming-curve (CFC) analysis. The CFC were obtained by counting the number of visible colonies on agar plates at successive intervals. The enrichment consisted of several groups with different colony-forming rates and the slow-growing bacteria appeared on cell extract/agar plates after 7 days of incubation. It was found that large numbers of slow-growing bacteria survived under starvation conditions. One of the slow colony-forming bacteria, strain TI-X7, was tentatively identified as being of the genus Micrococcus. The enrichment contained a large amount of Micrococcus-like tetrad cells. The dialysate fractions in excess cell extract, permeable through dialysis tubing, were extremely effective for growth of strain TI-X7. Received: 15 December 1995/Accepted: 20 February 1996