Sequential Growth of Bacteria on Crude Oil

By modification of the enrichment culture procedure three bacterial strains capable of degrading crude oil in sea water were isolated in pure culture, UP-2, UP-3, and UP-4. Strain UP-2 appears to be highly specialized for growth on crude oil in sea water since it showed strong preference for oil or oil degradation products as substrates for growth, converted 66% of the oil into a form no longer extractable by organic solvents, quantitatively degraded the paraffinic fraction (gas chromatographic analysis), emulsified the oil during exponential growth, and produced 1.6 × 10⁸ cells per mg of oil. After exhaustive growth of UP-2 on crude oil the residual oil supported the growth of UP-3 and UP-4, but not a previously isolated oil-degrading bacterium, RAG-1. Strains UP-2, UP-3, and UP-4 grew on RAG-1-degraded oil (specifically depleted of n-alkanes). The growth of UP-3 and UP-4 on UP-2 and RAG-1-degraded oil resulted in the production of new paraffinic compounds as revealed by gas chromatography. When the four strains were grown either together in a mixed culture or sequentially, there was over 75% oil conversion. By plating on selective media, growth of the individual strains was measured kinetically in the reconstituted mixed culture, revealing competition for common growth substances (UP-2 and RAG-1), enhanced die-off (UP-2), and stabilization (UP-4) during the stationary phase.     

Effect of Emulsan on Biodegradation of Crude Oil by Pure and Mixed Bacterial Cultures

Crude oil was treated with purified emulsan, the heteropolysaccharide bioemulsifier produced by Acinetobacter calcoaceticus RAG-1. A mixed bacterial population as well as nine different pure cultures isolated from various sources was tested for biodegradation of emulsan-treated and untreated crude oil. Biodegradation was measured both quantitatively and qualitatively. Recovery of ¹⁴CO₂ from mineralized ¹⁴C-labeled substrates yielded quantitative data on degradation of specific compounds, and capillary gas chromatography of residual unlabeled oil yielded qualitative data on a broad spectrum of crude oil components. Biodegradation of linear alkanes and other saturated hydrocarbons, both by pure cultures and by the mixed population, was reduced some 50 to 90% after emulsan pretreatment. In addition, degradation of aromatic compounds by the mixed population was reduced some 90% in emulsan-treated oil. In sharp contrast, aromatic biodegradation by pure cultures was either unaffected or slightly stimulated by emulsification of the oil.     

Production of NO and N(inf2)O by Pure Cultures of Nitrifying and Denitrifying Bacteria during Changes in Aeration

Peak emissions of NO and N(inf2)O are often observed after wetting of soil. The reactions to sudden changes in the aeration of cultures of nitrifying and denitrifying bacteria with respect to NO and N(inf2)O emissions were compared to obtain more information about the microbiological aspects of peak emissions. In continuous culture, the nitrifier Nitrosomonas europaea and the denitrifiers Alcaligenes eutrophus and Pseudomonas stutzeri were cultured at different levels of aeration (80 to 0% air saturation) and subjected to changes in aeration. The relative production of NO and N(inf2)O by N. europaea, as a percentage of the ammonium conversion, increased from 0.87 and 0.17%, respectively, at 80% air saturation to 2.32 and 0.78%, respectively, at 1% air saturation. At 0% air saturation, ammonium oxidation and N(inf2)O production ceased but NO production was enhanced. Coculturing of N. europaea with the nitrite oxidizer Nitrobacter winogradskyi strongly reduced the relative levels of NO and N(inf2)O production, probably as an effect of the lowered nitrite concentration. After lowering the aeration, N. europaea produced large short-lasting peaks of NO and N(inf2)O emissions in the presence but not in the absence of nitrite. A. eutrophus and P. stutzeri began to denitrify below 1% air saturation, with the former accumulating nitrite and N(inf2)O and the latter reducing nitrate almost completely to N(inf2). Transition of A. eutrophus and P. stutzeri from 80 to 0% air saturation resulted in transient maxima of denitrification intermediates. Such transient maxima were not observed after transition from 1 to 0%. Reduction of nitrate by A. eutrophus continued 48 h after the onset of the aeration, whereas N(inf2)O emission by P. stutzeri increased for only a short period. It was concluded that only in the presence of nitrite are nitrifiers able to dominate the NO and N(inf2)O emissions of soils shortly after a rainfall event.     

Degradation and Utilization of Hemicellulose from Intact Forages by Pure Cultures of Rumen Bacteria

Several pure strains of rumen bacteria have previously been shown to degrade isolated hemicelluloses from a form insoluble in 80% acidified ethanol to a soluble form, regardless of the eventual ability of the organism to utilize the end products as energy sources. This study was undertaken to determine whether similar hemicellulose degradation or utilization, or both, occurs from intact forages. Fermentations by pure cultures were run to completion by using three maturity stages of alfalfa and two maturity stages of bromegrass as individual substrates. Organisms capable of utilizing xylan or isolated hemicelluloses could degrade and utilize intact forage hemicellulose, with the exception of two strains of Bacteroides ruminicola which were unable to degrade or utilize hemicellulose from grass hays. Intact forage hemicelluloses were extensively degraded by three cellulolytic strains that were unable to use the end products; in general, these strains degraded a considerably greater amount of hemicelluloses than the hemicellulolytic organisms. Hemicellulose degradation or utilization, or both, varied markedly with the different species and strains of bacteria, as well as with the type and maturity stage of the forage. Definite synergism was observed when a degrading nonutilizer was combined with either one of two hemicellulolytic strains on the bromegrass substrates. One hemicellulolytic strain, which could not degrade or utilize any of the intact bromegrass hemicellulose alone, almost completely utilized the end products solubilized by the nonutilizer. Similar synergism, although of lesser magnitude, was observed when alfalfa was used as a substrate.     

Effect of Four Dispersants on Biodegradation and Growth of Bacteria on Crude Oil

Four chemical dispersants, Corexit 8666, Gamlen Sea Clean, G. H. Woods Degreaser-Formula 11470, and Sugee 2 were examined singly and in individual combinations with Arabian Crude Oil (1:1 ratio) at 10 and 25 C for their effects on the growth of bacteria indigenous to local marine waters, the bacterial population composition, and biodegradation of crude oil; in addition, their emulsifying capacities, at approximately 24 C, were determined. None of the dispersants used alone were toxic even at relatively high concentrations (1.25%), although Gamlen Sea Clean and G. H. Woods Degreaser-Formula 11470 did cause an increase in the lag phase which was more pronounced at 10 than at 25 C; addition of the crude oil reduced the lag phase increase. All of the dispersants used alone supported good growth of microorganisms, but qualitative population shifts were caused by the dispersant-oil combinations. The degrees of degradation of the n-alkane fraction of the crude oil varied depending upon the dispersant used. Under these test conditions, only Sugee 2, which had the poorest emulsifying capacity, promoted n-alkane degradation compared with the values obtained by using the crude oil alone.     

Microbial Utilization of Crude Oil

The utilization of two crude oil samples of different quality at 4 and 30 C has been studied by using pure and mixed bacterial cultures obtained by enrichment procedures. Growth, emulsification, and utilization occurred readily at both temperatures. The crude oil residue is increased in specific gravity and readily sediments out of solution. A comparison of the chemical analysis of the oils by liquid and gas-liquid chromatographic procedures before and after growth showed that the n-saturate fraction had been preferentially used. Some utilization of the aromatic fraction also occurred. Enrichments obtained with a high-quality crude oil were not as effective in utilizing a lower quality crude oil as sole carbon source as a population enriched on the low-quality crude oil.     

Degradation of Phthalic Acids by Denitrifying, Mixed Cultures of Bacteria

Mixed cultures of bacteria, enriched from aquatic sediments, grew anaerobically on all three isomers of phthalic acid. Each culture grew anaerobically on only one isomer and also grew aerobically on the same isomer. Pure cultures were isolated from the phthalic acid (o-phthalic acid) and isophthalic acid (m-phthalic acid) enrichments that grew aerobically on phthalic and isophthalic acids. Cell suspension experiments indicated that protocatechuate is an intermediate of aerobic catabolism. Pure cultures which grew aerobically on terephthalic acid (p-phthalic acid) could not be isolated from the enrichments, and neither could pure cultures that grew anaerobically on any of the isomers. Cell suspension experiments suggested that separate pathways exist for the aerobic and anaerobic oxidation of phthalic acids. Each enrichment culture used only one phthalic acid isomer under anaerobic conditions, but all isomers were simultaneously adapted for the anaerobic catabolism of benzoate. Cells grown anaerobically on a phthalic acid immediately attacked the isomer under anaerobic conditions, whereas there was a lag before aerobic breakdown occurred, and, for phthalic and terephthalic acids, chloramphenicol stopped aerobic adaptation but had no effect on anaerobic catabolism. This work suggests that phthalic acids are biodegradable in anaerobic environments.     

Anaerobic Mineralization of Quaternary Carbon Atoms: Isolation of Denitrifying Bacteria on Dimethylmalonate

The microbial capacity to degrade simple organic compounds with quaternary carbon atoms was demonstrated by enrichment and isolation of five denitrifying strains on dimethylmalonate as the sole electron donor and carbon source. Quantitative growth experiments showed a complete mineralization of dimethylmalonate. According to phylogenetic analysis of the complete 16S rRNA genes, two strains isolated from activated sewage sludge were related to the genus Paracoccus within the α-Proteobacteria (98.0 and 98.2% 16S rRNA gene similarity to Paracoccus denitrificans^T), and three strains isolated from freshwater ditches were affiliated with the β-Proteobacteria (97.4 and 98.3% 16S rRNA gene similarity to Herbaspirillum seropedicae^T and Acidovorax facilis^T, respectively). Most-probable-number determinations for denitrifying populations in sewage sludge yielded 4.6 × 10⁴ dimethylmalonate-utilizing cells ml⁻¹, representing up to 0.4% of the total culturable nitrate-reducing population.     

Kinetics of Nitrate Utilization by Mixed Populations of Denitrifying Bacteria

Kinetics of nitrate utilization by mixed bacterial populations from two agricultural soils and a pond sediment in Kentucky were measured by using progress curves of nitrous oxide production. Nitrous oxide production from anaerobic soil and sediment slurries containing added nitrate and acetylene exhibited first-order kinetics. Nitrate affinity (K_m) for mixed populations of denitrifying bacteria in unfertilized agricultural soils and pond sediments ranged from 1.8 to 13.7 μM. The affinity of bacterial populations for nitrate did not vary with habitat, and the ability to use low concentrations of nitrate was retained by bacterial populations living in environments which received large inputs of nitrate.     

Inhibition of Methanogenesis by Methyl Fluoride: Studies of Pure and Defined Mixed Cultures of Anaerobic Bacteria and Archaea

Methyl fluoride (fluoromethane [CH(inf3)F]) has been used as a selective inhibitor of CH(inf4) oxidation by aerobic methanotrophic bacteria in studies of CH(inf4) emission from natural systems. In such studies, CH(inf3)F also diffuses into the anaerobic zones where CH(inf4) is produced. The effects of CH(inf3)F on pure and defined mixed cultures of anaerobic microorganisms were investigated. About 1 kPa of CH(inf3)F, similar to the amounts used in inhibition experiments, inhibited growth of and CH(inf4) production by pure cultures of aceticlastic methanogens (Methanosaeta spp. and Methanosarcina spp.) and by a methanogenic mixed culture of anaerobic microorganisms in which acetate was produced as an intermediate. With greater quantities of CH(inf3)F, hydrogenotrophic methanogens were also inhibited. At a partial pressure of CH(inf3)F of 1 kPa, homoacetogenic, sulfate-reducing, and fermentative bacteria and a methanogenic mixed culture of anaerobic microorganisms based on hydrogen syntrophy were not inhibited. The inhibition by CH(inf3)F of the growth and CH(inf4) production of Methanosarcina mazei growing on acetate was reversible. CH(inf3)F inhibited only acetate utilization by Methanosarcina barkeri, which is able to use acetate and hydrogen simultaneously, when both acetate and hydrogen were present. These findings suggest that the use of CH(inf3)F as a selective inhibitor of aerobic CH(inf4) oxidation in undefined systems must be interpreted with great care. However, by a careful choice of concentrations, CH(inf3)F may be useful for the rapid determination of the role of acetate as a CH(inf4) precursor.     

Anaerobic Naphthalene Degradation by Microbial Pure Cultures under Nitrate-Reducing Conditions

Pure bacterial cultures were isolated from a highly enriched denitrifying consortium previously shown to anaerobically biodegrade naphthalene. The isolates were screened for the ability to grow anaerobically in liquid culture with naphthalene as the sole source of carbon and energy in the presence of nitrate. Three naphthalene-degrading pure cultures were obtained, designated NAP-3-1, NAP-3-2, and NAP-4. Isolate NAP-3-1 tested positive for denitrification using a standard denitrification assay. Neither isolate NAP-3-2 nor isolate NAP-4 produced gas in the assay, but both consumed nitrate and NAP-4 produced significant amounts of nitrite. Isolates NAP-4 and NAP-3-1 transformed 70 to 90% of added naphthalene, and the transformation was nitrate dependent. No significant removal of naphthalene occurred under nitrate-limited conditions or in cell-free controls. Both cultures exhibited partial mineralization of naphthalene, representing 7 to 20% of the initial added ¹⁴C-labeled naphthalene. After 57 days of incubation, the largest fraction of the radiolabel in both cultures was recovered in the cell mass (30 to 50%), with minor amounts recovered as unknown soluble metabolites. Nitrate consumption, along with the results from the ¹⁴C radiolabel study, are consistent with the oxidation of naphthalene coupled to denitrification for NAP-3-1 and nitrate reduction to nitrite for NAP-4. Phylogenetic analyses based on 16S ribosomal DNA sequences of NAP-3-1 showed that it was closely related to Pseudomonas stutzeri and that NAP-4 was closely related to Vibrio pelagius. This is the first report we know of that demonstrates nitrate-dependent anaerobic degradation and mineralization of naphthalene by pure cultures.     

Dechlorination of Chlorocatechols by Stable Enrichment Cultures of Anaerobic Bacteria

Metabolically stable anaerobic cultures obtained by enrichment with 5-bromovanillin, 5-chlorovanillin, catechin, and phloroglucinol were used to study dechlorination of chlorocatechols. A high degree of specificity in dechlorination was observed, and some chlorocatechols were appreciably more resistant to dechlorination than others: only 3,5-dichlorocatechol, 4,5-dichlorocatechol, 3,4,5-trichlorocatechol, and tetrachlorocatechol were dechlorinated, and not all of them were dechlorinated by the same consortium. 3,5-Dichlorocatechol produced 3-chlorocatechol, 4,5-dichlorocatechol produced 4-chlorocatechol, and 3,4,5-trichlorocatechol produced either 3,5-dichlorocatechol or 3,4-dichlorocatechol; tetrachlorocatechol produced only 3,4,6-trichlorocatechol. Incubation of uncontaminated sediments without additional carbon sources brought about dechlorination of 3,4,5-trichlorocatechol to 3,5-dichlorocatechol. O-demethylation of chloroguaiacols was generally accomplished by enrichment cultures, except that catechin enrichment was unable to O-demethylate tetrachloroguaiacol. None of the enrichments dechlorinated any of the polychlorinated phenols examined. The results suggested that dechlorination was not dependent on enrichment with or growth at the expense of chlorinated compounds and that it would be premature to formulate general rules for the structural dependence of the dechlorination reaction.     

Microbial Degradation of Crude Oil: Factors Affecting the Dispersion in Sea Water by Mixed and Pure Cultures

By means of the enrichment culture technique, a mixed population of microorganisms was obtained which catalyzed the dispersion of crude oil in supplemented sea water. From this enrichment culture, eight pure cultures were isolated and studied. Only one of the isolates (RAG-1) brought about a significant dispersion of crude oil. RAG-1 has been tentatively characterized as a member of the genus Arthrobacter. The other seven isolates gave rise to colonies on supplemented oil agar, but were neither able to disperse oil nor to stimulate the dispersion catalyzed by RAG-1. The dispersion of crude oil by either RAG-1 or the enrichment culture was absolutely dependent on exogenous sources of nitrogen and phosphorous and completely inhibited by 10⁻²m azide. The increase in cell number of RAG-1 was directly proportional to the concentration of crude oil added to the medium over the range 0.05 to 1.0 mg/ml. Within this linear region, 1.0 mg of crude oil yielded 9 × 10⁷ cells and approximately 65% of the oil was converted into a nonbenzene extractable form. Accompanying the emulsification was a decrease in the pH from 7.6 to 5.0. Acidic conditions, however, were neither necessary nor sufficient for oil dispersion. When sea water was supplemented with 0.029 mm K₂HPO₄ and 3.8 mm (NH₄)₂SO₄ and inoculated with RAG-1, oil dispersion occurred within 1 day. This dispersion could also be brought about by the supernatant following separation of the cells from the medium. Similarly, the supernatant obtained following growth of RAG-1 on hexadecane was capable of emulsifying crude oil in 60 min.     

The Effect of Monensin on Pure and Mixed Cultures of Rumen Bacteria

The antibiotic monensin was added to pure cultures of Bacteroides ruminicola, Selenomonas ruminantium, Anaerovibrio lipolytica and Megasphaera elsdenii. These organisms, representing succinate- and propionate-producing rumen bacteria, were not affected by monensin up to 10 μg/ml. Methanobacterium ruminantium was slightly inhibited by monensin, Butyrivibrio fibrisolvens, Ruminococcus albus and Streptococcus bovis were inhibited to differing extents by monensin at concentrations between 0.1 and 10 μg/ml. Bacteroides succinogenes was inhibited at first by monensin at >0.5 μg/ml but after a prolonged lag phase adapted to grow in the presence of monensin at concentrations below 5 μg/ml.
Monensin (1 μg/ml) almost completely stopped the digestion of chopped straw and dewaxed cotton fibres by rumen contents incubated in vitro. The digestion of grass and powdered filter paper was not significantly reduced under these conditions, but when the concentration of monensin was increased to between 3 and 5 μg/ml, the digestion of these substrates was reduced.     

Growth Curves of Anaerobic Bacteria in Solid Media

Simple pour plate and spectrophotometric techniques for the evaluation of growth curves of several anaerobic bacteria on solid media are described. Three basic patterns of anaerobic growth were observed. The curves obtained were very reproducible when studied on separate occasions. The curves obtained by spectrophotometric measurement were comparable to those obtained by the pour plate method, especially when a large bacterial inoculum was used. Limitations in the interpretation of the results are discussed. The methods and principles reported could provide the basis for the determination of bacterial growth on solid media using other organisms and different experimental conditions.     

Bacteria in Crude Oil Survived Autoclaving and Stimulated Differentially by Exogenous Bacteria

Autoclaving of crude oil is often used to evaluate the hydrocarbon-degrading abilities of bacteria. This may be potentially useful for bioaugmentation and microbial enhanced oil recovery (MEOR). However, it is not entirely clear if “endogenous” bacteria (e.g., spores) in/on crude oil survive the autoclaving process, or influence subsequent evaluation of the hydrocarbon-degradation abilities of the “exogenous” bacterial strains. To test this, we inoculated autoclaved crude oil medium with six exogenous bacterial strains (three Dietzia strains, two Acinetobacter strains, and one Pseudomonas strain). The survival of the spore-forming Bacillus and Paenibacillus and the non-spore-forming mesophilic Pseudomonas, Dietzia, Alcaligenes, and Microbacterium was detected using a 16S rRNA gene clone library and terminal restriction fragment length polymorphism (T-RFLP) analysis. However, neither bacteria nor bacterial activity was detected in three controls consisting of non-inoculated autoclaved crude oil medium. These results suggest that detection of endogenous bacteria was stimulated by the six inoculated strains. In addition, inoculation with Acinetobacter spp. stimulated detection of Bacillus, while inoculation with Dietzia spp. and Pseudomonas sp. stimulated the detection of more Pseudomonas. In contrast, similar exogenous bacteria stimulated similar endogenous bacteria at the genus level. Based on these results, special emphasis should be applied to evaluate the influence of bacteria capable of surviving autoclaving on the hydrocarbon-degrading abilities of exogenous bacteria, in particular, with regard to bioaugmentation and MEOR. Bioaugmentation and MEOR technologies could then be developed to more accurately direct the growth of specific endogenous bacteria that may then improve the efficiency of treatment or recovery of crude oil.     

Straw and Xylan Utilization by Pure Cultures of Nitrogen-Fixing Azospirillum spp

Azospirillum spp. were shown to utilize both straw and xylan, a major component of straw, for growth with an adequate combined N supply and also under N-limiting conditions. For most strains examined, a semisolid agar medium was satisfactory, but several strains appeared to be capable of slow metabolism of the agar. Subsequently, experiments were done with acid-washed sand supplemented with various carbon sources. In these experiments, authenticated laboratory strains, and all 16 recent field isolates from straw-amended soils, of both A. brasilense and A. lipoferum possessed the ability to utilize straw and xylan as energy sources for nitrogen fixation. Neither carboxymethyl cellulose nor cellulose was utilized. The strains and isolates differed in their abilities to utilize xylan and straw and in the efficiency of nitrogenase activity (CO₂/C₂H₂ ratio). Reasonable levels of activity could be maintained for at least 14 days in the sand cultures. Nitrogenase activity (acetylene reduction) was confirmed by ¹⁵N₂ incorporation. The level of nitrogenase activity observed was dependent on the time of the addition of acetylene to the culture vessels.     

Growth Response and Modifications of Organic Nitrogen Compounds in Pure and Mixed Cultures of Lactic Acid Bacteria from Wine

The interactions between the proteolytic X₂L strain of Oenococcus oeni and the non-proteolytic 12p strain of Pediococcus pentosaceus were assayed. The characteristics of cell growth, protein degradation, and amino acid production of both strains were determined in pure and mixed cultures. O. oeni showed poor cell growth and greater ability in the release of amino acids to the extracellular medium, whereas P. pentosaceus showed a higher yield in cell production with a decrease in the amino acid concentration in the medium. P. pentosaceus especially consumed essential amino acids for growth, and O. oeni released several of the essential amino acids important for growth of P. pentosaceus. In the mixed culture, mutualism was observed. The higher activity of the proteolytic system of O. oeni in mixed culture produced an increase in cell growth and in the amount of essential amino acids released. These findings provide new knowledge about the metabolic interactions between lactic acid bacteria isolated from wine when proteins are degraded in mixed bacterial populations.