Effects of Corn Steep Liquor on Growth Rate and Pyrene Degradation by Pseudomonas strains

The growth rates and pyrene degradation rates of Pseudomonas sp. LP1 and Pseudomonas aeruginosa LP5 were increased in corn steep liquor (CSL) supplemented. On pyrene alone the highest specific growth rate of LP1 was 0.018 h⁻¹, while on CSL-supplemented pyrene MSM, the value was 0.026 h⁻1. For LP5 the highest growth rate on CSL-supplemented pyrene-MSM was 0.034 h⁻¹. Conversely, on pyrene alone the highest rate was 0.024 h⁻¹. CSL led to marked reduction in residual pyrene. In the case of Pseudomonas sp. LP1 values of residual pyrene were 58.54 and 45.47%, respectively, for the unsupplemented and supplemented broth cultures, showing a difference of 13.09%. For LP5 the corresponding values were 64.01 and 26.96%, respectively, showing a difference of 37.05%. The rate of pyrene utilization by LP1 were 0.08 and 0.11 mg l⁻¹ h⁻¹ on unsupplemented and supplemented media, respectively. The corresponding values for LP5 were 0.07 and 0.015 mg l⁻¹ h⁻¹, respectively. These results suggest that CSL, a cheap and readily available waste product, could be very useful in the bioremediation of environments contaminated with pyrene.     

Degradation of hydrocarbons and biosurfactant production by Pseudomonas sp. strain LP1

Pseudomonas sp. strain LP1, an organism isolated on the basis of its ability to grow on pyrene, was assayed for its degradative and biosurfactant production potentials when growing on crude, diesel and engine oils. The isolate exhibited specific growth rate and doubling time of 0.304 days⁻¹ and 2.28 days, respectively on crude oil (Escravos Light). The corresponding values on diesel were 0.233 days⁻¹ and 2.97 days, while on engine oil, were 0.122 days⁻¹ and 5.71 days. The organism did not show significant biosurfactant production towards crude oil and diesel, but readily produced biosurfactant on engine oil. The highest Emulsification index (E₂₄) value for the biosurfactant produced by LP1 on engine oil was 80.33 ± 1.20, on day 8 of incubation. Biosurfactant production was growth-associated. The surface-active compound which exhibited zero saline tolerance had its optimal activity at 50°C and pH 2.0.     

Enhanced degradation of phenol by <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. CP4 entrapped in agar and calcium alginate beads in batch and continuous processes

Phenol is one of the major toxic pollutants in the wastes generated by a number of industries and needs to be eliminated before their discharge. Although microbial degradation is a preferred method of waste treatment for phenol removal, the general inability of the degrading strains to tolerate higher substrate concentrations has been a bottleneck. Immobilization of the microorganism in suitable matrices has been shown to circumvent this problem to some extent. In this study, cells of Pseudomonas sp. CP4, a laboratory isolate that degrades phenol, cresols, and other aromatics, were immobilized by entrapment in Ca-alginate and agar gel beads, separately and their performance in a fluidized bed bioreactor was compared. In batch runs, with an aeration rate of 1 vol⁻¹ vol⁻¹ min⁻¹, at 30°C and pH 7.0 ± 0.2, agar-encapsulated cells degraded up to 3000 mg l⁻¹ of phenol as compared to 1500 mg l⁻¹ by Ca-alginate-entrapped cells whereas free cells could tolerate only 1000 mg l⁻¹. In a continuous process with Ca-alginate entrapped cells a degradation rate of 200 mg phenol l⁻¹ h⁻¹ was obtained while agar-entrapped cells were far superior and could withstand and degrade up to 4000 mg phenol l⁻¹ in the feed with a maximum degradation rate of 400 mg phenol l⁻¹ h⁻¹. The results indicate a clear possibility of development of an efficient treatment technology for phenol containing waste waters with the agar-entrapped bacterial strain, Pseudomonas sp. CP4.     

Optimization of production of caffeine demethylase by <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. in a bioreactor

The effect of pH, aeration rate, and agitation rate on specific productivity of caffeine demethylase from Pseudomonas sp. was studied in a bioreactor. Maximum specific productivity of caffeine demethylase of 2,214 U g cell dry weight⁻¹ h⁻¹ was obtained at 0.27 vvm, 700 rpm, and pH 7.0. Under these conditions, volumetric oxygen transfer coefficient was 74.2 h⁻¹, indicating that caffeine demethylase production by Pseudomonas sp. was highly oxygen-dependent. Different metabolite formation at different agitation and aeration rates can be used as a strategy for recovery of pharmaceutically important metabolites from caffeine by manipulation of conditions in a bacterial culture. This is the first report on production of high levels of caffeine demethylase in bioreactors.     

Continuous cultures of <Emphasis Type="Italic">Pseudomonas putida</Emphasis> mt-2 overcome catabolic function loss under real case operating conditions

The long-term performance and stability of Pseudomonas putida mt-2 cultures, a toluene-sensitive strain harboring the genes responsible for toluene biodegradation in the archetypal plasmid pWW0, was investigated in a chemostat bioreactor functioning under real case operating conditions. The process was operated at a dilution rate of 0.1 h⁻¹ under toluene loading rates of 259 ± 23 and 801 ± 78 g m⁻³ h⁻¹ (inlet toluene concentrations of 3.5 and 10.9 g m⁻³, respectively). Despite the deleterious effects of toluene and its degradation intermediates, the phenotype of this sensitive P. putida culture rapidly recovered from a 95% Tol⁻ population at day 4 to approx. 100% Tol⁺ cells from day 13 onward, sustaining elimination capacities of 232 ± 10 g m⁻³ h⁻¹ at 3.5 g Tol m⁻³ and 377 ± 13 g m⁻³ h⁻¹ at 10.9 g Tol m⁻³, which were comparable to those achieved by highly tolerant strains such as P. putida DOT T1E and P. putida F1 under identical experimental conditions. Only one type of Tol⁻ variant, harboring a TOL-like plasmid with a 38.5 kb deletion (containing the upper and meta operons for toluene biodegradation), was identified.     

Oxidation of arsenite by two β-proteobacteria isolated from soil

Two heterotrophic As(III)-oxidizing bacteria, SPB-24 and SPB-31 were isolated from garden soil. Based on 16S rRNA gene sequence analysis, strain SPB-24 was closely related to genus Bordetella, and strain SPB-31 was most closely related to genus Achromobacter. Both strains exhibited high As(III) (15 mM for SPB-24 and 40 mM for SPB-31) and As(V) (>300 mM for both strains) resistance. Both strains oxidized 5 mM As(III) in minimal medium with oxidation rate of 554 and 558 μM h⁻¹ for SPB-24 and SPB-31, respectively. Washed cells of both strains oxidized As(III) over broad pH and temperature range with optimum pH 6 and temperature 42°C for both strains. The As(III) oxidation kinetic by washed cells showed K _m and V _max values of 41.7 μM and 1,166 μM h⁻¹ for SPB-24, 52 μM and 1,186 μM h⁻¹ for SPB-31. In the presence of minimal amount of carbon source, the strains showed high As(III) oxidation rate and high specific arsenite oxidase activity. The ability of strains to resist high concentration of arsenic and oxidize As(III) with highest rates reported so far makes them potential candidates for bioremediation of arsenic-contaminated environment.     

Enhanced fructooligosaccharides and inulinase production by a <Emphasis Type="Italic">Xanthomonas campestris</Emphasis> pv. <Emphasis Type="Italic">phaseoli</Emphasis> KM 24 mutant

Xanthomonas campestris pv phaseoli produced an extracellular endoinulinase (9.24 ± 0.03 U mL⁻¹) in an optimized medium comprising of 3% sucrose and 2.5% tryptone. X. campestris pv. phaseoli was further subjected to ethylmethanesulfonate mutagenesis and the resulting mutant, X. campestris pv. phaseoli KM 24 demonstrated inulinase production of 22.09 ± 0.03 U mL⁻¹ after 18 h, which was 2.4-fold higher than that of the wild type. Inulinase production by this mutant was scaled up using sucrose as a carbon source in a 5-L fermenter yielding maximum volumetric (21,865 U L⁻¹ h⁻¹) and specific (119,025 U g⁻¹ h⁻¹) productivities of inulinase after 18 h with an inulinase/invertase ratio of 2.6. A maximum FOS production of 11.9 g L⁻¹ h⁻¹ and specific productivity of 72 g g⁻¹ h⁻¹ FOS from inulin were observed in a fermenter, when the mutant was grown on medium containing 3% inulin and 2.5% tryptone. The detection of mono- and oligosaccharides in inulin hydrolysates by TLC analysis indicated the presence of an endoinulinase. This mutant has potential for large-scale production of inulinase and fructooligosaccharides.     

Alcoholic fermentation of xylose and mixed sugars using recombinant <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> engineered for xylose utilization

Previously, a Saccharomyces cerevisiae strain was engineered for xylose assimilation by the constitutive overexpression of the Orpinomyces xylose isomerase, the S. cerevisiae xylulokinase, and the Pichia stipitis SUT1 sugar transporter genes. The recombinant strain exhibited growth on xylose, under aerobic conditions, with a specific growth rate of 0.025 h⁻¹, while ethanol production from xylose was achieved anaerobically. In the present study, the developed recombinant yeast was adapted for enhanced growth on xylose by serial transfer in xylose-containing minimal medium under aerobic conditions. After repeated batch cultivations, a strain was isolated which grew with a specific growth rate of 0.133 h⁻¹. The adapted strain could ferment 20 g l⁻¹ of xylose to ethanol with a yield of 0.37 g g⁻¹ and production rate of 0.026 g l⁻¹ h⁻¹. Raising the fermentation temperature from 30°C to 35°C resulted in a substantial increase in the ethanol yield (0.43 g g⁻¹) and production rate (0.07 g l⁻¹ h⁻¹) as well as a significant reduction in the xylitol yield. By the addition of a sugar complexing agent, such as sodium tetraborate, significant improvement in ethanol production and reduction in xylitol accumulation was achieved. Furthermore, ethanol production from xylose and a mixture of glucose and xylose was also demonstrated in complex medium containing yeast extract, peptone, and borate with a considerably high yield of 0.48 g g⁻¹.     

Biodegradation of tributyl phosphate by novel bacteria isolated from enrichment cultures

Tributyl phosphate (TBP) is an organophosphorous compound, used extensively (3000–5000 tonnes/annum) as a solvent for nuclear fuel processing and as a base stock in the formulation of fire-resistant aircraft hydraulic fluids and other applications. Because of its wide applications and relative stability in the natural environment TBP poses the problem of pollution and health hazards. In the present study, fifteen potent bacterial strains capable of using tributyl phosphate (TBP) as sole carbon and phosphorus source were isolated from enrichment cultures. These isolates were identified on the basis of biochemical and morphological characteristics and 16S rRNA gene sequence analysis. Phylogenetic analysis of 16S rRNA gene sequences revealed that two isolates belonged to class Bacilli and thirteen to β and γ-Proteobacteria. All these isolates were found to be members of genera Alcaligenes, Providencia, Delftia, Ralstonia, and Bacillus. These isolates were able to tolerate and degrade up to 5 mM TBP, the highest concentration reported to date. The GC–MS method was developed to monitor TBP degradation. Two strains, Providencia sp. BGW4 and Delftia sp. BGW1 showed respectively, 61.0 ± 2.8% and 57.0 ± 2.0% TBP degradation within 4 days. The degradation rate constants, calculated by first order kinetic model were between 0.0024 and 0.0099 h⁻¹. These bacterial strains are novel for TBP degradation and could be used as an important bioresource for efficient decontamination of TBP polluted waste streams.     

Continuous nisin production with bioengineered <Emphasis Type="Italic">Lactococcus lactis</Emphasis> strains

Nisin production in continuous cultures of bioengineered Lactococcus lactis strains that incorporate additional immunity and regulation genes was studied. Highest nisin activities were observed at 0.2 h^–1 dilution rate and 12.5 g l^–1 fructose concentration for all strains. Recombinant strains were able to produce greater amounts of nisin at dilution rates below 0.3 h⁻¹ compared to the control strain. However, this significant difference disappeared at dilution rates of 0.4 and 0.5 h^–1. For the strains LL27, LAC338, LAC339, and LAC340, optimum conditions for nisin production were determined to be at 0.29, 0.26, 0.27, and 0.27 h^–1 dilution rates and 11.95, 12.01, 11.63, and 12.50 g l^–1 fructose concentrations, respectively. The highest nisin productivity, 496 IU ml^–1 h^–1, was achieved with LAC339. The results of this study suggest that low dilution rates stabilize the high specific nisin productivity of the bioengineered strains in continuous fermentation. Moreover, response surface methodology analysis showed that regulation genes yielded high nisin productivity at wide ranges of dilution rates and fructose concentrations.     

Succinic acid production by a newly isolated bacterium

A new bacterial strain producing succinic acid was enriched from bovine rumen content. It is facultatively anaerobic, belongs to the family Pasteurellaceae and has similarity to the genus Mannheimia. In batch cultivations with D-glucose or sucrose the strain produced up to 5.8 g succinic acid l⁻¹ with a productivity and a yield of up to 1.5 g l⁻¹ h⁻¹ and 0.6 g g⁻¹, respectively. With crude glycerol up to 8.4 g l⁻¹, 0.9 g l⁻¹ h⁻¹ and 1.2 g g⁻¹ were obtained. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Bacterial degradation of styrene in waste gases using a peat filter

A biofiltration process was developed for styrene-containing off-gases using peat as filter material. The average styrene reduction ratio after 190 days of operation was 70% (max. 98%) and the mean styrene elimination capacity was 12 g m⁻³ h⁻¹ (max. 30 g m⁻³ h⁻¹). Efficient styrene degradation required addition of nutrients to the peat, adjustment of the pH to a neutral level and efficient control of the humidity. Maintenance of the water balance was easier in a down-flow than in an up-flow process, the former consequently resulting in much better filtration efficiency. The optimum operation temperature was around 23 °C, but the styrene removal was still satisfactory at 12 °C. Seven different bacterial isolates belonging to the genera Tsukamurella, Pseudomonas, Sphingomonas, Xanthomonas and an unidentified genus in the γ group of the Proteobacteria isolated from the microflora of active peat filter material were capable of styrene degradation. The isolates differed in their capacity to decompose styrene to carbon dioxide and assimilate it to biomass. No toxic intermediate degradation products of styrene were detected in the filter outlet gas or in growing cultures of isolated bacteria. The use of these isolates in industrial biofilters is beneficial at low styrene concentrations and is safe from both the environmental and public health points of view. Received: 30 May 1997 / Received revision: 22 August 1997 / Accepted: 25 August 1997     

An unusual cyanobacterium from saline thermal waters with relatives from unexpected habitats

Cyanobacteria that grow above seawater salinity at temperatures above 45°C have rarely been studied. Cyanobacteria of this type of thermo-halophilic extremophile were isolated from siliceous crusts at 40–45°C in a geothermal seawater lagoon in southwest Iceland. Iceland Clone 2e, a Leptolyngbya morphotype, was selected for further study. This culture grew only at 45–50°C, in medium ranging from 28 to 94 g L⁻¹ TDS, It showed 3 doublings 24 h⁻¹ under continuous illumination. This rate at 54°C was somewhat reduced, and death occurred at 58°C. A comparison of the 16S rDNA sequence with all others in the NCBI database revealed 2 related Leptolyngbya isolates from a Greenland hot spring (13–16 g L⁻¹ TDS). Three other similar sequences were from Leptolyngbya isolates from dry, endolithic habitats in Yellowstone National Park. All 6 formed a phylogenetic clade, suggesting common ancestry. These strains shared many similarities to Iceland Clone 2e with respect to temperature and salinity ranges and optima. Two endolithic Leptolyngbya isolates, grown previously at 23°C in freshwater medium, grew well at 50°C but only in saline medium. This study shows that limited genotypic similarity may reveal some salient phenotypic similarities, even when the related cyanobacteria are from vastly different and remote habitats.     

Metabolite repression inhibits degradation of benzo[a]pyrene and dibenz[a,h]anthracene by Stenotrophomonas maltophilia VUN 10,003

Large inocula of Stenotrophomonas maltophilia VUN 10,003 were used to investigate bacterial degradation of benzo[a]pyrene and dibenz[a,h]anthracene. Although strain VUN 10,003 was capable of degrading 10–15 mg l⁻¹ of the five-ring compounds in the presence of pyrene after 63 days, further addition of pyrene after degradation of the five-ring polycyclic aromatic hydrocarbons (PAHs) ceased did not stimulate significant decreases in the concentration of benzo[a]pyrene or dibenz[a,h]anthracene. However, pyrene was degraded to undetectable levels 21 days after its addition. The amount of benzo[a]pyrene and dibenz[a,h]anthracene degraded by strain VUN 10,003 was not affected by the initial concentration of the compounds when tested at 25–100 mg l⁻¹, by the accumulation of by-products from pyrene catabolism or a loss of ability by the cells to catabolise benzo[a]pyrene or dibenz[a,h]anthracene. Metabolite or by-product repression was suspected to be responsible for the inhibition: By-products from the degradation of the five-ring compounds inhibited their further degradation. Journal of Industrial Microbiology & Biotechnology (2002) 28, 88–96 DOI: 10.1038/sj/jim/7000216 Received 30 January 2001/ Accepted in revised form 10 October 2001     

Quasi steady state growth of <Emphasis Type="Italic">Lactococcus lactis</Emphasis> in glucose-limited acceleration stat (A-stat) cultures

Quasi steady state growth of Lactococcus lactis IL 1403 was studied in glucose-limited A-stat cultivation experiments with acceleration rates (a) from 0.003 to 0.06 h⁻² after initial stabilization of the cultures in chemostat at D = 0.2–0.3 h⁻¹. It was shown that the high limit of quasi steady state growth rate depended on the acceleration rate used—at an acceleration rate 0.003 h⁻² the quasi steady state growth was observed until μ _crit = 0.59 h⁻¹, which is also the μ _max value for the culture. Lower values of μ _crit were observed at higher acceleration rates. The steady state growth of bacteria stabilized at dilution rate 0.2 h⁻¹ was immediately disrupted after initiating acceleration at the highest acceleration rate studied—0.06 h⁻². Observation was made that differences [Δ(μ − D)] of the specific growth rates from pre-programmed dilution rates were the lowest using an acceleration rate of 0.003 h⁻² (< 4% of preset changing growth rate). The adaptability of cells to follow preprogrammed growth rate was found to decrease with increasing dilution rate—it was shown that lower acceleration rates should be applied at higher growth rates to maintain the culture in the quasi steady state. The critical specific growth rate and the biomass yields based on glucose consumption were higher if the medium contained S ₀ = 5 g L⁻¹ glucose instead of S ₀ = 10 g L⁻¹. It was assumed that this was due to the inhibitory effect of lactate accumulating at higher concentrations in the latter cultures. Parallel A-stat experiments at the same acceleration and dilution rates showed good reproducibility—Δ(μ − D) was less than 5%, standard deviations of biomass yields per ATP produced (Y _ATP), and biomass yields per glucose consumed (Y _XS) were less than 15%.     

Adaptation of a <Emphasis Type="Italic">Mycobacterium</Emphasis> strain to phenanthrene degradation in a biphasic culture system: influence on interfacial area and droplet size

A phenanthrene-degrading Mycobacterium sp. strain 6PY1 was grown in an aqueous/organic biphasic culture system with phenanthrene as sole carbon source. Its capacity of degradation was studied during sequential inoculum enrichments, reaching complete phenanthrene degradation at a maximim rate of 7 mg l⁻¹ h⁻¹. Water–oil emulsions and biofilm formation were observed in biphasic cultures after four successive enrichments. The factors influencing interfacial area in the emulsions were: the initial phenanthrene concentration, the initial inoculum size, and the silicone oil volume fraction. The results showed that the interfacial area was mainly dependent on the silicone oil/mineral salts medium ratio and the inoculum size.     

Degradation of polycyclic aromatic hydrocarbons in soil by a two-step sequential treatment

The objectives of this work were to isolate the microorganisms responsible for a previously observed degradation of polycyclic aromatic hydrocarbons (PAH) in soil and to test a method for cleaning a PAH-contaminated soil. An efficient PAH degrader was isolated from an agricultural soil and designated as Mycobacterium LP1. In liquid culture, it degraded phenanthrene (58%), pyrene (24%), anthracene (21%) and benzo(a)pyrene (10%) present in mixture (initial concentration 50 μg ml⁻¹ each) and phenanthrene (92%) and pyrene (94%) as sole carbon sources after 14 days of incubation at 30°C. In soil, Mycobacterium LP1 mineralised ¹⁴C-phenanthrene (45%) and ¹⁴C-pyrene (65%) after 10 days. The good ability of this Mycobacterium was combined with the benzo(a)pyrene oxidation effect obtained by 1% w/w rapeseed oil in a sequential treatment of a PAH-spiked soil (total PAH concentration 200 mg kg⁻¹). The first step was incubation with the bacterium for 12 days and the second step was the addition of the rapeseed oil after this time and a further incubation of 22 days. Phenanthrene (99%), pyrene (95%) and anthracene (99%) were mainly degraded in the first 12 days and a total of 85% of benzo(a)pyrene was transformed during the whole process. The feasibility of the method is discussed.     

Heterologous expression of polyhydroxyalkanoate depolymerase from <Emphasis Type="Italic">Thermobifida</Emphasis> sp. in <Emphasis Type="Italic">Pichia pastoris</Emphasis> and catalytic analysis by surface plasmon resonance

A polyhydroxyalkanote depolymerase gene from Thermobifida sp. isolate BCC23166 was cloned and expressed as a C-terminal His₆-tagged fusion in Pichia pastoris. Primary structure analysis revealed that the enzyme PhaZ-Th is a member of a proposed new subgroup of SCL-PHA depolymerase containing a proline–serine repeat linker. PhaZ-Th was expressed as two glycosylated forms with apparent molecular weights of 61 and 70 kDa, respectively. The enzyme showed esterase activity toward p-nitrophenyl alkanotes with V _max and K _m of 3.63 ± 0.16 μmol min⁻¹ mg⁻¹ and 0.79 ± 0.12 mM, respectively, on p-nitrophenyl butyrate with optimal activity at 50–55°C and pH 7–8. Surface plasmon resonance (SPR) analysis demonstrated that PhaZ-Th catalyzed the degradation of poly-[(R)-3-hydroxybutyrate] (PHB) films, which was accelerated in (R)-3-hydroxyvalerate copolymers with a maximum degradation rate of 882 ng cm⁻² h⁻¹ for poly[(R)-3-hydroxybutyrate-co-3-hydroxyvalerate] (12 mol% V). Surface deterioration, especially on the amorphous regions of PHB films was observed after exposure to PhaZ-Th by atomic force microscopy. The use of P. pastoris as an alternative recombinant system for bioplastic degrading enzymes in secreted form and a sensitive SPR analytical technique will be of utility for further study of bioplastic degradation.     

Rapid biodegradation of metsulfuron-methyl by a soil fungus in pure cultures and soil

Summary Four strains of bacteria, 9 strains of fungi and 20 strains of actinomycetes capable of utilizing metsulfuron-methyl as sole carbon and energy source were isolated from a metsulfuron-methyl-treated soil by the enrichment culture method. A fungus named DS11F was selected as the most highly effective one according to the maximum tolerance concentration of 1,200 mg l⁻¹ and metsulfuron-methyl-degrading rate of 0.0716 g g⁻¹ cells h⁻¹, and was identified as an unknown strain of Penicillium sp. on the basis of colony growth, morphology and biochemical characteristics.␣Through liquid pure culture, the optimal metsulfuron-methyl-degrading conditions of DS11F were determined to be metsulfuron-methyl concentration 22.6 mg l⁻¹, inoculum concentration 12.25 mg l⁻¹, pH 7.0 and temperature 30°C. As additional C sources, supernatant of soaked compost could increase metsulfuron-methyl degradation by 8%, but glucose was ineffective. DS11F inoculation was found to significantly enhance the degradation of metsulfuron-methyl in soil, with the reduction of the concentration reaching 50% in 6 days. Admixture of compost could promote metsulfuron-methyl degradation to some extent. The growth of the inocula in the soils remained dominant and degradation resumed immediately when metsulfuron-methyl was applied again. The results show that addition of the isolated Penicillium sp. enhances the degradation of metsulfuron-methyl in water and soil.     

The most-probable-number enumeration of dichlobenil and 2,6-dichlorobenzamide (BAM) degrading microbes in Finnish aquifers

In groundwater subsurface deposits and a topsoil from five aquifers having 2,6-dichlorobenzamide (BAM) in water, we determined the most-probable-number (MPN) of 2,6-dichlorobenzonitrile (dichlobenil) and metabolite BAM degrading microorganisms. Dichlobenil and BAM were combined nitrogen sources in the MPN tubes, which were scored positive at concentrations <75% after 1 month incubation. Aerobic and anaerobic microbes degrading dichlobenil and BAM were common in samples in low numbers of 3.6–210 MPN g dw⁻¹. Additional degradation occurred in high MPN dilutions of some samples, the microbial numbers being 0.11–120 × 10⁵ MPN g dw⁻¹. The strains were isolated from low and high dilutions of one deposit, and degradation in pure cultures was confirmed by HPLC. According to the 16S rDNA sequencing, strains were from genera Zoogloea, Pseudomonas, Xanthomonas, Rhodococcus, Nocardioides, Sphingomonas, and Ralstonia. Dichlobenil (45.5 ± 18.3%) and BAM (37.6 ± 14%) degradation was low in the MPN tubes. Despite of microbial BAM degradation activity in subsurface deposits, BAM was measured from groundwater.