Non-selective precultivation of bacteria able to degrade different polycyclic aromatic hydrocarbons (PAH)

The influence of the precultivation with different carbon sources on the ability of three different bacterial strains (Sphingomonas sp. strain BA2, Gordona sp. strain BP9, Mycobacterium sp. strain VF1) to grow on phenanthrene. anthracene, pyrene or fluoranthene as the sole source of carbon and energy were studied. The strains were found to maintain their ability to grow on two of the four PAH after 30 serial transfers in liquid nutrient broth medium without selective pressure. The ability to grow on these PAH as the sole carbon and energy source was also maintained after curing experiments with acridine orange. The high stability of the PAH-degradation phenotype suggests that the tested strains carry at least parts of the PAH-degradation pathway genes on the chromosome. The PAH-degradation versatility of the strains was also influenced by the carbon source being used for precultivation. Possible reasons for the particularly good impact of the precultivation on hexadecane on the PAH degradation are discussed in this paper.     

Isolation of Adherent Polycyclic Aromatic Hydrocarbon (PAH)-Degrading Bacteria Using PAH-Sorbing Carriers

Two different procedures were compared to isolate polycyclic aromatic hydrocarbon (PAH)-utilizing bacteria from PAH-contaminated soil and sludge samples, i.e., (i) shaken enrichment cultures in liquid mineral medium in which PAHs were supplied as crystals and (ii) a new method in which PAH degraders were enriched on and recovered from hydrophobic membranes containing sorbed PAHs. Both techniques were successful, but selected from the same source different bacterial strains able to grow on PAHs as the sole source of carbon and energy. The liquid enrichment mainly selected for Sphingomonas spp., whereas the membrane method exclusively led to the selection of Mycobacterium spp. Furthermore, in separate membrane enrichment set-ups with different membrane types, three repetitive extragenic palindromic PCR-related Mycobacterium strains were recovered. The new Mycobacterium isolates were strongly hydrophobic and displayed the capacity to adhere strongly to different surfaces. One strain, Mycobacterium sp. LB501T, displayed an unusual combination of high adhesion efficiency and an extremely high negative charge. This strain may represent a new bacterial species as suggested by 16S rRNA gene sequence analysis. These results indicate that the provision of hydrophobic sorbents containing sorbed PAHs in the enrichment procedure discriminated in favor of certain bacterial characteristics. The new isolation method is appropriate to select for adherent PAH-degrading bacteria, which might be useful to biodegrade sorbed PAHs in soils and sludge.     

红树林湿地多环芳烃的微生物降解

红树林(mangrove)是海陆交汇带重要的湿地生态系统,也是环境污染物蓄积与转化的热区.多环芳烃(polycyclic aromatic hydrocarbons,PAHs)因其环境蓄积特点在红树林生境中广泛分布,威胁生态系统健康,其降解转化是近年的研究重点.本文聚焦红树林湿地多环芳烃的微生物降解研究现状,从红树林生...     

Isolation and Characterization of Polycyclic Aromatic Hydrocarbon–Degrading Mycobacterium Isolates from Soil

Bioremediation of soils contaminated with wood preservatives containing polycyclic aromatic hydrocarbons (PAHs) is desired because of their toxic, mutagenic, and carcinogenic properties. Creosote wood preservative–contaminated soils at the Champion International Superfund Site in Libby, Montana currently undergo bioremediation in a prepared-bed land treatment unit (LTU) process. Microbes isolated from these LTU soils rapidly mineralized the ¹⁴C-labeled PAH pyrene in the LTU soil. Gram staining, electron microscopy, and 16S rDNA-sequencing revealed that three of these bacteria, JLS, KMS, and MCS, were Mycobacterium strains. The phylogeny of the 16S rDNA showed that they were distinct from other Mycobacterium isolates with PAH-degrading activities. Catalase and superoxide dismutase (SOD) isozyme profiles confirmed that each isolate was distinct from each other and from the PAH-degrading mycobacterium, Mycobacterium vanbaalenii sp. nov, isolated from a petroleum-contaminated soil. We find that dioxygenase genes nidA and nidB are present in each of the Libby Mycobacterium isolates and are adjacent to each other in the sequence nidB-nidA, an order that is unique to the PAH-degrading mycobacteria.This revised version was published online in November 2004 with corrections to Volume 48.     

Comparison of mineralization of solid-sorbed phenanthrene by polycyclic aromatic hydrocarbon (PAH)-degrading Mycobacterium spp. and Sphingomonas spp.

The mineralization of ¹⁴C-phenanthrene, sorbed to porous synthetic amberlite sorbents, i.e., IRC50, XAD7-HP, and XAD2, by three phenanthrene-degrading Mycobacterium soil isolates, i.e., strains VM552, VM531, and VM451 and three phenanthrene-degrading Sphingomonas soil isolates, i.e., strains LH162, EPA505 and LH227, was compared. In P-buffer and in the presence of IRC50, for all strains the maximum rate of mineralization of ¹⁴C-phenanthrene was significantly higher (1.1–1.9 ng ml⁻¹ h⁻¹) than the initial abiotic desorption rate (0.2 ng ml⁻¹ h⁻¹), indicating that both Mycobacterium and Sphingomonas utilize sorbed phenanthrene with a higher rate than can be explained by abiotic desorption. Because all Mycobacterium and Sphingomonas strains belonged to different species, it can be suggested that this feature is intrinsic to those genera rather than a specific feature of a particular strain. The final mineralization extent in P-buffer in the presence of IRC50 was about a factor of two higher for the Mycobacterium strains compared to the Sphingomonas strains. Moreover, a significantly higher normalized phenanthrene mineralization ratio in the presence of IRC50 to the control (without IRC50) was found for the Mycobacterium strains compared to the normalized ratio found for the Sphingomonas strains. Addition of minimal nutrients had a more beneficial effect on phenanthrene mineralization by Sphingomonas compared to Mycobacterium, resulting into similar mineralization extents and rates for both types of strains in the presence of IRC50. Our results show that Mycobacterium is better adapted to utilization of sorbed phenanthrene compared to Sphingomonas, especially in nutrient-poor conditions.     

Isolation and characterization of indigenous soil bacteria for bioaugmentation of PAH contaminated soil of semiarid Patagonia, Argentina

The aim of this work was to isolate PAH degrading-bacteria from contaminated Patagonia soil with the ability to tolerate the usual environmental stresses (oligotrophic and dryness conditions). Two approaches were utilized to obtain PAH-degrading bacteria from the Patagonian soil. With a traditional enrichment approach only the PAH- degrading strain 36 was isolated. Using a direct isolation approach three PAH-degrading strains (1A, 22A and 22B) were isolated. The phylogenetic analysis revealed that all isolates belonged to Sphingomonas genus. The PAH degrading activity and the resistance to stress conditions of the strains were determined and compared with those of the exogenous PAH-degrading Sphingomonas paucimobilis 20006FA. The strains 1A, 22A and 36 were phylogenetically closely related between them and with the strain 20006FA. The strain 22B, that showed a different phylogenetic position, was more resistant to C-starvation and drying conditions than other Patagonian strains. The effect of the inoculation of these strains on phenanthrene-induced mineralization and elimination was studied in Patagonian soil artificially contaminated, at different environmental conditions. The results suggest that strain 22B is the most suitable strain for bioaugmentation in PAH-contaminated soils of Central Patagonia, due to its adaptation to the usual environmental conditions. Our results show the importance of a detailed physiological characterization of isolates for autochthonous bioaugmentation strategies success.     

Distribution and location of polycyclic aromatic hydrocarbons (PAHs) and PAH-degrading bacteria within polluted soil aggregates

A study was conducted to determine the location and distribution of PAH and PAH-degrading bacteria in different aggregate size fractions of an industrially polluted soil. The estimation of PAH-degrading bacteria using an MPN microplate technique indicated that these bacteria are most numerous in the aggregate size fractions corresponding to fine silt (2–20m) and clay(<2m) compared to larger fractions or unfractionated soil.PAH concentrations were also highest in the aggregate size fraction corresponding to fine silt. Similar results were found in a spiked soil (incubated for 6 months) with similar carbonated minerals. Transmission electron microscopy observations showed that the autochtonous PAH-degrading bacteria were embedded in the aggregates where PAHs were abundant. In spite of this extensive co-localisation PAH degradation was limited during 6 months incubation. This indicates that factors other than spatial distribution and PAH degrading ability control degradation rates. The fine silt fraction of the industrial soil had an elevated C/N ratio (35) compared to the clay fraction (C/N: 16). Thus the fraction which assumably had the highest specific surface area contained less PAH but similar numbers of PAH-degraders. N thus seem to play an important role in the long term, but as PAH degradation was low in fine size fractions, other sources/factors were probably limiting (easily degradable C, P org, O₂ etc.). Based on these findings, soil particle organization and structure of soil aggregates appear to be important for the characterization of a polluted soil (localization and sequestration). Manipulations that modify aggregation in polluted soils could thus potentially influence the accessibility and biodegradability of PAHs.     

REDUCTION IN SOIL POLYCYCLIC AROMATIC HYDROCARBONS BY ARBUSCULAR MYCORRHIZAL LEEK PLANTS

The effect of arbuscular mycorrhizal fungi (AMF) on the reduction of soil polycyclic aromatic hydrocarbon (PAH), nutrient uptake, and growth of leek (Allium porrum L. cv. Musselburgh) plants was studied under greenhouse conditions. This experiment was a 3 × 2 × 2 × 4 factorial design including three mycorrhizal treatments (non-AMF, Glomus intraradices, and G. versiforme strains), two microorganism statuses (with and without soil bacteria), two PAH chemicals (anthracene and phenanthrene), and four PAH concentrations (three concentrations added and one control). Leek growth was reduced significantly in soils spiked with anthracene or phenanthrene. Inoculation with either Glomus intraradices or G. versiforme not only increased N and P uptake and plant growth, but also enhanced PAH disappearance in soil. After 12 weeks of potcultures, the anthracene and phenanthrene concentrations in soils were decreased as compared to their initial level, 9%–31% versus 43%–88%, respectively. Reductions in concentration were larger for phenanthrene than anthracene. The addition of a soil microorganism (SM) extract in potcultures accelerated the disappearance of PAHs. The decrease of PAHs in soil was mainly attributed to the enhanced nutrient uptake by AMF, leading to improved plant growth, which, in turn, may stimulate soil microbial activity. This study shows the interrelationships between AMF, plants, other SMs, and PAH disappearance in soil. The phytoremediation of soil contaminated with PAHs can be accelerated through inoculation with AMF and other SMs.     

Nutrient-limited biodegradation of PAH in various soil strata at a creosote contaminated site

The effects of nutrient addition on the in situ biodegradation of polycyclic aromatic hydrocarbons in creosote contaminated soil were studied in soil columns taken from various soil strata at a wood preserving plant in Norway. Three samples were used: one from the topsoil (0–0.5 m), one from an organic rich layer (2–2.5 m) and one from the sandy aquifer (4.5–5 m). The addition of inorganic nitrogen and phosphorous stimulated the degradation of polycyclic aromatic hydrocarbons (PAHs) in the top soil and the aquifer sand. These two soils, which differed strongly in contamination levels, responded similarly to nutrient addition with the corresponding degradation of 4-ring PAHs. The ratio between available nitrogen (N) and phosphorous (P) might explain the degree of degradation observed for the 4-ring PAHs. However, the degree of degradation of 3-ring PAHs did not significantly increase after nutrient addition. An increase in the respiration rate, after nutrient addition, could only be observed in the topsoil. In the aquifer sand, 4-ring PAH degradation was not accompanied by an increase in the respiration rate or the number of heterotrophic micro-organisms. PAH degradation in the organic layer did not respond to nutrient addition. This was probably due to the low availability of the contaminants for micro-organisms, as a result of sorption to the soil organic matter. Our data illustrate the need for a better understanding of the role of nutrients in the degradation of high molecular weight hydrocarbons for the successful application of bioremediation at PAH contaminated sites.     

Culture Independent Detection of Sphingomonas sp. EPA 505 Related Strains in Soils Contaminated with Polycyclic Aromatic Hydrocarbons (PAHs)

The Sphingomonas genus hosts many interesting pollutant-degrading strains. Sphingomonas sp. EPA505 is the best studied polycyclic aromatic hydrocarbon (PAH)-degrading Sphingomonas strain. Based on 16S rRNA gene sequence analysis, Sphingomonas sp. strain EPA505 forms a separate branch in the Sphingomonas phylogenetic tree grouping exclusively PAH-degrading isolates. For specific PCR detection and monitoring of Sphingomonas sp. EPA505 and related strains in PAH-contaminated soils, a new 16S rRNA gene-based primer set was designed. The new primer set was shown to be highly selective for Sphingomonas sp. strain EPA505 as it only amplified DNA from strain EPA505 and not from other tested Sphingomonas strains or soil bacteria not belonging to the Sphingomonas genus. Using DNA extracts of a variety of inoculated PAH-contaminated soils, the primer pair was able to detect EPA505 in concentrations as low as 10² cells per gram of soil. Applying the new primer set, 16S rRNA gene fragments which were 99–100% similar to the corresponding gene of strain EPA505 were amplified from four of five PAH-contaminated soils. On the other hand, no PCR products were obtained from any of five tested uncontaminated soils. The preferential presence of EPA505 related Sphingomonas strains in PAH-contaminated soils with very different contamination profiles and different origin suggests an important role of this type of Sphingomonas in the natural Sphingomonas community colonizing PAH-contaminated sites.     

Characterization of fluoranthene- and pyrene-degrading bacteria isolated from PAH-contaminated soils and sediments

Sixteen environmental samples, from the United States, Germany and Norway, with histories of previous exposure to either creosote, diesel fuel or coal tar materials, were screened for bacteria which could degrade high molecular weight (HMW) polycyclic aromatic hydrocarbons (PAHs). A modified version of the spray plate technique was used for the isolations. Using fluoranthene (FLA) and pyrene (PYR) as model HMW PAHs, we isolated 28 strains on FLA and 21 strains on PYR. FLA degraders were defined as able to grow on FLA but not PYR. PYR degraders grew on both PAHs. All PYR degraders were found to be Gram-positive and all FLA degraders were Gram-negative. GC-FAME analysis showed that many of the PYR degraders were Mycobacterium spp and many of the FLA degraders were Sphingomonas spp. Comparison of the metabolic characteristics of the strains using the spray plate technique and direct growth studies revealed that more than half of the FLA degraders (59%) were able to cometabolize PYR (ie, they produced clearing zones or colored metabolites on spray plates but did not grow on the PAH) and the ability of many of these strains to cometabolize fluorene, anthracene, benzo[b]fluorene, benzo[a]anthracene and benzo[a]pyrene was significantly affected by pre-exposure to phenanthrene. Studies on the metabolic products produced from PYR cometabolism by strain EPA 505 suggested the possibility of attack at two different sites on the PYR molecule. However, the inability to derive degradable carbon from initial opening of one of the PYR rings probably accounted for the lack of growth on this PAH by the FLA-degrading strains. The PYR degraders on the other hand, were less able to cometabolize HMW PAHs, even following pre-exposure to PHE. Characterization of the FLA degradation pathway for several of the Sphingomonas isolates indicated oxidation and ring opening through to acenaphthenone as the principle metabolite. Strain CO6, however, also oxidized FLA through fluorenone, suggesting a dual attack on the FLA molecule, similar to that observed by others in Mycobacterium spp. Journal of Industrial Microbiology & Biotechnology (2000) 24, 100–112. Received 01 May 1999/ Accepted in revised form 01 November 1999     

Factors Affecting Predation by Cyclidium sp. and Euplotes sp. on PAH-Degrading and Nondegrading Bacteria

Abstract If predators select for or against contaminant-degrading bacteria, it will affect bacterial survival and has important implications for bioremediation. Protozoa are important predators of bacteria. In order to determine whether protozoa preyed differentially on bacteria with different degradation abilities, two ciliates (Euplotes sp. and Cyclidium sp.) and three strains of PAH-degrading bacteria (Vibrio spp., degrading naphthalene, anthracene, or phenanthrene) were isolated from sediment from New York/New Jersey Harbor. By manipulating growth conditions, bacterial strains with different PAH-degradation abilities and different cell properties were produced. Stepwise regression models were used to analyze how clearance rates on suspended bacteria and grazing rates on bacteria attached to particles were affected by bacterial size, hydrophobicity, C:N ratio, protein content, and PAH-degradation ability. Clearance rates ranged from 0 to 49 nl ciliate⁻¹ h⁻¹ for Euplotes sp. and from 0 to 1.7 nl ciliate⁻¹ h⁻¹ for Cyclidium sp. Clearance rates of both ciliates were positively correlated with bacterial size, hydrophobicity, and protein content, and negatively correlated with C:N ratio. PAH degradation ability had no (for Euplotes sp.) or small (for Cyclidium sp.) effects on clearance rates. The models accounted for 63–75% of the variation in clearance rates on different bacteria. Only Euplotes sp. grazed on attached bacteria, at rates from 3 to 176 bacteria ciliate⁻¹ h⁻¹. A regression model with only C:N ratio and protein content explained 45% of the variation in grazing rates. These models indicate that multiple properties of bacteria affect their susceptibility to predation by ciliates, but PAH-degradation ability per se has little effect. Received: 5 May 1998; Accepted: 14 September 1998     

Influence of growth medium on cometabolic degradation of polycyclic aromatic hydrocarbons by <Emphasis Type="Italic">Sphingomonas</Emphasis> sp. strain PheB4

The influence of growth medium on cometabolic degradation of polycyclic aromatic hydrocarbons (PAHs) was investigated when Sphingomonas sp. strain PheB4 isolated from surface mangrove sediments was grown in either phenanthrene-containing mineral salts medium (PMSM) or nutrient broth (NB). The NB-grown culture exhibited a more rapid cometabolic degradation of single and mixed non-growth substrate PAHs compared to the PMSM-grown culture. The concentrations of PAH metabolites were also lower in NB-grown culture than in PMSM-grown culture, suggesting that NB-grown culture removed metabolites at a faster rate, particularly, for metabolites produced from cometabolic degradation of a binary mixture of PAHs. Cometabolic pathways of single PAH (anthracene, fluorene, or fluoranthene) in NB-grown culture showed similarity to that in PMSM-grown culture. However, cometabolic pathways of mixed PAHs were more diverse in NB-grown culture than that in PMSM-grown culture. These results indicated that nutrient rich medium was effective in enhancing cometabolic degradation of mixed PAHs concomitant with a rapid removal of metabolites, which could be useful for the bioremediation of mixed PAHs contaminated sites using Sphingomonas sp. strain PheB4.     

Strong Impact on the Polycyclic Aromatic Hydrocarbon (PAH)-Degrading Community of a PAH-Polluted Soil but Marginal Effect on PAH Degradation when Priming with Bioremediated Soil Dominated by Mycobacteria

Bioaugmentation of soil polluted with polycyclic aromatic hydrocarbons (PAHs) is often disappointing because of the low survival rate and low activity of the introduced degrader bacteria. We therefore investigated the possibility of priming PAH degradation in soil by adding 2% of bioremediated soil with a high capacity for PAH degradation. The culturable PAH-degrading community of the bioremediated primer soil was dominated by Mycobacterium spp. A microcosm containing pristine soil artificially polluted with PAHs and primed with bioremediated soil showed a fast, 100- to 1,000-fold increase in numbers of culturable phenanthrene-, pyrene-, and fluoranthene degraders and a 160-fold increase in copy numbers of the mycobacterial PAH dioxygenase gene pdo1. A nonpolluted microcosm primed with bioremediated soil showed a high rate of survival of the introduced degrader community during the 112 days of incubation. A nonprimed control microcosm containing pristine soil artificially polluted with PAHs showed only small increases in the numbers of culturable PAH degraders and no pdo1 genes. Initial PAH degradation rates were highest in the primed microcosm, but later, the degradation rates were comparable in primed and nonprimed soil. Thus, the proliferation and persistence of the introduced, soil-adapted degraders had only a marginal effect on PAH degradation. Given the small effect of priming with bioremediated soil and the likely presence of PAH degraders in almost all PAH-contaminated soils, it seems questionable to prime PAH-contaminated soil with bioremediated soil as a means of large-scale soil bioremediation.     

Microbial populations related to PAH biodegradation in an aged biostimulated creosote-contaminated soil

A previous bioremediation survey on a creosote-contaminated soil showed that aeration and optimal humidity promoted depletion of three-ringed polycyclic aromatic hydrocarbons (PAHs), but residual concentrations of four-ringed benzo(a)anthracene (B(a)A) and chrysene (Chry) remained. In order to explain the lack of further degradation of heavier PAHs such as four-ringed PAHs and to analyze the microbial population responsible for PAH biodegradation, a chemical and microbial molecular approach was used. Using a slurry incubation strategy, soil in liquid mineral medium with and without additional B(a)A and Chry was found to contain a powerful PAH-degrading microbial community that eliminated 89% and 53% of the added B(a)A and Chry, respectively. It is hypothesized that the lack of PAH bioavailability hampered their further biodegradation in the unspiked soil. According to the results of the culture-dependent and independent techniques Mycobacterium parmense, Pseudomonas mexicana, and Sphingobacterials group could control B(a)A and Chry degradation in combination with several microorganisms with secondary metabolic activity.     

Pyrosequence analysis of bacterial communities in aerobic bioreactors treating polycyclic aromatic hydrocarbon-contaminated soil

Two aerobic, lab-scale, slurry-phase bioreactors were used to examine the biodegradation of polycyclic aromatic hydrocarbons (PAHs) in contaminated soil and the associated bacterial communities. The two bioreactors were operated under semi-continuous (draw-and-fill) conditions at a residence time of 35 days, but one was fed weekly and the other monthly. Most of the quantified PAHs, including high-molecular-weight compounds, were removed to a greater extent in the weekly-fed bioreactor, which achieved total PAH removal of 76%. Molecular analyses, including pyrosequencing of 16S rRNA genes, revealed significant shifts in the soil bacterial communities after introduction to the bioreactors and differences in the abundance and types of bacteria in each of the bioreactors. The weekly-fed bioreactor displayed a more stable bacterial community with gradual changes over time, whereas the monthly-fed bioreactor community was less consistent and may have been more strongly influenced by the influx of untreated soil during feeding. Phylogenetic groups containing known PAH-degrading bacteria previously identified through stable-isotope probing of the untreated soil were differentially affected by bioreactor conditions. Sequences from members of the Acidovorax and Sphingomonas genera, as well as the uncultivated “Pyrene Group 2” were abundant in the bioreactors. However, the relative abundances of sequences from the Pseudomonas, Sphingobium, and Pseudoxanthomonas genera, as well as from a group of unclassified anthracene degraders, were much lower in the bioreactors compared to the untreated soil.     

Autecological properties of soil sphingomonads involved in the degradation of polycyclic aromatic hydrocarbons

Autecological properties that are thought to be important for polycyclic aromatic hydrocarbon (PAH)-degradation by bacteria in contaminated soils include the ability to utilize a broad range of carbon sources, efficient biofilm formation, cell-surface hydrophobicity, surfactant production, motility, and chemotaxis. Sphingomonas species are common PAH-degraders, and a selection of PAH-degrading sphingomonad strains isolated from contaminated soils was therefore characterized in terms of these properties. All the sphingomonads tested were relatively hydrophilic and were able to grow as biofilms on a phenanthrene-coated surface, though biofilm formation under other conditions was variable. Sphingobium yanoikuyae B1 was able to utilize the greatest range of carbon sources, though it was not chemotaxic towards any of the substrates tested. Other sphingomonad strains were considerably less flexible in their catabolic range. None of the strains produced detectable surfactant and swimming motility varied between the strains. Examination of the total Sphingomonas community in the soils tested showed that one of the isolates studied was present at significant levels, suggesting that it can thrive under PAH-contaminated conditions despite the lack of many of the tested characteristics. We conclude that these properties are not essential for survival and persistence of Sphingomonas in PAH-contaminated soils.     

Pyrosequence analyses of bacterial communities during simulated in situ bioremediation of polycyclic aromatic hydrocarbon-contaminated soil

Barcoded amplicon pyrosequencing was used to generate libraries of partial 16S rRNA genes from two columns designed to simulate in situ bioremediation of polycyclic aromatic hydrocarbons (PAHs) in weathered, contaminated soil. Both columns received a continuous flow of artificial groundwater but one of the columns additionally tested the impact of biostimulation with oxygen and inorganic nutrients on indigenous soil bacterial communities. The penetration of oxygen to previously anoxic regions of the columns resulted in the most significant community changes. PAH-degrading bacteria previously determined by stable-isotope probing (SIP) of the untreated soil generally responded negatively to the treatment conditions, with only members of the Acidovorax and a group of uncharacterized PAH-degrading Gammaproteobacteria maintaining a significant presence in the columns. Additional groups of sequences associated with the Betaproteobacterial family Rhodocyclaceae (including those associated with PAH degradation in other soils), and the Thiobacillus, Thermomonas, and Bradyrhizobium genera were also present in high abundance in the biostimulated column. Similar community responses were previously observed during biostimulated ex situ treatment of the same soil in aerobic, slurry-phase bioreactors. While the low relative abundance of many SIP-determined groups in the column libraries may be a reflection of the slow removal of PAHs in that system, the similar response of known PAH degraders in a higher-rate bioreactor system suggests that alternative PAH-degrading bacteria, unidentified by SIP of the untreated soil, may also be enriched in engineered systems.     

Response of fluoranthene-degrading bacteria to surfactants

A prerequisite for surfactant-enhanced biodegradation is that the microorganisms survive, take up substrate and degrade it in the presence of the surfactant. Two Mycobacterium and two Sphingomonas strains, degrading fluoranthene, were investigated for their sensitivity towards non-ionic chemical surfactants. The effect of Triton X-100 and Tween 80 above their critical micelle concentration on mineralization of [¹⁴C]-glucose and [¹⁴C]-fluoranthene was measured in shaker cultures. Tween 80 had no toxic effect on any of the tested strains. The surfactant inhibited fluoranthene mineralization by the hydrophobic Mycobacterium spp. slightly, but more than doubled that by the two less hydrophobic Sphingomonas strains. Triton X-100 inhibited fluoranthene mineralization by all strains, yet this was more pronounced for the Sphingomonas spp. Both surfactants caused cell wall permeabilization, as shown by transient colouring of surfactant-containing media. Inhibition of glucose mineralization, indicating non-specific toxic effects of Triton X-100, was observed only for the Sphingomonas strains and the toxicity was caused by micelle-to-cell interactions. These strains, however, appeared to recover from initial Triton X-100 toxicity within 50–500 h of exposure. The ratio of surfactant concentration to initial cell density was found to determine critically the bacterial response to surfactants. For both Sphingomonas and Mycobacterium strains, this work indicates that fluoranthene solubilized in surfactant micelles is only partially available for mineralization by the bacteria tested. However, our results suggest that optimal conditions for polycyclic aromatic hydrocarbon mineralization can be developed by selection of the proper surfactant, bacterial strains, cell density and incubation conditions. Received: 6 February 1998 / Received revision: 19 June 1998 / Accepted: 19 June 1998