Chemotaxis Towards Aromatic Compounds: Insights from Comamonas testosteroni.

Chemotaxis is an important physiological adaptation that allows many motile bacteria to orientate themselves for better niche adaptation. Chemotaxis is best understood in Escherichia coli. Other representative bacteria, such as Rhodobacter sphaeroides, Pseudomonas species, Helicobacter pylori, and Bacillus subtilis, also have been deeply studied and systemically summarized. These bacteria belong to α-, γ-, ε-Proteobacteria, or Firmicutes. However, ß-Proteobacteria, of which many members have been identified as holding chemotactic pathways, lack a summary of chemotaxis. Comamonas testosteroni, belonging to ß-Proteobacteria, grows with and chemotactically responds to a range of aromatic compounds. This paper summarizes the latest research on chemotaxis towards aromatic compounds, mainly from investigations of C. testosteroni and other Comamonas species.

Cross Talk between Chemosensory Pathways That Modulate Chemotaxis and Biofilm Formation.

Complex chemosensory systems control multiple biological functions in bacteria, such as chemotaxis, gene regulation, and cell cycle progression. Many species contain more than one chemosensory system per genome, but little is known about their potential interplay. In this study, we reveal cross talk between two chemosensory pathways that modulate chemotaxis and biofilm formation in Comamonas testosteroni We demonstrate that some chemoreceptors that govern chemotaxis also contribute to biofilm formation and these chemoreceptors can physically interact with components of both pathways. Finally, we show that the chemotaxis histidine kinase CheA can phosphorylate not only its cognate response regulator CheY2 but also one of the response regulators from the pathway mediating biofilm formation, FlmD. The phosphoryl group transfer from CheA to CheY2 is much faster than that from CheA to FlmD, which is consistent with chemotaxis being a fast response and biofilm formation being a much slower developmental process. We propose that cross talk between chemosensory pathways may play a role in coordination of complex behaviors in bacteria.IMPORTANCE In many bacteria, two or more homologous chemosensory pathways control several cellular functions, such as motility and gene regulation, in response to changes in the cell's microenvironment. Cross talk between signal transduction systems is poorly understood; while generally it is considered to be undesired, in some instances it might be beneficial for coregulation of complex behaviors. We demonstrate that several receptors from the pathway controlling motility can physically interact with downstream components of the pathway controlling biofilm formation. We further show that a kinase from the pathway controlling motility can also phosphorylate a response regulator from the pathway controlling biofilm formation. We propose that cross talk between two chemosensory pathways might be involved in coordination of two types of cell behavior-chemotaxis and biofilm formation.

Carbon catabolite repression and cell dispersal affect degradation of the xenobiotic compound 3,4-dichloroaniline in Comamonas testosteroni WDL7 biofilms.

Organic pollutant degrading biofilms in natural ecosystems and water treatment systems are often exposed to other carbon sources in addition to the pollutant. The availability of auxiliary carbon sources can lead to surplus biomass growth, changes in biofilm structure and carbon catabolite repression (CCR) which together will affect pollutant degradation rate and efficiency of the system. To understand the interplay between these processes, continuous biofilms of the 3,4-dichloroaniline (3,4-DCA) degrading Comamonas testosteroni WDL7-RFP were grown in single- and dual-substrate conditions with 3,4-DCA and/or citrate and reciprocal effects on 3,4-DCA/citrate degradation, biofilm biomass and biofilm structure were examined. The main mechanism affecting 3,4-DCA degradation in biofilms in dual-substrate conditions was citrate-mediated CCR as reflected by a decrease in specific 3,4-DCA degrading activity. Growth on citrate partially compensated for the lowered specific 3,4-DCA degradation activity under dual substrate conditions but not to the extent expected from growth observed under single-substrate conditions with citrate. This was explained by higher residual 3,4-DCA concentrations in the presence of citrate that increased cell dispersal in the biofilms. Our results show hampered pollutant removal in biofilms due to a complex interplay of auxiliary organic C source utilization for growth affecting the specific pollutant degradation rate and changes in cell physiology due to increased exposure to the pollutant as a result of lowered pollutant degradation rates.

Influence of 3-Chloroaniline on the Biofilm Lifestyle of Comamonas testosteroni and Its Implications on Bioaugmentation.

UNLABELLED: Bioaugmentation has been frequently proposed in wastewater and soil treatment to remove toxic aromatic compounds. The performance of bioaugmentation is affected by a number of biological and environmental factors, including the interaction between the target pollutant and the augmented bacterial cells. In this study, using Comamonas testosteroni and 3-chloroaniline (3-CA) as the model organism and target pollutant, we explored the influence of toxic aromatic pollutants on the biofilm lifestyle of bacteria capable of degrading aromatic compounds toward a better understanding of cell-pollutant interaction in bioaugmentation. Our results showed that the exposure to 3-CA greatly reduced the retention of C. testosteroni cells in packed-bed bioreactors (from 22% to 15% after three pore volumes), which could be attributed to the altered bacterial motility and cell surface hydrophobicity. To further understand the molecular mechanisms, we employed an integrated genomic and transcriptomic analysis to examine the influence of 3-CA on the expression of genes important to the biofilm lifestyle of C. testosteroni We found that exposure to 3-CA reduced the intracellular c-di-GMP level by downregulating the expression of genes encoding c-di-GMP synthases and induced massive cell dispersal from the biofilms. Our findings provide novel environmental implications on bioaugmentation, particularly in biofilm reactors, for the treatment of wastewater containing recalcitrant industrial pollutants. IMPORTANCE: Bioaugmentation is a bioremediation approach that often has been described in the literature but has almost never been successfully applied in practice. Many biological and environmental factors influence the overall performance of bioaugmentation. Among these, the interaction between the target pollutant and the augmented bacterial cells is one of the most important factors. In this study, we revealed the influence of toxic aromatic pollutants on the biofilm lifestyle of bacteria capable of degrading aromatic compounds toward a better understanding of cell-pollutant interaction in bioaugmentation. Our findings provide novel environmental implications on bioaugmentation for the treatment of wastewater containing recalcitrant industrial pollutants; in particular, the exposure to toxic pollutants may reduce the retention of augmented organisms in biofilm reactors by reducing the c-di-GMP level, and approaches to elevating or maintaining a high c-di-GMP level may be promising to establish and maintain sustainable bioaugmentation activity.

Involvement in Denitrification is Beneficial to the Biofilm Lifestyle of Comamonas testosteroni: A Mechanistic Study and Its Environmental Implications.

Comamonas is one of the most abundant microorganisms in biofilm communities driving wastewater treatment. Little has been known about the role of this group of organisms and their biofilm mode of life. In this study, using Comamonas testosteroni as a model organism, we demonstrated the involvement of Comamonas biofilms in denitrification under bulk aerobic conditions and elucidated the influence of nitrate respiration on its biofilm lifestyle. Our results showed that C. testosteroni could use nitrate as the sole electron acceptor for anaerobic growth. Under bulk aerobic condition, biofilms of C. testosteroni were capable of reducing nitrate, and intriguingly, nitrate reduction significantly enhanced viability of the biofilm-cells and reduced cell detachment from the biofilms. Nitrate respiration was further shown to play an essential role in maintaining high cell viability in the biofilms. RNA-seq analysis, quantitative polymerase chain reaction, and liquid chromatography-mass spectrometry revealed a higher level of bis(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP) in cells respiring on nitrate than those grown aerobically (1.3 × 10(-4) fmol/cell vs 7.9 × 10(-6) fmol/cell; P < 0.01). C-di-GMP is one universal signaling molecule that regulates the biofilm mode of life, and a higher c-di-GMP concentration reduces cell detachment from biofilms. Taking these factors together, this study reveals that nitrate reduction occurs in mature biofilms of C. testosteroni under bulk aerobic conditions, and the respiratory reduction of nitrate is beneficial to the biofilm lifestyle by providing more metabolic energy to maintain high viability and a higher level of c-di-GMP to reduce cell detachment.

Comparative genome analysis reveals genetic adaptation to versatile environmental conditions and importance of biofilm lifestyle in Comamonas testosteroni.

Comamonas testosteroni is an important environmental bacterium capable of degrading a variety of toxic aromatic pollutants and has been demonstrated to be a promising biocatalyst for environmental decontamination. This organism is often found to be among the primary surface colonizers in various natural and engineered ecosystems, suggesting an extraordinary capability of this organism in environmental adaptation and biofilm formation. The goal of this study was to gain genetic insights into the adaption of C. testosteroni to versatile environments and the importance of a biofilm lifestyle. Specifically, a draft genome of C. testosteroni I2 was obtained. The draft genome is 5,778,710 bp in length and comprises 110 contigs. The average G+C content was 61.88 %. A total of 5365 genes with 5263 protein-coding genes were predicted, whereas 4324 (80.60 % of total genes) protein-encoding genes were associated with predicted functions. The catabolic genes responsible for biodegradation of steroid and other aromatic compounds on draft genome were identified. Plasmid pI2 was found to encode a complete pathway for aniline degradation and a partial catabolic pathway for chloroaniline. This organism was found to be equipped with a sophisticated signaling system which helps it find ideal niches and switch between planktonic and biofilm lifestyles. A large number of putative multi-drug-resistant genes coding for abundant outer membrane transporters, chaperones, and heat shock proteins for the protection of cellular function were identified in the genome of strain I2. In addition, the genome of strain I2 was predicted to encode several proteins involved in producing, secreting, and uptaking siderophores under iron-limiting conditions. The genome of strain I2 contains a number of genes responsible for the synthesis and secretion of exopolysaccharides, an extracellular component essential for biofilm formation. Overall, our results reveal the genomic features underlying the adaption of C. testosteroni to versatile environments and highlighting the importance of its biofilm lifestyle.

Bioaugmentation of a sequencing batch biofilm reactor with Comamonas testosteroni and Bacillus cereus and their impact on reactor bacterial communities.

The immobilization of microorganisms is essential for efficient bioaugmentation systems. The performance of Bacillus cereus G5 as biofilm-forming bacteria and Comamonas testosteroni A3 a 3,5 dinitrobenzoic acid (DNB)-degrading strain] in laboratory-scale sequencing batch biofilm reactors (SBBRs) treating DNB synthetic wastewater has been examined. The microbial diversity in the reactors was also explored. The reactor R3 inoculated with B. cereus G5 and C. testosteroni A3 together not only improved the removal of contaminants, but also exhibited obvious resistance to shock loading with DNB during later operations. Pyrosequencing was used to evaluate bacterial communities in three reactors. Comamonas was predominant in the reactor R3, indicating the effect of G5 in promoting immobilization of A3 cells in biofilms. Those microbial resources, e.g.G5, which can stimulate the self-immobilization of the degrading bacteria offer a novel strategy for immobilization of degraders in bioaugmentation systems and show broader application prospects.

Elevated level of the second messenger c-di-GMP in Comamonas testosteroni enhances biofilm formation and biofilm-based biodegradation of 3-chloroaniline.

The bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP) is a ubiquitous second messenger that determines bacterial lifestyle between the planktonic and biofilm modes of life. Although the role of c-di-GMP signaling in biofilm development and dispersal has been extensively studied, how c-di-GMP signaling influences environmental bioprocess activities such as biodegradation remains unexplored. To elucidate the impacts of elevating c-di-GMP level on environmental bioprocesses, we constructed a Comamonas testosteroni strain constitutively expressing a c-di-GMP synthase YedQ from Escherichia coli and examined its capability in biofilm formation and biodegradation of 3-chloroaniline (3-CA). The high c-di-GMP strain exhibited an increased binding to Congo red dye, a decreased motility, and an enhanced biofilm formation capability. In planktonic cultures, the strain with an elevated c-di-GMP concentration and the wild type could degrade 3-CA comparably well. However, under batch growth conditions with a high surface to volume ratio, an elevated c-di-GMP concentration in C. testosteroni significantly increased the contribution of biofilms in 3-CA biodegradation. In continuous submerged biofilm reactors, C. testosteroni with an elevated c-di-GMP level exhibited an enhanced 3-CA biodegradation and a decreased cell detachment rate. Taken together, this study provides a novel strategy to enhance biofilm-based biodegradation of toxic xenobiotic compounds through manipulating bacterial c-di-GMP signaling.

Nutrition effects on the biofilm immobilization and 3,5-DNBA degradation of Comamonas testosteroni A3 during bioaugmentation treatment.

The survival of inoculated microbes is critical for successful bioaugmentation in wastewater treatment. The influence of readily available nutrients (RANs) on the colonization of two functional bacteria, Pseudomonas putida M9, a strong biofilm-forming strain, and Comamonas testosteroni A3, a 3,5-dinitrobenzoic acid (3,5-DNBA)-degrading strain, in biofilms was studied with 3,5-dinitrobenoic acid synthetic wastewater (DCMM) complemented with various ratios of Luria-Bertani broth (LB). With the increase in LB rate, the biofilm biomass was increased, the percentage of gfp-labeled M9 measured in the mixed culture enhanced, and also M9 became dominant. In laboratory-scale sequencing batch biofilm reactors, with the increase in 3,5-DNBA concentration and extension of the running time, the 3,5-DNBA removal in DCMM wastewater complemented with RANs tended to be more efficient and its removal rates increased gradually over the experimental period. Our study demonstrated that supplementing RANs could be a useful strategy for enhancing colonization of degrading bacteria in wastewater treatment systems.

Comamonas testosteroni uses a chemoreceptor for tricarboxylic acid cycle intermediates to trigger chemotactic responses towards aromatic compounds.

Bacterial chemotaxis towards aromatic compounds has been frequently observed; however, knowledge of how bacteria sense aromatic compounds is limited. Comamonas testosteroni CNB-1 is able to grow on a range of aromatic compounds. This study investigated the chemotactic responses of CNB-1 to 10 aromatic compounds. We constructed a chemoreceptor-free, non-chemotactic mutant, CNB-1Δ20, by disruption of all 19 putative methyl-accepting chemotaxis proteins (MCPs) and the atypical chemoreceptor in strain CNB-1. Individual complementation revealed that a putative MCP (tagged MCP2201) was involved in triggering chemotaxis towards all 10 aromatic compounds. The recombinant sensory domain of MCP2201 did not bind to 3- or 4-hydroxybenzoate, protocatechuate, catechol, benzoate, vanillate and gentisate, but bound oxaloacetate, citrate, cis-aconitate, isocitrate, α-ketoglutarate, succinate, fumarate and malate. The mutant CNB-1ΔpmdF that lost the ability to metabolize 4-hydroxybenzoate and protocatechuate also lost its chemotactic response to these compounds, suggesting that taxis towards aromatic compounds is metabolism-dependent. Based on the ligand profile, we proposed that MCP2201 triggers taxis towards aromatic compounds by sensing TCA cycle intermediates. Our hypothesis was further supported by the finding that introduction of the previously characterized pseudomonad chemoreceptor (McpS) for TCA cycle intermediates into CNB-1Δ20 likewise triggered chemotaxis towards aromatic compounds.

Identification and characterization of the LysR-type transcriptional regulator HsdR for steroid-inducible expression of the 3α-hydroxysteroid dehydrogenase/carbonyl reductase gene in Comamonas testosteroni.

3α-Hydroxysteroid dehydrogenase/carbonyl reductase (3α-HSD/CR) from Comamonas testosteroni is a key enzyme in steroid degradation in soil and water. 3α-HSD/CR gene (hsdA) expression can be induced by steroids like testosterone and progesterone. Previously, we have shown that the induction of hsdA expression by steroids is a derepression where steroidal inducers bind to two repressors, RepA and RepB, thereby preventing the blocking of hsdA transcription and translation, respectively (G. Xiong and E. Maser, J. Biol. Chem. 276:9961-9970, 2001; G. Xiong, H. J. Martin, and E. Maser, J. Biol. Chem. 278:47400-47407, 2003). In the present study, a new LysR-type transcriptional factor, HsdR, for 3α-HSD/CR expression in C. testosteroni has been identified. The hsdR gene is located 2.58 kb downstream from hsdA on the C. testosteroni ATCC 11996 chromosome with an orientation opposite that of hsdA. The hsdR gene was cloned and recombinant HsdR protein was produced, as was anti-HsdR polyclonal antibodies. While heterologous transformation systems revealed that HsdR activates the expression of the hsdA gene, electrophoresis mobility shift assays showed that HsdR specifically binds to the hsdA promoter region. Interestingly, the activity of HsdR is dependent on decreased repression by RepA. Furthermore, in vitro binding assays indicated that HsdR can come into contact with RNA polymerase. As expected, an hsdR knockout mutant expressed low levels of 3α-HSD/CR compared to that of wild-type C. testosteroni after testosterone induction. In conclusion, HsdR is a positive transcription factor for the hsdA gene and promotes the induction of 3α-HSD/CR expression in C. testosteroni.

Establishment and characterization of dual-species biofilms formed from a 3,5-dinitrobenzoic-degrading strain and bacteria with high biofilm-forming capabilities.

In this study, the possibility of establishing a dual-species biofilm from a bacterium with a high biofilm-forming capability and a 3,5-dinitrobenzoic acid (3,5-DNBA)-degrading bacterium, Comamonas testosteroni A3, was investigated. Our results showed that the combinations of strain A3 with each of five strains with a high biofilm-forming capability (Pseudomonas sp. M8, Pseudomonas putida M9, Bacillus cereus M19, Pseudomonas plecoglossicida M21 and Aeromonas hydrophila M22) presented different levels of enhancement regarding biofilm-forming capability. Among these culture combinations, the 24-h dual-species biofilms established by C. testosteroni A3 with P. putida M9 and A. hydrophila M22 showed the strongest resistance to 3,5-DNBA shock loading, as demonstrated by six successive replacements with DMM2 synthetic wastewater. The degradation rates of 3,5-DNBA by these two culture combinations reached 63.3-91.6% and 70.7-89.4%, respectively, within 6 h of every replacement. Using the gfp-tagged strain M22 and confocal laser scanning microscopy, the immobilization of A3 cells in the dual-species biofilm was confirmed. We thus demonstrated that, during wastewater treatment processes, it is possible to immobilize degrader bacteria with bacteria with a high biofilm-forming capability and to enable them to develop into the mixed microbial flora. This may be a simple and economical method that represents a novel strategy for effective bioaugmentation.

Plasmid-mediated bioaugmentation of activated sludge bacteria in a sequencing batch moving bed reactor using pNB2.

AIMS: The applicability of plasmid pNB2 for bioaugmentation of bacteria in model wastewater treatment reactors receiving 3-chloroaniline (3-CA) was investigated. METHODS AND RESULTS: A setup of three biofilm reactors was studied, all initially inoculated with bacteria from activated sludge. Reactor PB received a Pseudomonas putida pNB2 donor strain not able to degrade 3-CA. Positive control reactor P received a 3-CA degrading Comamonas testosteroni pNB2-transconjugant. The negative control reactor N remained unchanged. Reactor P showed 3-CA degradation from the beginning of the experiment whereas in reactor PB, degradation started after an initial lag period. No degradation was observed in reactor N. PCR analysis showed that the P. putida donor abundance dropped in reactor PB, whereas the plasmid abundance did not, indicating transfer to other bacteria. A number of different 3-CA degrading C. testosteroni strains carrying pNB2 could be isolated from reactor PB. CONCLUSIONS: A successful plasmid-mediated bioaugmentation was achieved with C. testosteroni being the dominant 3-CA degrading pNB2 transconjugant species active in reactor PB. SIGNIFICANCE AND IMPACT OF THE STUDY: The study underlines the potential of gene transfer to contribute to establishment and spread of genetic information in general, particularly emphasizing the spread of xenobiotic degrading potential by dissemination of catabolic genes.

Characterization of phenol biodegradation by Comamonas testosteroni ZD4-1 and Pseudomonas aeruginosa ZD4-3.

OBJECTIVE: To investigate the characteristic and biochemical mechanism about the phenol biodegradation by bacterial strains ZD 4-1 and ZD 4-3. METHODS: Bacterial strains ZD 4-1 and ZD 4-3 were isolated by using phenol as the sole source of carbon and energy, and identified by 16S rDNA sequence analysis. The concentrations of phenol and total organic carbon (TOC) were monitored to explore the degradation mechanism. The biodegradation intermediates were scanned at 375 nm by using a uv-vis spectrophotometer. The enzyme assays were performed to detect the activities of dioxygenases. RESULTS: Bacterial strains ZD 4-1 and ZD 4-3 were identified as Comamonas testosteroni and Pseudomonas aeruginosa by 16S rDNA sequence analysis, respectively. The growth of the two strains was observed on a variety of aromatic hydrocarbons. The strains ZD 4-1 and ZD 4-3 metabolized phenol via ortho-pathways and meta-pathways, respectively. In addition, the results of enzyme assays showed that the biodegradation efficiency of phenol by meta-pathways was higher than that by ortho-pathways. Finally, the results of induction experiment indicated that the catechol dioxygenases, both catechol 1,2-dioxygenase (C120) and catechol 2,3-dioxygenase (C230), were all inducible. CONCLUSION: The strains ZD 4-1 and ZD 4-3 metabolize phenol through ortho-pathways and meta-pathway, respectively. Furthermore, the biodegradation efficiency of phenol by meta-pathways is higher than that by ortho-pathways.

Hydrodynamic characteristics of immobilized cell beads in a liquid-solid fluidized-bed bioreactor.

This study examined the hydrodynamic characteristics of a liquid-solid fluidized-bed bioreactor using elastic particles (PVA gel beads) of various diameters as carriers. The drag coefficient-Reynolds number, velocity-voidage, and expansion index-Reynolds number relationships observed during fluidization of PVA gel beads in a fluidized bed in our experiments were compared with the published results. Predictions made from previous correlations were examined with our new experimental findings, revealing the inadequacy of most of these correlations. Thus, new correlations describing the above-mentioned relationships are suggested. The drag coefficient of immobilized cell beads is larger than that of free cell ones at the same Reynolds number because the surface of the immobilized cell beads is rougher. For multiparticle systems, the correction factor, f(epsilon), is a function of the falling gel bead properties (Reynolds number) as well as the fluidized gel bead properties (Archimedes number), and depend strongly on the bed voidage (epsilon). A new simple relation was developed to predict easily the epsilon value from 0.5-0.9 at 4,986 < A(r) < 40,745 or 34 < Re(t) < 186. For all the immobilized cell beads used in this study, the prediction error of the bed voidage was less than 5% at epsilon > 0.5. The prediction equations in this study can be further applied to analyzing the hydrodynamic characteristics of a fluidized-bed reactor using similar entrapped elastic particles as carriers.

Bioaugmenting bioreactors for the continuous removal of 3-chloroaniline by a slow release approach.

The survival and activity of microbial degradative inoculants in bioreactors is critical to obtain successful biodegradation of non- or slowly degradable pollutants. Achieving this in industrial wastewater reactors is technically challenging. We evaluated a strategy to obtain complete and stable bioaugmentation of activated sludge, which is used to treat a 3-chloroaniline (3-CA) contaminated wastewater in a lab-scale semi-continuous activated sludge system. A 3-CA metabolizing bacterium, Comamonas testosteroni strain I2, was mixed with molten agar and encapsulated in 4 mm diameter open-ended silicone tubes of 3 cm long. The tubes containing the immobilized bacteria represented about 1% of the volume of the mixed liquor. The bioaugmentation activity of a reactor containing the immobilized cells was compared with a reactor with suspended I2gfp cells. From day 25-30 after inoculation, the reactor with only suspended cells failed to completely degrade 3-CA because of a decrease in metabolic activity. In the reactors with immobilized cells, however, 3-CA continued to be removed. A mass balance indicated that ca. 10% of the degradation activity was due to the immobilized cells. Slow release of the growing embedded cells from the agar into the activated sludge medium, resulting in a higher number of active 3-CA-degrading I2 cells, was responsible for ca. 90% of the degradation. Our results demonstrate that this simple immobilization procedure was effective to maintain a 3-CA-degrading population within the activated sludge community.

Rapid assessment of the physiological status of the polychlorinated biphenyl degrader Comamonas testosteroni TK102 by flow cytometry.

The viability of the polychlorinated biphenyl-degrading bacterium Comamonas testosteroni TK102 was assessed by flow cytometry (FCM) with the fluorogenic ester Calcein-AM (CAM) and the nucleic acid dye propidium iodide (PI). CAM stained live cells, whereas PI stained dead cells. When double staining with CAM and PI was performed, three physiological states, i.e., live (calcein positive, PI negative), dead (calcein negative, PI positive), and permeabilized (calcein positive, PI positive), were detected. To evaluate the reliability of this double-staining method, suspensions of live and dead cells were mixed in various proportions and analyzed by FCM. The proportion of dead cells measured by FCM directly correlated with the proportion of dead cells in the sample (y = 0.9872 x + 0.18; R(2) = 0.9971). In addition, the proportion of live cells measured by FCM inversely correlated with the proportion of dead cells in the sample (y = -0.9776 x + 98.36; R(2) = 0.9962). The proportion of permeabilized cells was consistently less than 2%. These results indicate that FCM in combination with CAM and PI staining is rapid (

The role of cell bioaugmentation and gene bioaugmentation in the remediation of co-contaminated soils.

Soils co-contaminated with metals and organics present special problems for remediation. Metal contamination can delay or inhibit microbial degradation of organic pollutants such that for effective in situ biodegradation, bioaugmentation is necessary. We monitored the degradation of 2,4-dichlorophenoxyacetic acid (2,4-D) or 3-chlorobenzoate (3-CB) in two different soils with and without cadmium (Cd) contamination. Additionally, we evaluated the ability of bioaugmentation to enhance organic degradation in these co-contaminated soils. Finally, we determined whether enhanced degradation was due to survival of the introduced organism (cell bioaugmentation) or plasmid transfer to indigenous microbial populations (gene bioaugmentation). In Brazito soil, dual inoculation with a Cd-resistant bacterium plus a known 2,4-D-degrading bacterium, Ralstonia eutropha JMP134, enhanced 2,4-D degradation. Escherichia coli D11, which lacks chromosomal genes necessary for complete 2,4-D mineralization, was used for gene bioaugmentation in Madera soil. Significant gene transfer of the plasmid to the indigenous populations was observed, and the rate of 2,4-D degradation was enhanced relative to that of controls. Cell bioaugmentation was further demonstrated when (Comamonas testosteroni was used to enhance biodegradation of 3-CB in Madera soil. In this case no transfer of plasmid pBRC60 to indigenous soil recipients was observed. For the Madera soil, nonbioaugmented samples ultimately showed complete 2,4-D degradation. In contrast, nonbioaugmented Brazito soils showed incomplete 2,4-D degradation. These studies are unique in showing that both cell bioaugmentation and gene bioaugmentation can be effective in enhancing organic degradation in co-contaminated soils. Ultimately, the bioaugmentation strategy may depend on the degree of contamination and the time frame available for remediation.