Correlation of biological activity and reactor performance in biofiltration of toluene with the fungus Paecilomyces variotii CBS115145

A biofiltration system inoculated with the mold Paecilomyces variotii CBS115145 showed a toluene elimination capacity (EC) of around 250 g/m3 of biofilter/h, which was higher than the values usually reported for bacteria. P. variotii assimilated m- and p-cresols but not the o isomer. Initial toluene hydroxylation occurred both on the methyl group and through the p-cresol pathway. These results were corroborated by detecting benzyl alcohol, benzaldehyde, and p-cresol as volatile intermediates. In liquid cultures with toluene as a substrate, the activity of toluene oxygenase (TO) was 5.6 nmol of O2/min/mg of biomass, and that of benzyl alcohol dehydrogenase was 16.2 nmol of NADH/min/mg of protein. Toluene biodegradation determined from the TO activity in the biofilter depended on the biomass distribution and the substrate concentration. The specific enzymatic activity decreased from 6.3 to 1.9 nmol of O2/min/mg of biomass along the reactor. Good agreement was found between the EC calculated from the TO activity and the EC measured on the biofilter. The results were confirmed by short-time biofiltration experiments. Average EC measured in different biofiltration experiments and EC calculated from the TO activity showed a linear relation, suggesting that in the biofilters, EC was limited by biological reaction. As the enzymatic activities of P. variotii were similar to those reported for bacteria, the high performance of the fungal biofilters can possibly be explained by the increased transfer of the hydrophobic compounds, including oxygen, from the gas phase to the mycelia, overcoming the transfer problems associated with the flat bacterial biofilms.     

Correlation of Biological Activity and Reactor Performance in Biofiltration of Toluene with the Fungus Paecilomyces variotii CBS115145

A biofiltration system inoculated with the mold Paecilomyces variotii CBS115145 showed a toluene elimination capacity (EC) of around 250 g/m³ of biofilter/h, which was higher than the values usually reported for bacteria. P. variotii assimilated m- and p-cresols but not the o isomer. Initial toluene hydroxylation occurred both on the methyl group and through the p-cresol pathway. These results were corroborated by detecting benzyl alcohol, benzaldehyde, and p-cresol as volatile intermediates. In liquid cultures with toluene as a substrate, the activity of toluene oxygenase (TO) was 5.6 nmol of O₂/min/mg of biomass, and that of benzyl alcohol dehydrogenase was 16.2 nmol of NADH/min/mg of protein. Toluene biodegradation determined from the TO activity in the biofilter depended on the biomass distribution and the substrate concentration. The specific enzymatic activity decreased from 6.3 to 1.9 nmol of O₂/min/mg of biomass along the reactor. Good agreement was found between the EC calculated from the TO activity and the EC measured on the biofilter. The results were confirmed by short-time biofiltration experiments. Average EC measured in different biofiltration experiments and EC calculated from the TO activity showed a linear relation, suggesting that in the biofilters, EC was limited by biological reaction. As the enzymatic activities of P. variotii were similar to those reported for bacteria, the high performance of the fungal biofilters can possibly be explained by the increased transfer of the hydrophobic compounds, including oxygen, from the gas phase to the mycelia, overcoming the transfer problems associated with the flat bacterial biofilms.     

Toluene removal efficiency, process robustness, and bacterial diversity of a biotrickling filter inoculated with Burkholderia sp. Strain T3

Microorganisms determine the overall biofilter performance under specific operating conditions. The toluene removal and process robustness of a laboratoryscale, ceramisite-based biotrickling filter inoculated with Burkholderia sp. strain T3 (BTFb) were compared with those of another biotrickling filter inoculated with activated sludge (BTFa) for 3 months under various operating conditions. Denaturing gradient gel electrophoresis was applied to visualise the bacterial community of the BTFa and BTFb. Real-time polymerase chain reaction was performed to determine the genes coding for toluenedegrading enzymes. Burkholderia sp. strain T3, which possesses the major toluene-degrading genes in BTFb, was traced in the BTFb bacterial community. The strain was found to stabilize the relative quantity steadily at higher than 60% during toluene biofiltration. Thus, BTFb performed more efficiently than BTFa as evidenced by achieving 98.86% toluene removal efficiency (RE) on 3 day, critical elimination capacity (EC) of 234.23 ± 10.54 g/m3/h, and rapid restoration of the initial RE and EC levels within 3 day of reoperation, even after 1 month of shutdown. The efficiency of BTFb is also evident by the stabilised RE and EC levels within a wide temperature range and a gradually decreasing system pH. Maintaining the pressure drop levels below 150 Pa during prolonged operation also contributed to the efficiency of BTFb. Thus, based on the study results, we propose that Burkholderia sp. strain T3 is a highly efficient and applicable inoculum for toluene biofiltration.     

Horizontal Gene Transfer to Endogenous Endophytic Bacteria from Poplar Improves Phytoremediation of Toluene

Poplar, a plant species frequently used for phytoremediation of groundwater contaminated with organic solvents, was inoculated with the endophyte Burkholderia cepacia VM1468. This strain, whose natural host is yellow lupine, contains the pTOM-Bu61 plasmid coding for constitutively expressed toluene degradation. Noninoculated plants or plants inoculated with the soil bacterium B. cepacia Bu61(pTOM-Bu61) were used as controls. Inoculation of poplar had a positive effect on plant growth in the presence of toluene and reduced the amount of toluene released via evapotranspiration. These effects were more dramatic for VM1468, the endophytic strain, than for Bu61. Remarkably, none of the strains became established at detectable levels in the endophytic community, but there was horizontal gene transfer of pTOM-Bu61 to different members of the endogenous endophytic community, both in the presence and in the absence of toluene. This work is the first report of in planta horizontal gene transfer among plant-associated endophytic bacteria and demonstrates that such transfer could be used to change natural endophytic microbial communities in order to improve the remediation of environmental insults.     

Dynamic responses of biofilters to changes in the operating conditions in the process of removing toluene and xylene from air

The dynamic behaviour of biofilters intended to remove toluene and xylene from air was studied during transient states. Laboratory scale biofilters were filled with a mixture of peat, bark and wood and inoculated with a mixed microbial population. Toluene and xylene were applied both as single pollutants and as mixtures. Attention was focused on the evaluation of the following transients: the response of biofilters to step changes and peaks in pollutant concentrations, the effect of changes between single and multiple pollutant loadings and the response to shutdown periods. The biofilters demonstrated a good dynamic stability during transient states induced by change in inlet pollutant concentrations. Their time periods did not exceed three hours. No interaction between xylene and toluene degradation was observed during changes in loading with single pollutants or their mixture. The performance interruptions lasting less than 24 hours were found to have no significant influence on the removal efficiency of biofilters. When the biofilters were reacclimated after longer starvation periods, a short temporary decrease in efficiency whose minimum and duration were proportional to the length of a preceding shutdown period was observed. The longest starvation period (7 days) resulted in a reacclimation lasting 7 hours only. Adaptations of a microbial population to new operating conditions as well as sorption/desorption processes were suggested as the main factors influencing the dynamic reponse characteristics.     

Horizontal gene transfer to endogenous endophytic bacteria from poplar improves phytoremediation of toluene

Poplar, a plant species frequently used for phytoremediation of groundwater contaminated with organic solvents, was inoculated with the endophyte Burkholderia cepacia VM1468. This strain, whose natural host is yellow lupine, contains the pTOM-Bu61 plasmid coding for constitutively expressed toluene degradation. Noninoculated plants or plants inoculated with the soil bacterium B. cepacia Bu61(pTOM-Bu61) were used as controls. Inoculation of poplar had a positive effect on plant growth in the presence of toluene and reduced the amount of toluene released via evapotranspiration. These effects were more dramatic for VM1468, the endophytic strain, than for Bu61. Remarkably, none of the strains became established at detectable levels in the endophytic community, but there was horizontal gene transfer of pTOM-Bu61 to different members of the endogenous endophytic community, both in the presence and in the absence of toluene. This work is the first report of in planta horizontal gene transfer among plant-associated endophytic bacteria and demonstrates that such transfer could be used to change natural endophytic microbial communities in order to improve the remediation of environmental insults.     

Microbial contamination in methanol biofilters inoculated with a pure strain of Pichia pastoris: A potential limitation for waste revalorization

Novel biotechnologies to valorize waste emissions are based on the use of specialized microbial groups that produce different compounds of industrial interest. On this scenario, the retention of such specific microorganisms in the system is of critical interest; however, the potential limitations of working with simplified cultures in a competitive open environment are neither fully explored nor well understood. In this work, a series of biofilters treating methanol vapors coupled with heterologous endochitinase production were used to evaluate the performance of a specialized microbial population during a typical open-to-environment operation. The biofilters were inoculated with a transformed strain of Pichia pastoris and were operated identically for about 90 days. The results showed that the biofiltration performance became diverse with time in terms of the elimination capacity (EC) shifting from a variation coefficient of 1.5% (EC = 274 ± 24, 279 ± 5, and 281.9 ± 25 g/[m³ h]) at the beginning of the operation to 33% (EC = 297 ± 9, 338 ± 7, and 341 ± 2 g/[m³ h]) at the end of operation. Epifluorescence analysis and cloning-sequencing suggested that P. pastoris remained as the dominant microorganism of methanol degradation, whereas diverse airborne bacteria, including Ochrobactrum spp. and Klebsiella oxytoca, played a secondary role possibly associated with the consumption of intermediates. Overall, this study found that low diversity systems operated under non-sterile conditions could be susceptible to contamination with external microorganisms causing a diversifying behavior at the performance and microbial community levels. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2715, 2019     

Biofiltration of waste gases with the fungi Exophiala oligosperma and Paecilomyces variotii

Two biofilters fed toluene-polluted air were inoculated with new fungal isolates of either Exophiala oligosperma or Paecilomyces variotii, while a third bioreactor was inoculated with a defined consortium composed of both fungi and a co-culture of a Pseudomonas strain and a Bacillus strain. Elimination capacities of 77 g m^–3 h^–1 and 55 g m^–3 h^–1 were reached in the fungal biofilters (with removal efficiencies exceeding 99%) in the case of, respectively, E. oligosperma and Paecilomyces variotii when feeding air with a relative humidity (RH) of 85%. The inoculated fungal strains remained the single dominant populations throughout the experiment. Conversely, in the biofilter inoculated with the bacterial–fungal consortium, the bacteria were gradually overgrown by the fungi, reaching a maximum elimination capacity around 77 g m^–3 h^–1. Determination of carbon dioxide concentrations both in batch assays and in biofiltration studies suggested the near complete mineralization of toluene. The non-linear toluene removal along the height of the biofilters resulted in local elimination capacities of up to 170 g m^–3 h^–1 and 94 g m^–3 h^–1 in the reactors inoculated, respectively, with E. oligosperma and P. variotii. Further studies with the most efficient strain, E. oligosperma, showed that the performance was highly dependent on the RH of the air and the pH of the nutrient solution. At a constant 85% RH, the maximum elimination capacity either dropped to 48.7 g m^–3 h^–1 or increased to 95.6 g m^–3 h^–1, respectively, when modifying the pH of the nutrient solution from 5.9 to either 4.5 or 7.5. The optimal conditions were 100% RH and pH 7.5, which allowed a maximum elimination capacity of 164.4 g m^–3 h^–1 under steady-state conditions, with near-complete toluene degradation.     

一株苯系物降解真菌筛选及降解特性

采用逐量分批驯化的方法以污水处理厂污泥作为菌源,苯、甲苯、二甲苯为唯一碳源,驯化、分离、筛选能够有效降解苯系物的真菌,命名为B1。采用单因素以及正交实验方法并对真菌降解环境影响因素及降解效率进行了测定和研究。结果表明:真菌B1对苯系物降解的最佳条件为C:N=5:1,pH5,温度30℃,菌种接种量为5.5ml(50ml培养基)。采用GC对初始液相浓度0~90mg/L范围内的苯系物降解效果进行测定,未发现苯系物对真菌降解活性产生抑制作用。真菌对苯系物的降解效率为:甲苯>苯>二甲苯,最高降解效率分别达到87.39%,85.21%,81.47%。混合物降解效果略高于单一底物的降解效果。     

Shift of microbial community in gas-phase biofilters with different inocula,inlet loads and nitrogen sources

The research on gaseous VOCs biofilters has often concentrated on process optimization. However, the microbial community change upon operating conditions is not well understood. In this study, three lab-scale biofilters treating gaseous toluene were operated for 66 days with different inocula under changes in inlet loads and nitrogen sources. Three biofilters were inoculated with activated sludge, river sediment or microbial consortia, respectively. The microbial community differed a lot initially but gradually deviated toward similar structures with the same dominant microorganisms, i.e. Proteobacteria, Actinobacteria (phylum level) and Rhodococcus,Pseudomonas(genus level). Among three biofilters, the two biofilters inoculated with activated sludge and river sediment showed higher microbial diversity, better VOCs removal performance and higher metabolic activity. Higher relative abundance of Alcanivorax (3% compared with lower than 0.03%), Pimelobacte (0.05% compared with lower than 0.01%)were detected under low inlet load, and Zoogloea(0.1%), Alkaliphilus(0.2%) were detected when the inlet load was increased. the abundance of Pseudomonasdecreased from 14% to 2% when ammonia was used as nitrogen source instead of nitrate, meanwhile the abundance of Bacillus and Gordoniaincreased from 0.01% to 0.05% and 0.8% to 5.8% respectively. Some special organisms were observed i.e. the intestinal microorganism.     

High throughput,colorimetric screening of microbial ester biosynthesis reveals high ethyl acetate production from Kluyveromyces marxianus on C5, C6, and C12 carbon sources

Advances in genome and metabolic pathway engineering have enabled large combinatorial libraries of mutant microbial hosts for chemical biosynthesis. Despite these advances, strain development is often limited by the lack of high throughput functional assays for effective library screening. Recent synthetic biology efforts have engineered microbes that synthesize acetyl and acyl esters and many yeasts naturally produce esters to significant titers. Short and medium chain volatile esters have value as fragrance and flavor compounds, while long chain acyl esters are potential replacements for diesel fuel. Here, we developed a biotechnology method for the rapid screening of microbial ester biosynthesis. Using a colorimetric reaction scheme, esters extracted from fermentation broth were quantitatively converted to a ferric hydroxamate complex with strong absorbance at 520 nm. The assay was validated for ethyl acetate, ethyl butyrate, isoamyl acetate, ethyl hexanoate, and ethyl octanoate, and achieved a z‐factor of 0.77. Screening of ethyl acetate production from a combinatorial library of four Kluyveromyces marxianus strains on seven carbon sources revealed ethyl acetate biosynthesis from C5, C6, and C12 sugars. This newly adapted method rapidly identified novel properties of K. marxianus metabolism and promises to advance high throughput microbial strain engineering for ester biosynthesis.     

Fungal/bacterial interactions during the biodegradation of TEX hydrocarbons (toluene, ethylbenzene and p-xylene) in gas biofilters operated under xerophilic conditions

The treatment of air contaminated with toluene, ethylbenzene, and p-xylene was assayed in three laboratory-scale biofilters, each consisting of two modules connected in series, packed with a pelletized organic fertilizer and inoculated with a toluene-degrading liquid enrichment culture. Biofilters were operated in parallel for 185?days in which the volumetric organic loading rate was progressively increased. The operation regime was subjected to drying out, so that packing humidity generally remained below 40%. Significant process failure occurred with ethylbenzene and p-xylene, but the toluene biofilter comparatively sustained a significant elimination capacity. Microbial community characterization by quantitative PCR and denaturing gradient gel electrophoresis showed substantial fungal enrichment in the toluene biofilter. Ribotypes identical to the well-known toluene-degrading black yeast Exophiala oligosperma (Chaetotyriales) were found among the dominant species. The microbial community structure was similar in the biofilters loaded with toluene and ethylbenzene but with p-xylene was quite specific and encompassed other chaetothyrialean fungi. Several species of Actinomycetales were found in the packing while the inoculum was dominated by representatives of the Burkholderiales and Xanthomonadales. One single fungal ribotype homologous to Acremonium kiliense was detected in the inoculum. The implications of xerophilic biofilter operation on process biosafety and efficiency are discussed.     

Long-term performance of peat biofilters treating ethyl acetate, toluene, and its mixture in air

Three laboratory-scale peat biofilters were operated at 90 s empty bed residence time (EBRT) for over a year. Biodegradation of ethyl acetate, toluene, or a 1:1 mixture were investigated. In first stage, inlet concentration was progressively increased from 0.4 to 4.5 g/m(3). The maximum elimination capacity (EC) found for ethyl acetate was 190 gC/m(3).h, and it was not affected by toluene. The maximum EC found for toluene as a sole contaminant was 150 gC/m(3).h, but the presence of ethyl acetate decreased the toluene maximum EC to 80 gC/m(3).h. From respirometry monitoring, values of 3.19 g CO(2)/gC and 3.06 g CO(2)/gC for pure ethyl acetate and pure toluene, respectively, were found, with overall yield coefficients of 0.13 g dry biomass produced per gram ethyl acetate consumed and 0.28 g dry biomass produced per gram toluene consumed. CO(2) production in the 1:1 mixture was successfully simulated. Dynamics of living and dead cells were monitored in four sections of the biofilters. Concentrations ranged between 2.6 x 10(9) and 3.0 x 10(10) cells per gram-dry peat for total bacteria, and 2.4 x 10(9)-1.9 x 10(10) cells per gram-dry peat for living bacteria. At high loads loss of bacterial density in the inlet zones, and increase in the dead cells percentages up to 60% was observed. In second stage, long-term performance at an inlet concentration of 1.5 g/m(3) was evaluated to show the process feasibility. Good agreement with previous data was obtained in terms of EC and CO(2) production. Restoration of living cells proportion was also observed.     

Assessment of microbial populations in methyl ethyl ketone degrading biofilters by denaturing gradient gel electrophoresis

Denaturing gradient gel electrophoresis (DGGE) analysis of polymerase chain reaction-amplified genes coding for 16S rRNA was used to assess differences in bacterial community structure as a function of spatial location along the height of two biofilters used to treat a model waste gas stream containing methyl ethyl ketone (MEK). One of the laboratory-scale biofilters was operated as a conventional continuous-flow biofilter (CFB) and the other was operated as a sequencing batch biofilter (SBB). Both biofilters, inoculated with an identical starting culture and operated over a period lasting more than 300 days, received the same influent MEK concentration and same mass of MEK on a daily basis. The systems differed, however, in terms of the fraction of time during which contaminated air was supplied and the overall operating strategy employed. DGGE analysis indicated that microbial community structures differed as a function of height in each of the biofilters. The DGGE banding patterns also differed between the two biofilters, suggesting that operating strategies imposed on the biofilters imparted a sufficiently large selective pressure to influence microbial community structures. This may explain, in part, the superior performance of the SBB over the CFB during model transient loading conditions, and it may open new possibilities for purposely manipulating the microbial populations in biofilters treating gas-phase contaminants in a manner that leads to more favorable treatment performance.Electronic Supplementary Material Supplementary material is available in the online version of this article at     

Analysis of the anaerobic microbial community capable of degrading p-toluene sulphonate

Three strains of Clostridium sp., 14 (VKM B-2201), 42 (VKM B-2202), and 21 (VKM B-2279), two methanogens, Methanobacterium formicicum MH (VKM B-2198) and Methanosarcina mazei MM (VKM B-2199), and one sulfate-reducing bacterium, Desulfovibrio sp. SR1 (VKM B-2200), were isolated in pure cultures from an anaerobic microbial community capable of degrading p-toluene sulfonate. Strain 14 was able to degrade p-toluene sulfonate in the presence of yeast extract and bactotryptone and, like strain 42, to utilize p-toluene sulfonate as the sole sulfur source with the production of toluene. p-Toluene sulfonate stimulated the growth of Ms. mazei MM on acetate. The sulfate-reducing strain Desulfovibrio sp. SR1 utilized p-toluene sulfonate as an electron acceptor. The putative scheme of p-toluene sulfonate degradation by the anaerobic microbial community is discussed.     

Effect of Availability of Nitrogen Compounds on Community Structure of Aquatic Bacteria in Model Systems

To test if the quality and concentration of dissolved nitrogen (N) species could be a selective force in shaping bacterioplankton community structure, competition for various N compounds among five heterotrophic marine bacteria (Pseudomonas strains B, B25, and AX; Bacillus strain A6; Erythrobacter strain F19) was examined. Two of the five strains (AX and B25) were capable of utilizing urea for growth. The five strains were inoculated into dilute (1/1,000 strength) ZoBell medium enriched with various N sources (free amino acids, casein, ammonium, nitrate, or urea). Regardless of the added N source, the communities were either dominated by strain B (at 50 μM N) or strain AX (at 250 μM N). Without any addition of N, strain F19 dominated. If F19 was not included in the community, strain B25 dominated. Despite these differences in community structure, consumption of the added N compounds was surprisingly similar and no advantages of urea for the urea-utilizing bacterium B25 were obvious. To examine if urea could be of selective advantage to the urea-degrading strains B25 and AX, communities with and without B25 were amended with urea N. As expected, strain B25 became dominant when present, but without this strain the non-urea-utilizing strain B outcompeted the urea-utilizing strain AX. Possibly, strain B benefited from N released during catabolism of urea by strain AX. Changes in community composition did not result in major changes in the nitrogen dynamics. The results indicate that dissolved N species can be a selective force in shaping microbial communities. Relative to nutrient generalists, nutrient specialists may either have competitive advantages or stimulate growth of other species by synergetic interactions. Results from the model communities suggest that there may be a large degree of unpredictability in the making of microbial communities, whereas major ecosystem functions such as N cycling appear relatively stable.     

Bioaugmentation of activated sludge by an indigenous 3-chloroaniline-degrading Comamonas testosteroni strain, I2gfp

A strain identified as Comamonas testosteroni I2 was isolated from activated sludge and found to be able to mineralize 3-chloroaniline (3-CA). During the mineralization, a yellow intermediate accumulated temporarily, due to the distal meta-cleavage of chlorocatechol. This strain was tested for its ability to clean wastewater containing 3-CA upon inoculation into activated sludge. To monitor its survival, the strain was chromosomally marked with the gfp gene and designated I2gfp. After inoculation into a lab-scale semicontinuous activated-sludge (SCAS) system, the inoculated strain maintained itself in the sludge for at least 45 days and was present in the sludge flocs. After an initial adaptation period of 6 days, complete degradation of 3-CA was obtained during 2 weeks, while no degradation at all occurred in the noninoculated control reactor. Upon further operation of the SCAS system, only 50% 3-CA removal was observed. Denaturing gradient gel electrophoresis (DGGE) of 16S rRNA genes revealed a dynamic change in the microbial community structure of the activated sludge. The DGGE patterns of the noninoculated and the inoculated reactors evolved after 7 days to different clusters, which suggests an effect of strain inoculation on the microbial community structure. The results indicate that bioaugmentation, even with a strain originating from that ecosystem and able to effectively grow on a selective substrate, is not permanent and will probably require regular resupplementation.     

Thermophilic biofiltration of benzene and toluene

In the current studies, we characterized the degradation of a hot mixture of benzene and toluene (BT) gases by a thermophilic biofilter using polyurethane as packing material and high-temperature compost as a microbial source. We also examined the effect of supplementing the biofilter with yeast extract (YE). We found that YE substantially enhanced microbial activity in the thermophilic biofilter. The degrading activity of the biofilter supplied with YE was stable during long-term operation (approximately 100 d) without accumulating excess biomass. The maximum elimination capacity (1,650 g x m(-3) h(-1)) in the biofilter supplemented with YE was 3.5 times higher than that in the biofilter without YE (470 g g x m(-3) h(-1)). At similar retention times, the capacity to eliminate BT for the YE-supplemented biofilter was higher than for previously reported mesophilic biofilters. Thus, thermophilic biofiltration can be used to degrade hydrophobic compounds such as a BT mixture. Finally, 16S rDNA polymerase chain reaction-DGGE (PCR-DGGE) fingerprinting revealed that the thermophilic bacteria in the biofilter included Rubrobacter sp. and Mycobacterium sp.     

Active compost biofiltration of toluene

Composting of leaves and alfalfa (i.e. active compost) was used for thebiofiltration of toluene-contaminated air in a 6-L biofilter (initial bedheight: 180 mm). During the thermophilic phase (45 to 55 °C), toluenebiodegradation rates reached 110 gtoluene.m^-3.h^-1 at an inlet concentration ofabout 5 g.m^-3.h^-1 and a gas residence time of 90 seconds. Thehighest rates were obtained late in the thermophilic phase suggesting amicrobial adaptation was occurring. Biodegradation rates decreased rapidly(50% in 48h) in the cooling stage. Under mesophilic conditions, themaximum biodegradation rates that could be obtained by increasing the inlettoluene concentration were near 89 gtoluene.m^-3.h^-1 which issimilar to that reported in the literature for mature compost biofilters. Novolatile by-product was detected by gas chromatography. Mineralization of¹⁴C-toluene and benzene showed that they were completelydegraded into CO₂ and H₂O under boththermophilic and mesophilic conditions. Bacteria isolated from latemesophilic stage had the capacity to degrade all BTEX compounds but were notable to transform chlorinated compounds. No organisms were isolated whichcould use toluene as their sole source of carbon and energy at 50 °C.Active compost biofiltration should be an excellent process for thetreatment of gaseous BTEX by biofiltration. This is the first report ofthermophilic biofiltration of toluene.     

Bioaugmentation of Activated Sludge by an Indigenous 3-Chloroaniline-Degrading Comamonas testosteroni Strain, I2gfp

A strain identified as Comamonas testosteroni I2 was isolated from activated sludge and found to be able to mineralize 3-chloroaniline (3-CA). During the mineralization, a yellow intermediate accumulated temporarily, due to the distal meta-cleavage of chlorocatechol. This strain was tested for its ability to clean wastewater containing 3-CA upon inoculation into activated sludge. To monitor its survival, the strain was chromosomally marked with the gfp gene and designated I2gfp. After inoculation into a lab-scale semicontinuous activated-sludge (SCAS) system, the inoculated strain maintained itself in the sludge for at least 45 days and was present in the sludge flocs. After an initial adaptation period of 6 days, complete degradation of 3-CA was obtained during 2 weeks, while no degradation at all occurred in the noninoculated control reactor. Upon further operation of the SCAS system, only 50% 3-CA removal was observed. Denaturing gradient gel electrophoresis (DGGE) of 16S rRNA genes revealed a dynamic change in the microbial community structure of the activated sludge. The DGGE patterns of the noninoculated and the inoculated reactors evolved after 7 days to different clusters, which suggests an effect of strain inoculation on the microbial community structure. The results indicate that bioaugmentation, even with a strain originating from that ecosystem and able to effectively grow on a selective substrate, is not permanent and will probably require regular resupplementation.