Selection of Microorganisms Capable of Degrading Petroleum and Its Products at Low Temperatures

Of 150 cultures capable of degrading petroleum at +6°C, 40 strains growing in a liquid mineral nutrient medium containing petroleum (2%) as the sole source of carbon were selected. Of them, 13 cultures displaying a petroleum degradation rate exceeding 25% were selected. Abilities of these cultures and their associations to utilize fuel oil and its components—oils and benzene resins—were studied. A culture exhibiting degradation rates of fuel oil, its oils, benzene resins, and petroleum amounting to 17, 26, 10, and 51%, respectively, was selected. This culture can be used for cleanup of petroleum pollution under cold climatic conditions.     

Selection of microorganisms capable of degrading petroleum and petroleum products at decreased temperatures

Of 150 cultures capable of degrading petroleum at +6 degrees C, 40 strains growing in the liquid mineral nutrient medium containing petroleum (2%) as a sole source of carbon were selected. Of them, 13 cultures displaying a petroleum degradation rate exceeding 25% were selected. Abilities of these cultures and their associations to utilize fuel oil and its components--oils and benzene resins--were studied. The culture exhibiting degradation rates of fuel oil, its oils, benzene resins, and petroleum amounting to 17, 26, 10, and 51%, respectively, was selected. This culture can be used for cleanup of petroleum pollution under cold climatic conditions.     

Phenol utilisation by fungi isolated from activated sludge

The paper presents the efficiency of phenol removal (concentrations from 500 to 2000 mg/l) by fungi isolated from activated sludge purifying wastewater with high phenol concentration. Five fungal strains were isolated and identified. All isolated strains appeared to be Moniliales from the class of Fungi Imperfecti (Candida sp., Monosporium sp., Trichosporon sp.) Stationary cultures of the individual strains and their mixtures were maintained in Czapek medium containing phenol in concentration from 500 to 2000 mg/l. All isolated strains (except one) were capable of utilising phenol up to a concentration of 1500 mg/l. Depending on investigated strain, phenol in concentration of 500 mg/l was decomposed during 4-25 days, 750 mg/l during 4-14 days. After 20 days, a phenol decline of 1000 mg/l was observed. After 16 days, the phenol decline was 1500 mg/l. Higher phenol concentrations (1500 mg/l) were utilised only by a mixture of two strains. The investigated fungal strains showed good efficiency of phenol removal from high phenol concentration in wastewater and they may be proposed for use in the process of purifying wastewater of this type.     

Comparison of autochthonous bacteria and commercially available cultures with respect to their effectiveness in fuel oil degradation

Summary This study examined the microbial degradation of fuel oil by nine highly adapted different commercially available mixed bacterial cultures (DBC-plus, Flow Laboratories, Meckenheim, F.R.G.) and a bacterial community from a domestic sewage sludge sample. All mixed cultures were cultivated under aerobic batch conditions shaking (110 rpm) at 20°C in a mineral base medium containing 1 or 5% (v/v) fuel oil as the sole carbon source. Percent degradation of fuel oil and the n-alkane fraction was recorded for the nine DBC-plus cultures and the mixed population of the activated sludge sample. The increase in colony counts, protein, and optical density was studied during a 31-day incubation period for DBC-plus culture A, DBC-plus culture A2 and the activated sludge sample. The activated sludge mixed culture was most effective in degrading fuel oil, but various isolated bacterial strains from this bacterial community were not able to grow on fuel oil as the sole carbon source. In contrast, the n-alkane degradation rates of the DBC-cultures were lower, but single strains from the commercially available mixed cultures were able to mineralize fuel oil hydrocarbons. Strains ofPseudomonas aeruginosa were isolated most frequently and these organisms were able to grow very rapidly on fuel oil as a complex sole carbon source. The results indicate that fuel oil degradation in domestic sewage sludge is performed by mixed populations of naturally occurring bacteria and does not depend on the application of highly adapted commercially available cultures.     

Role of anaerobic spore-forming bacteria in the acidogenesis of glucose: Changes induced by discontinuous or low-rate feed supply

A mineral salts medium containing 1% (w/v) glucose providing carbon-limited growth conditions was subjected to anaerobic acidogenesis by mixed populations of bacteria in chemostat cultures. The formation of butyrate was shown to be dependent on the presence of saccharolytic anaerobic sporeformers in the acid-forming population. By the use of pasteurized activated sludge as an inoculum a culture was obtained consisting solely of anaerobic sporeformers that gave rise to the formation of butyrate, acetate, hydrogen and carbon dioxide as the main fermentation products. No formation of propionate could be detected. In this culture, the role of sporulation was investigated by applying periods of starvation and a single-step lowering of dilution rate (shift-down). In an experiment using a mineral salts medium supplemented with 1% (w/v) glucose and 0.5% (w/v) casein hydrolysate formation of refractile forespores as well as cell lysis could be demonstrated after 6 h starvation.In mixed cultures, initially inoculated with non-pasteurized activated sludge, a regular interruption of feed supply for 1 h per day resulted in selection of non-sporulatiog anaerobes. The fermentation pattern changed to a production of propionate and acetate, with a concomitant reduction of gas production. Similar results were obtained with shift-down in dilution rate.     

The effect of aromatic compounds on the work of activated sludge.

The resistance of bacteria occurring in activated sludge purifying refining-petrochemical wastewaters to phenol, cresol and pirocatechin as well as the possibility of the purification of synthetic wastewaters carrying high concentrations of these compounds by sludge composed of resistant strains was studied. The most toxic of the studied compounds was phenol. Six out of 29 strains were resistant to high concentration of phenol (1000 mg/l). Activated sludge synthesized from strains resistant to phenol, cresol and pyrocatechin was tolerant to high concentrations of these compounds. Worsening of the work of the sludge, expressed by decrease of GOD, was observed at the concentrations of phenol, cresol and pyrocatechin of 2000, 400 and 300 mg/l, respectively.     

阿特拉津降解菌株的分离、鉴定和工业废水生物处理试验

用液体无机盐培养基富集培养法和无机盐平板直接分离法, 从生产阿特拉津的农药厂的废水和污泥混合物中分离到13个能以阿特拉津为唯一氮源生长的细菌菌株。通过16S rRNA基因序列分析, 11个菌株被鉴定为Arthrobacter spp., 2个菌株被鉴定为Pseudomonas spp.。对阿特拉津降解活力最高的Arthrobacter sp. AD30和Pseudomonas sp. AD39的降解基因组成和降解特性进行了详细研究。降解基因的PCR扩增表明, AD30和AD39都含有trzN-atzBC基因, 能将有毒的阿特拉津降解成无毒的氰尿酸。降解实验表明, 向阿特拉津浓度为200 mg/L的无机盐培养基中分别接种等量的AD30、AD39和这两个菌株的混合菌液, 30°C振荡培养48 h以后, 阿特拉津去除率分别为92.5%、97.9%和99.6%, 表明混合菌的降解效果好于单菌。用AD30和AD39的混合菌液接种阿特拉津浓度为176 mg/L的工业废水, 30°C振荡培养72 h以后, 99.1%的阿特拉津被去除, 表明混合菌株在阿特拉津工业废水的生物处理中有很好的应用潜力。     

Biodegradation of crude oil by soil microorganisms in the tropic

Five microorganisms, three bacteria and two yeasts, capable of degrading Tapis light crude oil were isolated from oil-contaminated soil in Bangkok, Thailand. Soil enrichment culture was done by inoculating the soil in mineral salt medium with 0.5% v/v Tapis crude oil as the sole carbon source. Crude oil biodegradation was measured by gas chromatography method. Five strains of pure microorganisms with petroleum degrading ability were isolated: three were bacteria and the other two were yeasts. Candida tropicalis strains 7Y and 15Y were identified as efficient oil degraders. Strain 15Y was more efficient, it was able to reduce 87.3% of the total petroleum or 99.6% of n-alkanes within the 7-day incubation period at room temperature of 25 ± 2 °C.     

Alga-derived substrates select for distinct Betaproteobacterial lineages and contribute to niche separation in Limnohabitans strains

We examined the proportions of major Betaproteobacteria subgroups within bacterial communities in diverse nonaxenic, monospecific cultures of algae or cyanobacteria: four species of cryptophyta (genera Cryptomonas and Rhodomonas), four species of chlorophyta (genera Pediastrum, Staurastrum, and Chlamydomonas), and two species of cyanobacteria (genera Dolichospermum and Aphanizomenon). In the cryptophyta cultures, Betaproteobacteria represented 48 to 71% of total bacteria, the genus Limnohabitans represented 18 to 26%, and the Polynucleobacter B subcluster represented 5 to 16%. In the taxonomically diverse chlorophyta group, the genus Limnohabitans accounted for 7 to 45% of total bacteria. In contrast, cyanobacterial cultures contained significantly lower proportions of the Limnohabitans bacteria (1 to 3% of the total) than the cryptophyta and chlorophyta cultures. Notably, largely absent in all of the cultures was Polynucleobacter necessarius (Polynucleobacter C subcluster). Subsequently, we examined the growth of Limnohabitans strains in the presence of different algae or their extracellular products (EPP). Two strains, affiliated with Limnohabitans planktonicus and Limnohabitans parvus, were separately inoculated into axenic cultures of three algal species growing in an inorganic medium: Cryptomonas sp., Chlamydomonas noctigama, and Pediastrum boryanum. The Limnohabitans strains cocultured with these algae or inoculated into their EPP consistently showed (i) pronounced population growth compared to the control without the algae or EPP and (ii) stronger growth stimulation of L. planktonicus than of L. parvus. Overall, growth responses of the Limnohabitans strains cultured with algae were highly species specific, which suggests a pronounced niche separation between two closely related Limnohabitans species likely mediated by different abilities to utilize the substrates produced by different algal species.     

Denitrification of high concentrations of nitrites and nitrates in synthetic medium with different sources of organic carbon. III. Methanol

The denitrification of nitrites and nitrates (1000 mg N/l) in medium containing methanol as a source of organic carbon was studied. Continuous cultures of mixed population of autochtonic microflora from bottom sludge of nitrogenous wastewater reservoir were set up in a chemostat-type column and packed bed reactor. The efficiency of denitrification of nitrates in packed bed reactor was 506.7 mg N/l/h whereas denitrification of nitrites was from 8.7 to 16.0 mg N/l/h depending on the granulation of the filing material. In the latter case 83% nitrogen was removed from the medium. One of the factors causing low efficiency of denitrification of nitrites is excessive alkalization of the medium in the bed. The use of a three-step bed with adjusted pH resulted in complete denitrification of nitrites with efficiency 60 mg N/l/h. The bacteria inside the bed were dominated by Paracoccus denitrificans and by Pseudomonas aeruginosa when nitrates were present. The sensitivity of P. denitrificans to high concentrations of nitrites seems to be the second factor contributing to low efficiency of denitrification with methanol as organic substrate.     

Low-temperature microbial degradation of crude oil products differing in the extent of condensation

Out of the 30 strains capable of oil degradation at 4-6 degrees C, four were selected by the ability to degrade 40% of the oil substrate present in the growth medium: Rhodococcus spp. DS-07 and DS-21 and Pseudomonas spp. DS-09 and DS-22. We studied the activity of these strains as degraders of oil products of various condensation degrees (crude oil, masut, petroleum oils, benzene resins and ethanol-benzene resins) at 4-6 degrees C. The maximum degrees of degradation of masut and ethanol-benzene resins were observed in Pseudomonas spp. DS-22 (17.2% and 5.2%, respectively). The maximum degradation of petroleum oils and benzene resins was observed in Rhodococcus spp. DS-07 (40% and 16.6%, respectively). The strains provide a basis for developing biodegrader preparations applicable to bioremediation of oil-polluted sites under the conditions of cold climate.     

Microbial degradation of nitrobenzene and mono-nitrophenol by bacteria enriched from municipal activated sludge.

Using a mixture of three mono nitrophenols as sole carbon, nitrogen and energy sources, mixed cultures were enriched from municipal activated sludge to degrade both nitrophenols and nitrobenzene. Bacterial growth and degradation rate could be increased by supplementing the medium with 0.1% YE. Microorganisms were isolated from the nitrophenols enrichment, and they were identified as strains of Comamonas testosteroni and Acidovorax delafieldii. These strains showed broad degradation ability toward nitrophenols and nitrobenzene.     

Biodegradation of petroleum hydrocarbons by filamentous fungi (Aspergillus ustus and Purpureocillium lilacinum) isolated from used engine oil contaminated soil

The use of microorganisms for remediation and restoration of hydrocarbons contaminated soils is an effective and economic solution. The current study aims to find out efficient telluric filamentous fungi to degrade petroleum hydrocarbons pollutants. Six fungal strains were isolated from used engine (UE) oil contaminated soil. Fungi were screened for their ability to degrade crude oil, diesel and UE oil using 2.6-dichlorophenol indophenol (DCPIP). Two isolates were selected, identified and registered at NCBI as Aspergillus ustus HM3.aaa and Purpureocillium lilacinum HM4.aaa. Fungi were tested for their tolerance to different concentration of petroleum oils using radial growth diameter assay. Hydrocarbons removal percentage was evaluated gravimetrically. The degradation kinetic of crude oil was studied at a time interval of 10 days. A.ustus was the most tolerant fungi to high concentration of petroleum oils in solid medium. Quantitative analysis showed that crude oil was the most degraded oil by both isolate; P. lilacinium and A. ustus removed 44.55% and 30.43% of crude oil, respectively. The two fungi were able to degrade, respectively, 27.66 and 21.27% of diesel and 14.39 and 16.00% of UE oil. As compared to the controls, these fungi accumulated high biomass in liquid medium with all petroleum oils. Likewise, crude oil removal rate constant (K) and half-lives (t_1/2) were 0.02 day⁻¹, 34.66 day and 0.015 day⁻¹, 46.21 day for P. lilacinium and A. ustus, respectively. The selected fungi appear interesting for petroleum oils biodegradation and their application for soil bioremediation require scale-up studies.     

Characterisation of biodegradation capacities of environmental microflorae for diesel oil by comprehensive two-dimensional gas chromatography

In contaminated soils, efficiency of natural attenuation or engineered bioremediation largely depends on biodegradation capacities of the local microflorae. In the present study, the biodegradation capacities of various microflorae towards diesel oil were determined in laboratory conditions. Microflorae were collected from 9 contaminated and 10 uncontaminated soil samples and were compared to urban wastewater activated sludge. The recalcitrance of hydrocarbons in tests was characterised using both gas chromatography (GC) and comprehensive two-dimensional gas chromatography (GC×GC). The microflorae from contaminated soils were found to exhibit higher degradation capacities than those from uncontaminated soil and activated sludge. In cultures inoculated by contaminated-soil microflorae, 80% of diesel oil on an average was consumed over 4-week incubation compared to only 64% in uncontaminated soil and 60% in activated sludge cultures. As shown by GC, n-alkanes of diesel oil were totally utilised by each microflora but differentiated degradation extents were observed for cyclic and branched hydrocarbons. The enhanced degradation capacities of impacted-soil microflorae resulted probably from an adaptation to the hydrocarbon contaminants but a similar adaptation was noted in uncontaminated soils when conifer trees might have released natural hydrocarbons. GC×GC showed that a contaminated-soil microflora removed all aromatics and all branched alkanes containing less than C₁₅. The most recalcitrant compounds were the branched and cyclic alkanes with 15–23 atoms of carbon.     

Detection and isolation of chloromethane-degrading bacteria from the Arabidopsis thaliana phyllosphere, and characterization of chloromethane utilization genes

Chloromethane gas is produced naturally in the phyllosphere, the compartment defined as the aboveground parts of vegetation, which hosts a rich bacterial flora. Chloromethane may serve as a growth substrate for specialized aerobic methylotrophic bacteria, which have been isolated from soil and water environments, and use cmu genes for chloromethane utilization. Evidence for the presence of chloromethane-degrading bacteria on the leaf surfaces of Arabidopsis thaliana was obtained by specific quantitative PCR of the cmuA gene encoding the two-domain methyltransferase corrinoid protein of chloromethane dehalogenase. Bacterial strains were isolated on a solid mineral medium with chloromethane as the sole carbon source from liquid mineral medium enrichment cultures inoculated with leaves of A. thaliana. Restriction analysis-based genotyping of cmuA PCR products was used to evaluate the diversity of chloromethane-degrading bacteria during enrichment and after strain isolation. The isolates obtained, affiliated to the genus Hyphomicrobium based on their 16S rRNA gene sequence and the presence of characteristic hyphae, dehalogenate chloromethane, and grow in a liquid culture with chloromethane as the sole carbon and energy source. The cmu genes of these isolates were analysed using new PCR primers, and their sequences were compared with those of previously reported aerobic chloromethane-degrading strains. The three isolates featured a colinear cmuBCA gene arrangement similar to that of all previously characterized strains, except Methylobacterium extorquens CM4 of known genome sequence.     

Treatment of lipid-containing wastewater using bacteria which assimilate lipids

Bacteria which grew in a medium containing olive oil as a sole source of carbon were isolated from two meat plants in the Sendai district of Japan. All of the isolates tested assimilated beef tallow, lard, olive oil and used salad oil as a sole carbon source in shaking cultures. One of the isolates, strain 351, digested lipids most efficiently, as shown by the amount of n-hexane extracts that remained. This bacterium was identified as Bacillus sp. A new and efficient laboratory-scale apparatus for the biological treatment of lipid-containing wastewater was devised using strain 351. The apparatus consisted of a water circulation system for the primary treatment of the water, in which strain 351 was inoculated, and an ordinary aeration tank using activated sludge as a secondary treatment. Lipids in the wastewater could be almost completely removed by this apparatus without physical treatment. On the other hand, an ordinary aeration system in the laboratory using an air stone and air pump resulted in the floating of lipids, and was not successful in digesting lipids even in the presence of strain 351.     

Bactericidal activities of essential oils of basil and sage against a range of bacteria and the effect of these essential oils on Vibrio parahaemolyticus

Basil and sage essential oils were examined for bactericidal activity against a range of Gram-positive and Gram-negative bacteria by viable count determinations. Generally, Gram-positive bacteria showed higher resistance to basil and sage essential oils than Gram-negative bacteria. Vibrio species showed a high sensitivity to both essential oils. Stationary growth phase cells of selected bacteria showed higher resistance to these essential oils than exponential growth phase cells. Basil-resistant (b21) and sage-resistant (s20) strains of Vibrio parahaemolyticus were isolated. Both strains showed higher resistance to heat and H2O2 than parent strain. Conversely, heat-adapted V. parahaemolyticus also showed a higher resistance to these essential oils than nonadapted cells.     

Formation of cellulose fibrils by gram-negative bacteria and their role in bacterial flocculation

Summary Exocellular fibrils, consisting of true cellulose, were found to be produced by many bacteria. These bacteria have been selected out of a large number of strains isolated from activated sludge on the basis of their flocculent growth habit in liquid medium.The amount of cellulose, present in the bacterial flocs, varied from 1.0 to 4.0%. In addition to strains isolated from activated sludge, like Pseudomonas, Achromobacter, Alcaligenes and Aerobacter, also strains of the genera Rhizobium, Agrobacterium and Azotobacter were found to give flocculent growth due to the formation of cellulose fibrils.Bacterial flocculation in pure cultures of the strains examined was mainly caused by the production of exocellular fibrils. Apparently, the formation of cellulose fibrils seems to be a common property of Gram-negative, floc-forming bacteria, and may not be restricted to Acetobacter xylinum.     

Biodegradation of heavy oily sludge by a two-step inoculation composting process using synergistic effect of indigenous isolated bacteria

The impact of two-step inoculation of indigenous strains and their synergistic effect in the scaling-up of petroleum hydrocarbons biodegradation from a mineral-based medium (MBM) to a two-phase composting process were investigated. After isolating the strains KA3 and KA4 from heavy oily sludge (HOS), their emulsification index (E₂₄), bacterial adhesion to hydrocarbon (BATH), and oil degradation efficiency were evaluated in the MBM. Then, they were inoculated twice into the composting bioreactors lasted for the primary 8 weeks as the first phase (FP) and subsequent 8 weeks as the second phase (SP). The results indicated that the consortium of the two strains degraded 16-61% of crude oil (1-5% concentration) in the MBM. In the composting reactors, removals of 20 g kg⁻¹ initial concentration of total petroleum hydrocarbons (TPH) were found to be 63.95, 61.00, and 89.35% for the strains KA3, KA4, and their consortium, respectively. The computed biodegradation constants indicated the synergistic effect of the two strains and the effectiveness of the second-step inoculation. The study demonstrated the successful scaling-up of HOS biodegradation from MBM to the two-phase composting process through two-step inoculation of the isolated strains.     

Comparison of solid‐phase cytometry and the plate count method for the evaluation of the survival of bacteria in pharmaceutical oils

Aim: To compare the survival of four bacterial strains (Escherichia coli, Proteus mirabilis, Staphylococcus aureus, Pseudomonas aeruginosa) in pharmaceutical oils, including jojoba oil/tea tree oil, carbol oil, jojoba oil and sesame oil. Methods and Results: Oils were spiked with the test bacteria in a concentration of 10⁴ CFU ml^?1. Bacteria were extracted from oils with phosphate‐buffered saline containing 0·5% Tween 20. Aliquots of the pooled water layers were analysed by solid‐phase cytometry and plate counting. Plate counts dropped to zero for all test strains exposed for 24 h to three of the four oils. In contrast, significant numbers of viable cells were still detected by SPC, except in the jojoba oil/tea tree oil mixture and partly in sesame oil. Conclusions: Exposure of bacteria for 24 h to the two oils containing an antimicrobial led to a loss of their culturability but not necessarily of their viability. The antibacterial activity of the jojoba oil/tea tree oil mixture supersedes that of carbol oil. Significance and Impact of the Study: These in vitro data suggest that the jojoba oil/tea tree oil mixture more than carbol oil inhibits bacterial proliferation when used for intermittent self‐catherization.