The Effects of Cyanobacterial Exudates on Bacterial Growth and Biodegradation of Organic Contaminants

The pulp and paper industry largely depends on the biodegradation activities of heterotrophic bacteria to remove organic contaminants in wastewater prior to discharge. Our recent discovery of extensive cyanobacterial communities in pulp and paper waste treatment systems led us to investigate the potential impacts of cyanobacterial exudates on growth and biodegradation efficiency of three bacterial heterotrophs. Each of the three assessed bacteria represented different taxa commonly found in pulp and paper waste treatment systems: a fluorescent Pseudomonad, an Ancylobacter aquaticus strain, and a Ralstonia eutropha strain. They were capable of utilizing phenol, dichloroacetate (DCA), or 2,4-dichlorophenoxyacetic acid (2,4-D), respectively. Exudates from all 12 cyanobacterial strains studied supported the growth of each bacterial strain to varying degrees. Maximum biomass of two bacterial strains positively correlated with the total organic carbon content of exudate treatments. The combined availability of exudate and a known growth substrate (i.e., phenol, DCA, or 2,4-D) generally had a synergistic affect on the growth of the Ancylobacter strain, whereas mixed effects were seen on the other two strains. Exudates from four representative cyanobacterial strains were assessed for their impacts on phenol and DCA biodegradation by the Pseudomonas and Ancylobacter strains, respectively. Exudates from three of the four cyanobacterial taxa repressed phenol biodegradation, but enhanced DCA biodegradation. These dissimilar impacts of cyanobacterial exudates on bacterial degradation of contaminants suggest a species-specific association, as well as a significant role for cyanobacteria during the biological treatment of wastewaters.     

Utilization of glucoisosaccharinic acid by a bacterial isolate unable to metabolize glucose

Summary Enrichment cultivation was used to select a bacterial strain capable of metabolizing glucoisosaccharinic acid (GISA), the major organic acid component of black liquor from the sulphate cellulose (Kraft) process. The bacterium was isolated from mud at the bottom of a lake polluted by pulping effluents. It grew well on GISA as the sole carbon source at pH 6.0±0.5, the absolute limits for growth on this substrate being 5.1-1 was consumed within 24 h in batch laboratory fermentations, the maximum observed rate of consumption after the lag phase being almost 10 g l^-1 d^-1. The strain appeared to be independent of organic nitrogen.     

Fermentation of Inulin by Clostridium thermosuccinogenes sp. nov., a Thermophilic Anaerobic Bacterium Isolated from Various Habitats

Four closely related strains of thermophilic bacteria were isolated via enrichment in batch and continuous culture with inulin as the sole source of carbon and energy by using inoculations from various sources. These new strains were isolated from beet pulp from a sugar refinery, soil around a Jerusalem artichoke, fresh cow manure, and mud from a tropical pond in a botanical garden. The cells of this novel species of strictly anaerobic, gram-positive bacteria were rod shaped and nonmotile. Growth on inulin was possible between 40 and 65°C, with optimum growth at 58°C. All strains were capable of fermenting a large number of sugars. Formate, acetate, ethanol, lactate, H₂, and succinate were the main organic fermentation products after growth on fructose, glucose, or inulin. Synthesis of inulinase in batch culture closely paralleled growth, and the enzyme was almost completely cell bound. Strain IC is described as the type strain of a new species, Clostridium thermosuccinogenes sp. nov., with a G+C content of 35.9 mol%.     

A new aerobic chemolithoautotrophic arsenic oxidizing microorganism isolated from a high Andean watershed

Biological arsenic oxidation has been suggested as a key biogeochemical process that controls the mobilization and fate of this metalloid in aqueous environments. To the best of our knowledge, only four aerobic chemolithoautotrophic arsenite-oxidizing (CAO) bacteria have been shown to grow via direct arsenic oxidation and to have the essential genes for chemolithoautotrophic arsenite oxidation. In this study, a new CAO bacterium was isolated from a high Andean watershed evidencing natural dissolved arsenic attenuation. The bacterial isolate, designated TS-1, is closely related to the Ancylobacter genus, in the Alphaproteobacteria class. Results showed that TS-1 has genes for arsenite oxidation and carbon fixation. The dependence of bacterial growth from arsenite oxidation was demonstrated. In addition, a mathematical model was suggested and the kinetic parameters were obtained by simultaneously fitting the biomass growth, arsenite depletion curves, and arsenate production. This research increases the knowledge of chemolithoautotrophic arsenic oxidizing microorganisms and its potential role as a driver for natural arsenic attenuation.     

Evaluation of chitoligosaccharides effect upon probiotic bacteria

The main objective of the present study was to evaluate the antibacterial effect - through the determination of minimum inhibitory (and lethal) concentrations, as well as the possible prebiotic potential of chitooligosaccharides (COS) - through the determination of growth curves, on Bifidobacterium animalis Bb12, Bifidobacterium animalis Bo and Lactobacillus acidophilus Ki. Atomic force microscopy was further used to obtain high resolution images of COS effects upon the cell morphology. Our results demonstrate that COS do not stimulate the growth of those strains, neither the strains are capable of using COS as a primary source of carbon. Analysis of morphology when exposed to inhibitory/bactericidal concentrations, suggested that COS do not exert any direct damage upon the bacteria structure, instead the bacteria are apparently covered by COS, which likely prevent nutrient uptake.     

Isolation and Characterization of Strains CVO and FWKO B, Two Novel Nitrate-Reducing, Sulfide-Oxidizing Bacteria Isolated from Oil Field Brine

Bacterial strains CVO and FWKO B were isolated from produced brine at the Coleville oil field in Saskatchewan, Canada. Both strains are obligate chemolithotrophs, with hydrogen, formate, and sulfide serving as the only known energy sources for FWKO B, whereas sulfide and elemental sulfur are the only known electron donors for CVO. Neither strain uses thiosulfate as an energy source. Both strains are microaerophiles (1% O₂). In addition, CVO grows by denitrification of nitrate or nitrite whereas FWKO B reduces nitrate only to nitrite. Elemental sulfur is the sole product of sulfide oxidation by FWKO B, while CVO produces either elemental sulfur or sulfate, depending on the initial concentration of sulfide. Both strains are capable of growth under strictly autotrophic conditions, but CVO uses acetate as well as CO₂ as its sole carbon source. Neither strain reduces sulfate; however, FWKO B reduces sulfur and displays chemolithoautotrophic growth in the presence of elemental sulfur, hydrogen, and CO₂. Both strains grow at temperatures between 5 and 40°C. CVO is capable of growth at NaCl concentrations as high as 7%. The present 16s rRNA analysis suggests that both strains are members of the epsilon subdivision of the division Proteobacteria, with CVO most closely related to Thiomicrospira denitrifcans and FWKO B most closely related to members of the genus Arcobacter. The isolation of these two novel chemolithotrophic sulfur bacteria from oil field brine suggests the presence of a subterranean sulfur cycle driven entirely by hydrogen, carbon dioxide, and nitrate.     

Acidophilic, Heterotrophic Bacteria of Acidic Mine Waters

Obligately acidophilic, heterotrophic bacteria were isolated both from enrichment cultures developed with acidic mine water and from natural mine drainage. The bacteria were grouped by the ability to utilize a number of organic acids as sole carbon sources. None of the strains were capable of chemolithotrophic growth on inorganic reduced iron and sulfur compounds. All bacteria were rod shaped, gram negative, nonencapsulated, motile, capable of growth at pH 2.6 but not at pH 6.0, catalase and oxidase positive, strictly aerobic, and capable of growth on citric acid. The bacteria were cultivatable on solid nutrient media only if agarose was employed as the hardening agent. Bacterial densities in natural mine waters ranged from approximately 20 to 250 cells per ml, depending upon source and culture medium. Ferric hydrates and stream vegetation contained from 1,500 to over 7 × 10⁶ cells per g.     

Depolymerization and decolorization of kraft lignin by bacterium Comamonas sp. B-9

There is no commercial or industrial-scale process for the remediation of black liquor using microorganisms to date. One of the most important causes is that most microorganisms are not able to use lignin as their principal metabolic carbon or energy source. The bacterial strain Comamonas sp. B-9 has shown remarkable ability to degrade kraft lignin and decolorize black liquor using lignin as its principal metabolic carbon and energy source. This report looks at the depolymerization and decolorization of kraft lignin by Comamonas sp. B-9. The degradation, decolorization, and total carbon removal reached 45, 54, and 47.3 %, respectively, after 7 days treatment. Comamonas sp. B-9 was capable of depolymerizing kraft lignin effectively as analyzed by gel permeation chromatography and decolorization via degrading benzene ring structures as shown using Fourier transform infrared spectroscopy analysis.     

Oxalate utilisation is widespread in the actinobacterial genus Kribbella

The type strains of all 33 species in the genus Kribbella were tested for growth on oxalate (^?OOC-COO^?) as sole carbon source. Media were initially formulated to contain sodium oxalate, but even a concentration as low as 7.5 mM oxalate prevented growth. A modified medium based on calcium oxalate was very successful in characterising oxalate utilisation by Kribbella strains (metabolism of oxalate by oxalotrophic bacteria results in visible zones of clearing around the growth streaks on the opaque plates). To assess the variability of oxalate utilisation in Kribbella species, we also tested eight non-type strains for their ability to use oxalate. Thirty of 33 type strains (90.9%) and six of eight non-type strains (75%) were able to use oxalate as a sole carbon source. Based on these results, we propose that oxalate would be an excellent carbon source for the selective isolation of Kribbella strains. Based on the oxalate-utilisation phenotype and analyses of the 19 publicly available Kribbella type-strain genome sequences, we propose a pathway for oxalate metabolism in Kribbella. This pathway is significantly different from those previously proposed for oxalate metabolism in other bacteria, involving the indirect catabolism of oxalate to formate. Formate production is proposed to be involved in energy generation and to be crucial for oxalate import via an oxalate:formate antiporter. To our knowledge, this is the first report of an oxalate:formate antiporter in an aerobic, Gram-positive bacterium.     

Oxidation of Gaseous and Volatile Hydrocarbons by Selected Alkene-Utilizing Bacteria

Eleven strains of alkene-utilizing bacteria belonging to the genera Mycobacterium, Nocardia, and Xanthobacter were tested for their ability to grow with C₁ to C₆ alkanes, C₂ to C₆ alkenes, alkadienes, and monoterpenes furnished individually as sole sources of carbon and energy in a mineral salts medium. A limited number of alkenes and alkanes supported growth of the bacteria; some bacteria were unable to grow on any of the saturated hydrocarbons tested. Monoterpenes were frequently used as carbon and energy sources by alkene-utilizing bacteria belonging to the genera Mycobacterium and Nocardia. Washed cell suspensions of alkene-grown bacteria attacked the whole range of alkenes tested, whereas only three strains were able to oxidize alkanes as well. The alkenes tested were oxidized either to water and carbon dioxide or to epoxyalkanes. Few epoxides accumulated in stoichiometric amounts from the corresponding alkenes, because most epoxides formed were further converted to other compounds like alkanediols.     

Immunolocalization of Dinitrogenase Reductase Produced by Klebsiella pneumoniae in Association with Zea mays L.

The endophytic lifestyle of Klebsiella pneumoniae is described, including the production of dinitrogenase reductase by bacteria residing in maize root tissue. The green fluorescent protein (GFP) was used to detect the colonization of maize by K. pneumoniae strains 2028 and 342. These strains were found to reside in intercortical layers of the stem and within the region of maturation in the root. The production of dinitrogenase reductase by GFP-tagged bacteria was visualized using immunolocalization. This activity was only apparent when bacteria were supplied with an exogenous carbon source. The results suggest that maize provides a suitable habitat for K. pneumoniae and that this species is capable of producing nitrogenase under the appropriate plant cultivation conditions.     

Isolation of Halotolerant Thermus spp. from Submarine Hot Springs in Iceland

Thermophilic, aerobic bacteria of the genus Thermus were isolated from submarine alkaline hot springs in Iceland. Five submarine hot springs were sampled, and all had viable counts of Thermus spp. of about 10³ CFU/ml. All submarine strains grew in the presence of NaCl at 3% or higher, but no strains from terrestrial hot springs would grow at concentrations higher than 1% NaCl. The growth rate of submarine Thermus strains was not stimulated by NaCl and was reduced at NaCl concentrations higher than 1%. The pattern of growth of these isolates on single carbon sources was similar to that of terrestrial isolates.     

The study of mycolytic properties of aerobic spore-forming bacteria producing extracellular chitinases

The mycolytic activity of 27 strains of antagonistic bacilli belonging to two taxonomic groups (18 strains of Bacillus subtilis and 9 strains of Paenibacillus ehimensis) capable of induced synthesis of chitinolytic enzymes was studied. Most of the B. subtilis strains neither displayed visible mycolytic effects on the phytopathogenic fungus Bipolaris sorokiniana in vitro, nor produced chitinases in the presence of an autoclaved mycelium. On the contrary, P. ehimensis strains grown under conditions favorable for induction of chitinases and other hydrolases exhibited a pronounced lytic effect on B. sorokiniana and actively grew by utilizing mycelium as the sole source of carbon and nitrogen. Comparison of the mycolytic activities of extracellular hydrolases in the studied strains demonstrated low correlation between chitinase production and the ability of the strains to degrade the cell walls of B. sorokiniana. Characterization of enzyme profiles in the studied strains revealed that β-1,3-glucanase was a more significant factor than chitinase for determining the mycolytic potential of bacteria and their ability to utilize the mycelium of phytopathogenic fungi as a growth substrate.     

Growth of Candida famata and Trichosporon cutaneum on uric acid as the sole source of carbon and energy,a hitherto unknown property of yeasts

Yeast strains capable of utilizing uric acid as the sole source of carbon and energy were isolated from soil by the enrichment culture method. The strains were identified as Candida famata (Harrison) Meyer et Yarrow and Trichosporon cutaneum (De Beurm., Gougerot et Vaucher) Ota. On the subcellular level growth of yeasts on uric acid was accompanied with the development of a number of large microbodies in the cells.     

Comparative Growth of Natural Bacterial Isolates on Various Lignin-Related Compounds

Bacterial strains were isolated on the basis of their ability to proliferate in a minimal medium containing one of a series of lignin-related compounds as the sole carbon and energy source. These included the aromatic monomers guaiacol, vanillic and coumaric acids, a dimer and a trimer possessing the arylglycerol-β-aryl ether linkage, anisoin, and both the ether-soluble and -insoluble fractions of kraft lignin. The growth of the strains on each of these compounds was measured. The results showed that the metabolic properties of the strains varied according to the structure of the carbon sources used for their selection. Spectrophotometric tracings of the culture medium during the log phase of growth of one of the strains on the β-O-4 dimer revealed decomposition with the release of guaiacol.     

Effects of Bacterial Host and Dichloromethane Dehalogenase on the Competitiveness of Methylotrophic Bacteria Growing with Dichloromethane

Methylobacterium sp. strain DM4 and Methylophilus sp. strain DM11 can grow with dichloromethane (DCM) as the sole source of carbon and energy by virtue of homologous glutathione-dependent DCM dehalogenases with markedly different kinetic properties (the k_cat values of the enzymes of these strains are 0.6 and 3.3 s⁻¹, respectively, and the K_m values are 9 and 59 μM, respectively). These strains, as well as transconjugant bacteria expressing the DCM dehalogenase gene (dcmA) from DM11 or DM4 on a broad-host-range plasmid in the background of dcmA mutant DM4-2cr, were investigated by growing them under growth-limiting conditions and in the presence of an excess of DCM. The maximal growth rates and maximal levels of dehalogenase for chemostat-adapted bacteria were higher than the maximal growth rates and maximal levels of dehalogenase for batch-grown bacteria. The substrate saturation constant of strain DM4 was much lower than the K_m of its associated dehalogenase, suggesting that this strain is adapted to scavenge low concentrations of DCM. Strains and transconjugants expressing the DCM dehalogenase from strain DM11, on the other hand, had higher growth rates than bacteria expressing the homologous dehalogenase from strain DM4. Competition experiments performed with pairs of DCM-degrading strains revealed that a strain expressing the dehalogenase from DM4 had a selective advantage in continuous culture under substrate-limiting conditions, while strains expressing the DM11 dehalogenase were superior in batch culture when there was an excess of substrate. Only DCM-degrading bacteria with a dcmA gene similar to that from strain DM4, however, were obtained in batch enrichment cultures prepared with activated sludge from sewage treatment plants.     

Isolation and characterization of thermophilic bacteria capable of degrading dehydroabietic acid.

Using a semi-continuous enrichment method, we isolated two thermophilic bacterial strains, which could completely degrade abietane resin acids, including dehydroabietic acid (DhA). Strain DhA-73, isolated from a laboratory-scale bioreactor treating bleached kraft mill effluent at 55 degrees C, grew on DhA as sole carbon source; while DhA-71, isolated from municipal compost, required dilute tryptic soy broth for growth on DhA. DhA-71 grew on DhA from 30 degrees C to 60 degrees C with maximum growth at 50 degrees C; while, DhA-73 grew on DhA from 37 degrees C to 60 degrees C with maximum growth at 55 degrees C. At 55 degrees C, the doubling times for DhA-71 and DhA-73 were 3.3 and 3.7 h, respectively. DhA-71 and DhA-73 had growth yields of 0.26 and 0.19 g of protein per g of DhA, respectively. During growth on DhA, both strains converted DhA to CO2, biomass, and dissolved organic carbon. Analyses of the 16S-rDNA sequences of these two strains suggest that they belong to two new genera in the Rubrivivax subgroup of the beta subclass of the Proteobacteria. Strains DhA-71 and DhA-73 are the first two bacteria isolated and characterized that are capable of biodegradation of resin acids at high temperatures. This study provided direct evidence for biodegradation of resin acids and feasibility for biotreatment of pulp mill effluent at elevated temperatures.     

A novel <Emphasis Type="Italic">Delftia</Emphasis> plant symbiont capable of autotrophic methylotrophy

A facultative methylotrophic bacterium, strain Lp-1, which was isolated from root nodules of lupine (Lupinus polyphyllus L.) on the medium with methanol as a carbon and energy source, exhibited high similarity of the 16S rRNA gene sequences to Delftia strains (94?99.9%). The cells of Delftia sp. Lp-1 were motile gram-negative rods dividing by binary fission. Predominant fatty acids were C_16:0 (34.2%), C_16:1ω9 (14.5%), and C_18:1ω7c (17.3%). Phosphatidylethanolamine, phosphatidylcholine, and phosphatidylglycerol were the dominant phospholipids. Q₈ was the major ubiquinone. Optimal growth occurred at 24?26°C and pH 7.1?7.3; growth was inhibited by 1% NaCl. The organism oxidized methanol with the classical methanol dehydrogenase and used the ribulose bisphosphate pathway of C₁ metabolism. Analysis of translated amino acid sequence of the large subunit of the MxaF methanol dehydrogenase revealed 85.5?94% similarity to the sequences of such autotrophic methylotrophs of the class Alphaproteobacteria as Angulomicrobium, Starkeya, and Ancylobacter, indicating the possible acquisition of the mxaF gene via horizontal gene transfer. Delftia sp. Lp-1 (VKM B-3039, DSM 24446), the first methylotrophic member of the genus Delftia, was shown to be a plant symbiont, stimulating plant growth and morphogenesis, increasing the level of photosynthetic pigments and specific leaf weight. It possesses the nifH gene of nitrogen fixation, is capable of phosphate solubilization, synthesis of auxins and siderophores, and is antagonistic to plant pathogenic fungi and bacilli.