Bacteria–algae association in batch cultures of phytoplankton from a tropical reservoir: the significance of algal carbohydrates

Summary 1. The dissolved organic matter, especially carbohydrates, released by phytoplanktonic organisms may be ecologically important, through its influence on carbon cycling and microbial diversity. Here axenic cultures of three phytoplanktonic species, Cryptomonas tetrapyrenoidosa (Cryptophyceae), Staurastrum orbiculare (Zygnematophyceae) and Thalassiosira duostra (Bacillariophyceae), were inoculated with a microbial community from the same habitat in which the algae had been isolated (a tropical reservoir). Replicate cultures were not inoculated.
2. In both axenic and co-inoculated cultures, phytoplanktonic density and extracellular carbohydrate production were monitored microscopically and by high performance liquid chromatography with a pulse amperometric detector, respectively. Bacterial population density was also monitored by epifluorescence microscope in the microbial co-inoculated cultures.
3. Both bacterial and phytoplanktonic densities increased for 11 days in all cases. The use of extracellular carbohydrates by bacteria was also showed for all phytoplanktonic species. Of the three species of phytoplankton, only T. duostra had a faster population growth in the presence of bacteria, and reached a higher biomass than in axenic culture.     

Morphological diversity of freshwater bacteria belonging to theBlastocaulis-planctomyces group as observed in natural populations and enrichments

The nonprosthecate, stalked, budding, aquatic bacteria presently thought to belong to theBlastocaulis-Planctomyces group are morphologically quite diverse—as can be seen in high resolution transmission electron micrographs of these organisms as they occur in freshwater samples and enrichments prepared from them. Stalk fine structure is of several different types: some of these organisms possess flattened (in some cases, possibly tubular) ribbon-like stalks made up of parallel arrays of subunit fibrils of varying dimensions; others have stalks composed of twisted (ropelike) bundles of relatively coarse individual fibrils. Diversity exists in various other appendages (flagella, spires, fimbriae, holdfasts), cell envelope features (including the peculiar crateriform structures), degree of rosette formation, and cell shape. Based on this diversity, four morphotypes have been delineated among the members of this group. The axenic cultures we have isolated are being characterized within the morphotype framework.     

Isolation and characterization of methanogenic bacteria from rice paddies

Abstract Enrichment cultures for H₂-CO₂, methanol- or acetate-utilizing methanogens were prepared from two rice field soil samples. All the cultures except one acetate enrichment showed significant methane production. Pure cultures of Methanobacterium - and Methanosarcina -like organisms were isolated from H₂-CO₂ and methanol enrichment cultures, respectively, and were characterized for various nutritional and growth conditions. The organisms had an optimal pH range of 6.4–6.6 and a temperature optimum of 37°C. The Methanobacterium isolates were able to utilize H₂-CO₂ but no other substrates as sole energy source, while the Methanosarcina isolates were able to utilize methanol, methylamines or H₂-CO₂ as sole energy sources. Both Methanobacterium isolates and one isolate of Methanosarcina were able to use dinitrogen as the sole source of nitrogen for growth. The isolates used several sulfur compounds as sole sources of sulfur.     

Characterization of plant polysaccharide- and mucin-fermenting anaerobic bacteria from human feces

Organisms able to grow on arabinogalactan, pectin, xylan, wheat bran, guar, apple cell walls, and mucin were isolated by enrichment from human feces. The number of polysaccharide fermenters and the properties of the predominant bacteria varied between subjects. The ability to use one polysaccharide was not related to the ability to use others. Some organisms (e.g., Bacteroides spp.) isolated on other substrates also utilized mucin, but were not isolated in the mucin enrichment. The mucin fermenters isolated by enrichment had a very restricted ability to utilize complex polysaccharides and their constituent monosaccharides, suggesting that the presence of plant polysaccharides in the human colon is unlikely to prevent the use of colonic mucin as an energy source by bacteria. Characterization with a range of biochemical tests showed that many of the isolates, but especially the mucin fermenters, did not resemble organisms described previously.     

Biochemical evidence for formate transfer in syntrophic propionate-oxidizing cocultures of Syntrophobacter fumaroxidans and Methanospirillum hungatei

The hydrogenase and formate dehydrogenase levels in Syntrophobacter fumaroxidans and Methanospirillum hungatei were studied in syntrophic propionate-oxidizing cultures and compared to the levels in axenic cultures of both organisms. Cells grown syntrophically were separated from each other by Percoll gradient centrifugation. In S. fumaroxidans both formate dehydrogenase and hydrogenase levels were highest in cells which were grown syntrophically, while the formate-H(2) lyase activities were comparable under the conditions tested. In M. hungatei the formate dehydrogenase and formate-H(2) lyase levels were highest in cells grown syntrophically, while the hydrogenase levels in syntrophically grown cells were comparable to those in cells grown on formate. Reconstituted syntrophic cultures from axenic cultures immediately resumed syntrophic growth, and the calculated growth rates of these cultures were highest for cells which were inoculated from the axenic S. fumaroxidans cultures that exhibited the highest formate dehydrogenase activities. The results suggest that formate is the preferred electron carrier in syntrophic propionate-oxidizing cocultures of S. fumaroxidans and M. hungatei.     

Characterization of plant polysaccharide- and mucin-fermenting anaerobic bacteria from human feces.

Organisms able to grow on arabinogalactan, pectin, xylan, wheat bran, guar, apple cell walls, and mucin were isolated by enrichment from human feces. The number of polysaccharide fermenters and the properties of the predominant bacteria varied between subjects. The ability to use one polysaccharide was not related to the ability to use others. Some organisms (e.g., Bacteroides spp.) isolated on other substrates also utilized mucin, but were not isolated in the mucin enrichment. The mucin fermenters isolated by enrichment had a very restricted ability to utilize complex polysaccharides and their constituent monosaccharides, suggesting that the presence of plant polysaccharides in the human colon is unlikely to prevent the use of colonic mucin as an energy source by bacteria. Characterization with a range of biochemical tests showed that many of the isolates, but especially the mucin fermenters, did not resemble organisms described previously.     

Hydrogen as an electron donor for dechlorination of tetrachloroethene by an anaerobic mixed culture.

Hydrogen served as an electron donor in the reductive dechlorination of tetrachloroethene to vinyl chloride and ethene over periods of 14 to 40 days in anaerobic enrichment cultures; however, sustained dechlorination for more extended periods required the addition of filtered supernatant from a methanol-fed culture. This result suggests a nutritional dependency of hydrogen-utilizing dechlorinators on the metabolic products of other organisms in the more diverse, methanol-fed system. Vancomycin, an inhibitor of cell wall synthesis in eubacteria, was found to inhibit acetogenesis when added at 100 mg/liter to both methanol-fed and hydrogen-fed cultures. The effect of vancomycin on dechlorination was more complex. Methanol could not sustain dechlorination when vancomycin inhibited acetogenesis, while hydrogen could. These results are consistent with a model in which hydrogen is the electron donor directly used for dechlorination by organisms resistant to vancomycin and with the hypothesis that the role of acetogens in methanol-fed cultures is to metabolize a portion of the methanol to hydrogen. Methanol and other substrates shown to support dechlorination in pure and mixed cultures may merely serve as precursors for the formation of an intermediate hydrogen pool. This hypothesis suggests that, for bioremediation of high levels of tetrachloroethene, electron donors that cause the production of a large hydrogen pool should be selected or methods that directly use H2 should be devised.     

MORPHOGENESIS OF MONOSTROMA OXYSPERMUM (KÜTZ.) DOTY (CHLOROPHYCEAE) IN AXENIC CULTURE,ESPECIALLY IN BIALGAL CULTURE1

The typical morphology of Monostroma oxyspermum (Kütz.) Doty is lost in axenic culture. In synthetic media of the ASP type, it grows as a colony-like mass composed of round cells with numerous rhizoids. Such a mass is a fragile structure which falls apart upon shaking, or slight touch, into small cell-groups and single cells or cells with a long rhizoid. Only temporary saccate or monostromatic fronds appear and reach 1–2 mm in length when grown in enriched seawater media, but disintegrate and become a colony-like mass. The typical morphology is easily restored by adding at specific intervals filtrates of bacterial cultures and supernatant medium from axenic brown and red algal cultures to the basal medium (ASP7), or by reinfecting the Monostroma with an appropriate bacterial flora. Furthermore, the typical morphology in also maintained by bialgal cultures between Monostroma and other axenic strains of various species of seaweeds except the species belonging to the Chlorophyceae. Monostroma thus appears to utilize some substances released by most species of brown and red algae for its typical growth. Active substances released by bacteria, brown and red algae have not yet been identified and purified. However, it is demonstrated that in axenic cultures many species of seaweeds produce active extracellular substances which play an important role in growth and Morphogenesis of other species of seaweeds.     

Hydrogen as an electron donor for dechlorination of tetrachloroethene by an anaerobic mixed culture.

Hydrogen served as an electron donor in the reductive dechlorination of tetrachloroethene to vinyl chloride and ethene over periods of 14 to 40 days in anaerobic enrichment cultures; however, sustained dechlorination for more extended periods required the addition of filtered supernatant from a methanol-fed culture. This result suggests a nutritional dependency of hydrogen-utilizing dechlorinators on the metabolic products of other organisms in the more diverse, methanol-fed system. Vancomycin, an inhibitor of cell wall synthesis in eubacteria, was found to inhibit acetogenesis when added at 100 mg/liter to both methanol-fed and hydrogen-fed cultures. The effect of vancomycin on dechlorination was more complex. Methanol could not sustain dechlorination when vancomycin inhibited acetogenesis, while hydrogen could. These results are consistent with a model in which hydrogen is the electron donor directly used for dechlorination by organisms resistant to vancomycin and with the hypothesis that the role of acetogens in methanol-fed cultures is to metabolize a portion of the methanol to hydrogen. Methanol and other substrates shown to support dechlorination in pure and mixed cultures may merely serve as precursors for the formation of an intermediate hydrogen pool. This hypothesis suggests that, for bioremediation of high levels of tetrachloroethene, electron donors that cause the production of a large hydrogen pool should be selected or methods that directly use H2 should be devised.     

UREASE GENE SEQUENCES FROM ALGAE AND HETEROTROPHIC BACTERIA IN AXENIC AND NONAXENIC PHYTOPLANKTON CULTURES1

While urea has long been recognized as an important form of nitrogen in planktonic ecosystems, very little is known about how many or which phytoplankton and bacteria can use urea as a nitrogen source. We developed a method, targeting the gene encoding urease, for the direct detection and identification of ureolytic organisms and tested it on seven axenic phytoplankton cultures (three diatoms, two prymnesiophytes, a eustigmatophyte, and a pelagophyte) and on three nonaxenic Aureococcus anophagefferens Hargraves et Sieburth cultures (CCMP1784 and two CCMP1708 cultures from different laboratories). The urease amplicon sequences from axenic phytoplankton cultures were consistent with genomic data in the three species for which both were available. Seven of 12 phytoplankton species have one or more introns in the amplified region of their urease gene(s). The 63 urease amplicons that were cloned and sequenced from nonaxenic A. anophagefferens cultures grouped into 17 distinct sequence types. Eleven types were related to α‐Proteobacteria, including three types likely belonging to the genus Roseovarius. Four types were related to γ‐Proteobacteria, including two likely belonging to the genus Marinobacter, and two types were related to β‐Proteobacteria. Terminal restriction fragment length polymorphism (TRFLP) analyses suggested that the sequenced amplicons represented approximately half of the diversity of bacterial urease genes present in the nonaxenic cultures. While many of the bacterial urease sequence types were apparently lab‐ or culture‐specific, others were found in all three nonaxenic cultures, suggesting the possibility of specific relationships between these bacteria and A. anophagefferens.     

Influence on plant growth of the breakdown of organic phosphorus compounds by micro-organisms

Summary Micro-organisms which break down lecithin or phytin have been isolated from soil by enrichment cultures. Only those organisms were investigated which on an agar medium containing lecithin or phytin as the sole source of P (in the case of lecithin, also as a source of N and energy), produced a clear area around their colonies. Certain of these organisms were inoculated into sterile cultures of radish plants grown on sand or agar substrates containing lecithin or phytin as the source of P, and the growth and N- and P-uptakes of the plants were compared with those of similar plants grown in non-sterile sand cultures or supplied with KH₂PO₄. Evidence was obtained that both lecithin and phytin can serve as P-sources for higher plants even under sterile conditions, phytin producing a greater effect than lecithin. Inoculation, however, had only in the phytin-containing medium sometimes a slight effect on the P-nutrition of the plants and further work in this field is therefore necessary.     

Degradation and mineralization of high-molecular-weight polycyclic aromatic hydrocarbons by defined fungal-bacterial cocultures

This study investigated the biodegradation of high-molecular-weight polycyclic aromatic hydrocarbons (PAHs) in liquid media and soil by bacteria (Stenotrophomonas maltophilia VUN 10,010 and bacterial consortium VUN 10,009) and a fungus (Penicillium janthinellum VUO 10, 201) that were isolated from separate creosote- and manufactured-gas plant-contaminated soils. The bacteria could use pyrene as their sole carbon and energy source in a basal salts medium (BSM) and mineralized significant amounts of benzo[a]pyrene cometabolically when pyrene was also present in BSM. P. janthinellum VUO 10,201 could not utilize any high-molecular-weight PAH as sole carbon and energy source but could partially degrade these if cultured in a nutrient broth. Although small amounts of chrysene, benz[a]anthracene, benzo[a]pyrene, and dibenz[a,h]anthracene were degraded by axenic cultures of these isolates in BSM containing a single PAH, such conditions did not support significant microbial growth or PAH mineralization. However, significant degradation of, and microbial growth on, pyrene, chrysene, benz[a]anthracene, benzo[a]pyrene, and dibenz[a,h]anthracene, each as a single PAH in BSM, occurred when P. janthinellum VUO 10,201 and either bacterial consortium VUN 10,009 or S. maltophilia VUN 10,010 were combined in the one culture, i.e., fungal-bacterial cocultures: 25% of the benzo[a]pyrene was mineralized to CO(2) by these cocultures over 49 days, accompanied by transient accumulation and disappearance of intermediates detected by high-pressure liquid chromatography. Inoculation of fungal-bacterial cocultures into PAH-contaminated soil resulted in significantly improved degradation of high-molecular-weight PAHs, benzo[a]pyrene mineralization (53% of added [(14)C]benzo[a]pyrene was recovered as (14)CO(2) in 100 days), and reduction in the mutagenicity of organic soil extracts, compared with the indigenous microbes and soil amended with only axenic inocula.     

Isolation and characterization of a fluoranthene-utilizing strain of Pseudomonas paucimobilis.

A soil bacterium capable of utilizing fluoranthene as the sole source of carbon and energy for growth was purified from a seven-member bacterial community previously isolated from a creosote waste site for its ability to degrade polycyclic aromatic hydrocarbons. By standard bacteriological methods, this bacterium was characterized taxonomically as a strain of Pseudomonas paucimobilis and was designated strain EPA505. Utilization of fluoranthene by strain EPA 505 was demonstrated by increase in bacterial biomass, decrease in aqueous fluoranthene concentration, and transient formation of transformation products in liquid cultures where fluoranthene was supplied as the sole carbon source. Resting cells grown in complex medium showed activity toward anthraquinone, benzo[b]fluorene, biphenyl, chrysene, and pyrene as demonstrated by the disappearance of parent compounds or changes in their UV absorption spectra. Fluoranthene-grown resting cells were active against these compound as well as 2,3-dimethylnaphthalene, anthracene, fluoranthene, fluorene, naphthalene, and phenanthrene. These studies demonstrate that organic compounds not previously reported to serve as growth substrates can be utilized by axenic cultures of microorganisms. Such organisms may possess novel degradative systems that are active toward other compounds whose biological degradation has been limited because of inherent structural considerations or because of low aqueous solubility.     

Isolation and characterization of a fluoranthene-utilizing strain of Pseudomonas paucimobilis

A soil bacterium capable of utilizing fluoranthene as the sole source of carbon and energy for growth was purified from a seven-member bacterial community previously isolated from a creosote waste site for its ability to degrade polycyclic aromatic hydrocarbons. By standard bacteriological methods, this bacterium was characterized taxonomically as a strain of Pseudomonas paucimobilis and was designated strain EPA505. Utilization of fluoranthene by strain EPA 505 was demonstrated by increase in bacterial biomass, decrease in aqueous fluoranthene concentration, and transient formation of transformation products in liquid cultures where fluoranthene was supplied as the sole carbon source. Resting cells grown in complex medium showed activity toward anthraquinone, benzo[b]fluorene, biphenyl, chrysene, and pyrene as demonstrated by the disappearance of parent compounds or changes in their UV absorption spectra. Fluoranthene-grown resting cells were active against these compound as well as 2,3-dimethylnaphthalene, anthracene, fluoranthene, fluorene, naphthalene, and phenanthrene. These studies demonstrate that organic compounds not previously reported to serve as growth substrates can be utilized by axenic cultures of microorganisms. Such organisms may possess novel degradative systems that are active toward other compounds whose biological degradation has been limited because of inherent structural considerations or because of low aqueous solubility.     

Reductive dehalogenation of brominated ethenes by Sulfurospirillum multivorans and Desulfitobacterium hafniense PCE‐S

Sulfurospirillum multivorans and Desulfitobacterium hafniense PCE‐S are anaerobes that can utilize tetrachloroethene (PCE) as an electron acceptor in their energy metabolism. The end‐product of PCE reduction for both organisms is cis‐1,2‐dichloroethene, which is formed via trichloroethene as the intermediate. The bacteria were able to dehalogenate cis‐ and trans‐1,2‐dibromoethene (cDBE and tDBE) in growing cultures and cell extracts. Dibromoethene supported growth of both organisms. The organisms debrominated cDBE and tDBE to vinyl bromide (VB); D. hafniense PCE‐S also produced ethene in addition to VB. The PCE reductive dehalogenases (PCE dehalogenases) of S. multivorans and D. hafniense PCE‐S mediated the debromination of tribromoethene (TBE) and both isomers of 1,2‐DBE, indicating that this enzyme was responsible for the reductive dehalogenation of brominated ethenes. cDBE, tDBE, 1,1‐DBE and VB were formed upon TBE debromination; VB was the major end‐product. The PCE dehalogenase of D. hafniense PCE‐S also formed ethene. With the purified enzymes from both organisms the kinetic properties of dehalogenation of brominated alkenes were studied and compared with those of their chlorinated analogues.     

Pentitol oxidoreductases inSclerotinia sclerotiorum

Summary All attempts to isolate microoganisms from soil that utilize unsubstituted cycloparaffinic hydrocarbons, e.g. cyclohexane, as sole source of carbon and energy have been unsuccessful. However, cyclohexane was degraded in fertile soil as measured by release of ¹⁴C-carbon dioxide on addition of UL-¹⁴C-cyclohexane. Hydrocarbon utilizing organisms isolated from the soil grew rapidly on cycloalkanones. Several cultures, after growth on propane, could oxidize cycloparaffins to the homologous cycloalkanone. These results suggest that degradation of cycloalkanes in nature may be via co-metabolism.     

Carbon metabolism of intracellular bacteria

Bacterial metabolism has been studied intensively since the first observations of these 'animalcules' by Leeuwenhoek and their isolation in pure cultures by Pasteur. Metabolic studies have traditionally focused on a small number of model organisms, primarily the Gram negative bacillus Escherichia coli, adapted to artificial culture conditions in the laboratory. Comparatively little is known about the physiology and metabolism of wild microorganisms living in their natural habitats. For approximately 500-1000 species of commensals and symbionts, and a smaller number of pathogenic bacteria, that habitat is the human body. Emerging evidence suggests that the metabolism of bacteria grown in vivo differs profoundly from their metabolism in axenic cultures.     

Monoxenic and axenic cultivation of carrier and patient strains of Entamoeba histolytica.

All of five strains of Entamoeba histolytica, isolated from symptomatic cases of amoebiasis, could be adapted to axenic growth on the TP-S-1 medium of Diamond (1968). Four axenic strains were started from amoeba-Crithidia cultures; one could be axenized directly after isolation from a case of cutaneous amoebiasis. Attempts to monoxenize, resp. axenize strains, isolated from Dutch, asymptomatic carriers, were less successful. Only three out of ten strains could be submitted to bacteria-free growth. These three strains, however, originated probably from a recent case of intestinal amoebiasis. The results, suggesting that highly virulent strains can be easier cultivated bacteria-free than those with low or no virulence, are further discussed. The yield of axenic amoebae per tube fluctuates largely depending on many factors such as the strain, the number of transfers (i.e. degree of establishment), the quality of Panmede liver digest and serum in the TP-S-1 medium, and the care of manipulating the cultures. For optimal growth, a more acid medium was required in an amoeba-Crithidia culture than in an axenic culture. Multinucleated, giant amoebae were frequently observed in axenic cultures.     

Specific detection of Dehalococcoides species by fluorescence in situ hybridization with 16S rRNA-targeted oligonucleotide probes

Dehalococcoides ethenogenes is the only known cultivated organism capable of complete dehalogenation of tetrachloroethene (PCE) to ethene. The prevalence of Dehalococcoides species in the environment and their association with complete dehalogenation of chloroethenes suggest that they play an important role in natural attenuation of chloroethenes and are promising candidates for engineered bioremediation of these contaminants. Both natural attenuation and bioremediation require reliable and sensitive methods to monitor the presence, distribution, and fate of the organisms of interest. Here we report the development of 16S rRNA-targeted oligonucleotide probes for Dehalococcoides species. The two designed probes together encompass 28 sequences of 16S rRNA genes retrieved from the public database. Except D. ethenogenes and CBDB1, all the others are environmental clones obtained from sites contaminated with chlorinated ethenes. They are all closely related and form a unique cluster of Dehalococcoides species. In situ hybridization of probe Dhe1259t with D. ethenogenes strain 195 and two enrichment cultures demonstrated the applicability of the probe to monitoring the abundance of active Dehalococcoides species in these enrichment samples.     

Expanding the solar spectrum used by photosynthesis

A limiting factor for photosynthetic organisms is their light-harvesting efficiency, that is the efficiency of their conversion of light energy to chemical energy. Small modifications or variations of chlorophylls allow photosynthetic organisms to harvest sunlight at different wavelengths. Oxygenic photosynthetic organisms usually utilize only the visible portion of the solar spectrum. The cyanobacterium Acaryochloris marina carries out oxygenic photosynthesis but contains mostly chlorophyll d and only traces of chlorophyll a. Chlorophyll d provides a potential selective advantage because it enables Acaryochloris to use infrared light (700-750 nm) that is not absorbed by chlorophyll a. Recently, an even more red-shifted chlorophyll termed chlorophyll f has been reported. Here, we discuss using modified chlorophylls to extend the spectral region of light that drives photosynthetic organisms.