Dynamic and structural response of a multispecies biofilm to environmental perturbations induced by the continuous increase of benzimidazole fungicides in a permeable reactive biobarrier.

Purpose: This work explores the dynamics of spatiotemporal changes in the taxonomic structure of biofilms and the degradation kinetics of three imidazole group compounds: carbendazim (CBZ), methyl thiophanate (MT), and benomyl (BN) by a multispecies microbial community attached to a fixed bed horizontal tubular reactor (HTR). This bioreactor mimics a permeable reactive biobarrier, which helps prevent the contamination of water bodies by pesticides in agricultural wastewater. Methods: To rapidly quantify the microbial response to crescent loading rates of benzimidazole compounds, a gradient system was used to transiently raise the fungicide volumetric loading rates, measuring the structural and functional dynamics response of a microbial community in terms of the volumetric removal rates of the HTR entering pollutants. Results: The loading rate gradient of benzimidazole compounds severely impacts the spatiotemporal taxonomic structure of the HTR biofilm-forming microbial community. Notable differences with the original structure in HTR stable conditions can be noted after three historical contingencies (CBZ, MT, and BN gradient loading rates). It was evidenced that the microbial community did not return to the composition prior to environmental disturbances; however, the functional similarity of microbial communities after steady state reestablishment was observed. Conclusions: The usefulness of the method of gradual delivery of potentially toxic agents for a microbial community immobilized in a tubular biofilm reactor was shown since its functional and structural dynamics were quickly evaluated in response to fungicide composition and concentration changes. The rapid adjustment of the contaminants' removal rates indicates that even with changes in the taxonomic structure of a microbial community, its functional redundancy favors its adjustment to gradual environmental disturbances.

Integrated adsorption and biological removal of the emerging contaminants ibuprofen, naproxen, atrazine, diazinon, and carbaryl in a horizontal tubular bioreactor.

Groundwater and surface water bodies may have contaminants from urban, industrial, or agricultural wastewater, including emerging contaminants (ECs) or micropollutants (MPs). Frequently, they are not efficiently removed by microbial action due to their minimal concentration in water and the low microbiota affinity for complex compounds. This work developed a process allowing the adsorption of contaminants and their simultaneous biodegradation using horizontal tubular fixed-bed biofilm reactors (HTR). Each HTR has two zones: an equalizer-aerator of the incoming liquid flow and a fixed bed zone. This zone was packed with a mixed support material consisting of granular bio-activated carbon (Bio-GAC) and porous material that increases the bed permeability, thus decreasing the pressure drop. Five microbial communities were acclimated and immobilized in granular activated carbon (GAC) to obtain different specialized Bio-GAC particles able to remove the micropollutants ibuprofen, naproxen, atrazine, carbaryl, and diazinon. The Bio-GAC particles were transferred to HTRs continuously run in microaerophilia at several MPs loading rates. Under these conditions, the removal efficiencies of MPs, except atrazine and carbaryl, were around 100.

Permeable reactive surface-biobarriers. Testing and evaluation of an ecotechnology for the removal of agrotoxic compounds carried by agricultural runoffs.

PURPOSE: The objective of the work is to determine the best operating conditions for variants of an ecological engineering tool (permeable reactive surface biobarrier -PRSB-) potentially useful for the protection of water resources, preventing the arrival of sediments and pesticides transported by runoffs and tile drainage from agricultural lands, to water bodies. METHODS: Four PRB-prototypes were constructed as fixed-bed horizontal channels packed with a porous material supporting an enriched microbial biofilm. Their dynamic and stoichiometric performance was evaluated in the presence or absence of granular activated carbon, with limiting or sufficient O2 supply. The removal of the pesticides and their leading catabolic derivatives were determined by HPLC. The most abundant cultivable microorganisms were isolated and identified by the sequencing of 16sDNA amplicons. RESULTS: The pollutant removal efficiencies obtained in the aerobic biobarriers or microaerophilia were similar. In addition, slight differences were observed in the presence of GAC as an adsorbent, meaning that the most economical and straightforward type of biobarrier was adequate to remove the pollutants studied. In addition, among the most abundant microorganisms isolated in the microbial biofilms colonizing the aerobic biobarriers, the microalgae Micractinium sp. showed the capacity to accumulate the insecticides permethrin and cypermethrin. CONCLUSIONS: The main observed role of Micractinium sp. in the aerobic barriers was the bioaccumulation of pyrethroids, meaning that biosorption is also a valuable removal mechanism operating in the aerobic PRBs. In this aspect, they behave analogously to subsurface constructed wetlands but, instead of superficial plant life, aerobic PRSBs host microalgae.

Degradation kinetics of carbendazim by Klebsiella oxytoca, Flavobacterium johnsoniae, and Stenotrophomonas maltophilia strains.

The fungicide carbendazim is an ecotoxic pollutant frequently found in water reservoirs. The ability of microorganisms to remove pollutants found in diverse environments, soil, water, or air is well documented. Although microbial communities have many advantages in bioremediation processes, in many cases, those with the desired capabilities may be slow-growing or have low pollutant degradation rates. In these cases, the manipulation of the microbial community through enrichment with specialized microbial strains showing high specific growth rates and high rates and efficiencies of pollutant degradation is desirable. In this work, bacteria of the genera Klebsiella, Flavobacterium, and Stenotrophomonas, isolated from the biofilm attached to the packed zones of a biofilm reactor, were able to grow individually in selective medium containing carbendazim. In the three bacteria studied, the mheI gene encoding the first enzyme involved in the degradation of the fungicide carbendazim was found. Studying the dynamics of growth and carbendazim degradation of the three bacteria, the effect of co-formulants was also evaluated. The pure compound and a commercial formulation of carbendazim were used as substrates. Finally, the study made it possible to define the biokinetic advantages of these strains for amendment of microbial communities.

The corrosion process caused by the activity of the anaerobic sporulated bacterium Clostridium celerecrescens on API XL 52 steel.

The microbial corrosion of oil and gas pipes is one of the problems occurring in the oil industry. Various mechanisms explaining microbial corrosion have been demonstrated. Commonly, biocorrosion is attributed to sulfate-reducing bacteria. Also, it has recently been reported that microbial species can connect their electron transport system to metal electrodes. In this research, two spore-forming bacteria isolated in different years from a gas pipeline were identified by biochemical techniques and by 16S rDNA amplification, sequencing, and comparison with the NCBI database. Isolates were also compared between them using molecular techniques as the restriction patterns, unique for 16S rDNA (ARDRA), and the profile of the amplified bit from the genomic DNA, using an unspecific primer (RAPD). The results obtained showed that both isolates corresponded to Clostridium celerecrescens with a 99% similarity according to the sequence reported on the NCBI database. Also, the ARDRA and RAPD electrophoretic profiles of both strains were identical, and no plasmids were found in the strains. Thus, it can be settled that this bacterium is persistent in the environment prevailing in gas pipelines. Also, it was demonstrated that the bacterial secretion of organic acids contributes to the pitting and general biocorrosion of API XL 52 steel. The rates of corrosion obtained, approximately after 40 days, were correlated with the presence and metabolic activity of C. celerecrescens on the metallic surfaces.

Simulation and experimental validation of a gradient feeding system for fast assessment of the kinetic behavior of a microbial consortium in a tubular biofilm reactor.

This study deals with the mathematical simulation and experimental validation of a gradient system for the gradual change of the imidacloprid loading rate to a tubular biofilm reactor (TBR). The strategy was used for fast studies of the kinetic and stoichiometric impact caused by the increase in the pesticide loading rate in a TBR, running in plug flow regime. Seemingly, this strategy has never been used for biokinetic and stoichiometric studies in biofilm reactors. For this purpose, a mathematical model describing the substrate transient behavior Sg(t) in a concentration gradient generator system using variable volume tanks is proposed. A second model, representing the temporary variation in the loading rate of imidacloprid to an aerated equalizer tank preceding the packed zone of the TBR, is also presented. Both models were experimentally confirmed. After the treatment of the experimental data, the kinetic and stoichiometric changes occurring in the TBR, caused by the gradual increase in the imidacloprid loading rate, were readily evaluated. Although the structure of the microbial community, at the phylum level, showed similar behavior along the tubular reactor, the stress produced by the gradual increase in imidacloprid concentration had functional consequences on the mixed microbial populations which were reflected on the stoichiometric and kinetic parameters. After increasing more than five times the imidacloprid loading rate to the TBR, the imidacloprid removal efficiency decayed about 40%, and the microbial-specific removal rate of the insecticide showed a decrease of about 30%.

Biodegradation of a commercial mixture of the herbicides atrazine and S-metolachlor in a multi-channel packed biofilm reactor.

Atrazine and S-metolachlor are two of the most widely used herbicides for agricultural purposes; consequently, residues of both compounds and their metabolites had been detected in ground and superficial waters. Unlike atrazine, the complete degradation of metolachlor has not been achieved. Hence, the purpose of this research is to study the biodegradation of a commercial mixture of atrazine and S-metolachlor in a prototype of a multi-channel packed-bed-biofilm reactor (MC-PBR) designed with the aim of solving the problems of pressure drop and oxygen transfer, typically found on this type of bioreactors.Because the removal efficiency of the herbicides was increased when Candida tropicalis was added to the original microbial community isolated, the reactor was inoculated with this enriched community. The operational conditions tested in batch and continuous mode did not affect the removal efficiency of atrazine; however, this was not the case for S-metolachlor. The removal rates and efficiencies showed a notable variation along the MC-PBR operation.

Effect of propanil, linuron, and dicamba on the degradation kinetics of 2,4-dichlorophenoxyacetic acid by Burkholderia sp. A study by differential analysis of 2,4-dichlorophenoxyacetic acid degradation data.

The successive application of distinct pesticides, or mixtures of them, is a frequent practice that could adversely affect the microbial species inhabiting soil and aquatic ecosystems. The ability of soil or aquatic microbiota to degrade a pesticide could be affected by the presence of another. If the degradation rate of the first compound is inhibited, its dissipation half-life in the environment could be hazardously enlarged. Few studies have been made to quantify the impact on the biodegradation rate of pesticides in soils or water by the presence of other pesticides. In this work, a method for assessing the effect of a pesticide on the biodegradation rate of another, measuring its effect on the biodegradation kinetics of a single bacterial strain is presented. The mathematical analysis is a powerful tool to study the stoichiometry and kinetics of microbial processes, which was used to evaluate independently, in detail, the effect of three pesticides (propanil, linuron, and dicamba) on the biodegradation kinetics of 2,4-dichlorophenoxyacetic acid by a strain of Burkholderia sp. It was evidenced that linuron and dicamba caused a decay of more than 40% in the top instantaneous degradation rate of 2,4-dichlorophenoxyacetic acid, while propanil showed a minimal effect.

Kinetics of carbendazim degradation in a horizontal tubular biofilm reactor.

The fungicide carbendazim is an ecotoxic agent affecting aquatic biota. Due to its suspected hormone-disrupting effects, it is considered a "priority hazard substance" by the Water Framework Directive of the European Commission, and its degradation is of major concern. In this work, a horizontal tubular biofilm reactor (HTBR) operating in plug-flow regime was used to study the kinetics of carbendazim removal by an acclimated microbial consortium. The reactor was operated in steady state continuous culture at eight different carbendazim loading rates. The concentrations of the fungicide were determined at several distances of the HTBR. At the loading rates tested, the highest instantaneous removal rates were observed in the first section of the tubular biofilm reactor. No evidence of inhibition of the catabolic activity of the microbial community was found. Strains of the genera Flectobacillus, Klebsiella, Stenotrophomonas, and Flavobacterium were identified in the biofilm; the last three degrade carbendazim in axenic culture.

Biodegradation of the herbicide Diuron in a packed bed channel and a double biobarrier with distribution of oxygenated liquid by airlift devices: influence of oxygen limitation.

From agricultural soils, where the herbicide Diuron has been frequently applied, a microbial community capable of degrading Diuron and 3,4-dichloroaniline was obtained. The volumetric rates and degradation efficiencies of Diuron and 3,4-DCA were evaluated in two distinct biofilm reactors, which differ in their operating conditions. One is a horizontal fixed bed reactor; plug-flow operated (PF-PBC) with severe limitation of oxygen. In this reactor, the air was supplied to an equalizer reservoir at the start of the PF-PBC reactor. The other is a compartmentalized aerobic biobarrier with internal recirculation of liquid aerated through airlift devices (ALB), continuously or intermittently operated. Both reactors were inoculated with a microbial community capable of degrading Diuron, isolated from a sugarcane field. In the oxygen-limited PF-PBC reactor, 3,4-DCA accumulation was detected, mainly in the middle zone of the packed channel. On the contrary, in the fully aerobic ALB reactor, minimal accumulation of catabolic byproducts was detected, and high Diuron removal efficiencies and removal rates were obtained when it was continuously operated in steady-state conditions. Additionally, the influence of oxygen limitation on the kinetic behavior of the PF-PBC reactor was determined, and a method to estimate the local removal rates of Diuron RV,CD along the plug-flow channel is described. It was observed that the local values of the instantaneous removal rate of Diuron dCD/dt are high in the aerobic region of the PF-PBC reactor; but, suddenly decay in the reactor zones limited by dissolved oxygen.

Steady-state inhibition model for the biodegradation of sulfonated amines in a packed bed reactor.

Aromatic amines are important industrial products having in their molecular structure one or more aromatic rings. These are used as precursors for the synthesis of dyes, adhesives, pesticides, rubber, fertilizers and surfactants. The aromatic amines are common constituents of industrial effluents, generated mostly by the degradation of azo dyes. Several of them are a threat to human health because they can by toxic, allergenic, mutagenic or carcinogenic. The most common are benzenesulfonic amines, such as 4-ABS (4-aminobenzene sulfonic acid) and naphthalene sulfonic amines, such as 4-ANS (4-amino naphthalene sulfonic acid). Sometimes, the mixtures of toxic compounds are more toxic or inhibitory than the individual compounds, even for microorganisms capable of degrading them. Therefore, the aim of this study was to evaluate the degradation of the mixture 4-ANS plus 4-ABS by a bacterial community immobilized in fragments of volcanic stone, using a packed bed continuous reactor. In this reactor, the amines loading rates were varied from 5.5 up to 69 mg L(-1) h(-1). The removal of the amines was determined by high-performance liquid chromatography and chemical oxygen demand. With this information, we have studied the substrate inhibition of the removal rate of the aromatic amines during the degradation of the mixture of sulfonated aromatic amines by the immobilized microorganisms. Experimental results were fitted to parabolic, hyperbolic and linear inhibition models. The model that best characterizes the inhibition of the specific degradation rate in the biofilm reactor was a parabolic model with values of RXM=58.15±7.95 mg (10(9) cells h)(-1), Ks=0.73±0.31 mg L(-1), Sm=89.14±5.43 mg L(-1) and the exponent m=5. From the microbial community obtained, six cultivable bacterial strains were isolated and identified by sequencing their 16S rDNA genes. The strains belong to the genera Variovorax, Pseudomonas, Bacillus, Arthrobacter, Nocardioides and Microbacterium. This microbial consortium could use the mixture of aromatic amines as sources of carbon, nitrogen, energy and sulfur.

Evaluating the degradation of the herbicides picloram and 2,4-D in a compartmentalized reactive biobarrier with internal liquid recirculation.

Tordon is a widely used herbicide formulation of 2,4-dichlorophenoxyacetic acid (2,4-D) and 4-amino-3,5,6-trichloropicolinic acid (picloram), and it is considered a toxic herbicide. The purposes of this work were to assess the feasibility of a microbial consortium inoculated in a lab-scale compartmentalized biobarrier, to remove these herbicides, and isolate, identify, and evaluate their predominant microbial constituents. Volumetric loading rates of herbicides ranging from 31.2 to 143.9 g m(-3) day(-1), for 2,4-D, and 12.8 to 59.3 g m(-3) day(-1) for picloram were probed; however, the top operational limit of the biobarrier, detected by a decay in the removal efficiency, was not reached. At the highest loading rates probed, high average removal efficiencies of 2,4-D, 99.56 ± 0.44; picloram, 94.58 ± 2.62; and chemical oxygen demand (COD), 89.42 ± 3.68, were obtained. It was found that the lab-scale biofilm reactor efficiently removed both herbicides at dilution rates ranging from 0.92 to 4.23 day(-1), corresponding to hydraulic retention times from 1.087 to 0.236 days. On the other hand, few microbial strains able to degrade picloram are reported in the literature. In this work, three of the nine bacterial strains isolated cometabolically degrade picloram. They were identified as Hydrocarboniphaga sp., Tsukamurella sp., and Cupriavidus sp.

Effect of herbicide adjuvants on the biodegradation rate of the methylthiotriazine herbicide prometryn.

A microbial community, selected by its ability to degrade triazinic herbicides was acclimatized by successive transfers in batch cultures. Initially, its ability to degrade prometryn, was evaluated using free cells or cells attached to fragments of a porous support. As carbon, nitrogen and sulfur sources, prometryn, (98.8 % purity), or Gesagard, a herbicide formulation containing 44.5 % prometryn and 65.5 % of adjuvants, were used. In batch cultures, a considerable delay in the degradation of prometryn, presumptively caused by the elevated concentration of inhibitory adjuvants, occurred. When pure prometryn was used, volumetric removal rates remarkably higher than those obtained with the herbicide formulation were estimated by fitting the raw experimental data to sigmoidal decay models, and differentiating them. When the microbial consortium was immobilized in a continuously operated biofilm reactor, the negative effect of adjuvants on the rate and removal efficiency of prometryn could not be detected. Using the herbicide formulation, the consortium showed volumetric removal rates greater than 20 g m(-3) h(-1), with prometryn removal efficiencies of 100 %. The predominant bacterial strains isolated from the microbial consortium were Microbacterium sp., Enterobacter sp., Acinetobacter sp., and Flavobacterium sp. Finally, by comparison of the prometryn removal rates with others reported in the literature, it can be concluded that the use of microbial consortia immobilized in a biofilm reactor operated in continuous regime offer better results than batch cultures of pure microbial strains.

Biodegradation of a mixture of the herbicides ametryn, and 2,4-dichlorophenoxyacetic acid (2,4-D) in a compartmentalized biofilm reactor.

In this work, an efficient degradation process for the removal of 2,4-D and ametryn, together with organic and inorganic adjuvants used in the commercial formulations of both herbicides, was developed. Although both compounds are toxic for microbial communities, ametryn is markedly more toxic than 2,4-D. In spite of this, the microbial consortium used could resist loading rates up to 31.5 mg L(-1) d(-1) of ametryn, with removal efficiencies up to 97% for both herbicides. Thus, an alternative use of this consortium could be bioaugmentation, as a tool to protect the structure and function of an activated-sludge biota against ametryn or 2,4-D shock loads. The process was carried out in a lab-scale prototype of aerobic biobarrier constructed as a compartmentalized fixed film reactor with airlift recirculation of oxygenated liquid.

Biodegradation of the herbicide propanil, and its 3,4-dichloroaniline by-product in a continuously operated biofilm reactor.

The persistence of propanil in soil and aquatic environments along with the possible accumulation of toxic degradation products, such as chloroanilines, is of environmental concern. In this work, a continuous small-scale bioprocess to degrade the herbicide propanil, its main catabolic by-product, 3,4-dichloroaniline (3,4-DCA), and the herbicide adjuvants is carried out. A microbial consortium, constituted by nine bacterial genera, was selected. The isolated strains, identified by amplification and sequencing of their 16S rDNA, were: Acidovorax sp., Luteibacter (rhizovicinus), Xanthomonas sp., Flavobacterium sp., Variovorax sp., Acinetobacter (calcoaceticus), Pseudomonas sp., Rhodococcus sp., and Kocuria sp. The ability of the microbial consortium to degrade the herbicide was evaluated in a biofilm reactor at propanil loading rates ranging from 1.9 to 36.8 mg L(-1) h(-1). Complete removal of propanil, 3,4-DCA, chemical oxygen demand and total organic carbon was obtained at propanil loading rates up to 24.9 mg L(-1) h(-1). At higher loading rates, the removal efficiencies decayed. Four of the identified strains could grow individually in propanil, and 3,4-DCA: Pseudomonas sp., Acinetobacter calcoaceticus, Rhodococcus sp., and Xanthomonas sp. The Kokuria strain grew on 3,4-DCA, but not on propanil. The first three bacteria have been related to biodegradation of phenyl urea herbicides or chlorinated anilines. Although some strains of the genera Xanthomonas and Kocuria have a role in the biodegradation of several xenobiotic compounds, as far as we know, there are no reports about degradation of propanil by Xanthomonas or 3,4-DCA by Kocuria species.

Aerobic biodegradation of a sulfonated phenylazonaphthol dye by a bacterial community immobilized in a multistage packed-bed BAC reactor.

A microbial community able to aerobically degrade the azo dye Acid Orange 7 was selected from riparian or lacustrine sediments collected at sites receiving textile wastewaters. Three bacterial strains, pertaining to the genera Pseudomonas, Arthrobacter, and Rhizobium, constitute the selected community. The biodegradation of AO7 was carried out in batch-suspended cell culture and in a continuously operated multistage packed-bed BAC reactor. The rapid decolorization observed in batch culture, joined to a delay of about 24 h in COD removal and cell growth, suggests that enzymes involved in biodegradation of the aromatic amines generated after AO7 azo-bond cleavage (1-amino-2-naphthol [1-A2N] and 4-aminobenzenesulfonic acid [4-ABS]), are inducible in this microbial consortium. After this presumptive induction period, the accumulated byproducts, measured through COD, were partially metabolized and transformed in cell mass. At all azo dye loading rates used, complete removal of AO7 and 1-A2N was obtained in the multistage packed-bed BAC reactor (PBR).; however, the overall COD (eta ( COD )) and 4-ABS (eta ( ABS )) removal efficiencies obtained in steady state continuous culture were about 90%. Considering the toxicity of 1-A2N, its complete removal has particular relevance. In the first stages of the packed-bed BAC reactor (Fig. 4a-c), major removal was observed. In the last stage, only a slight removal of COD and 4-ABS was obtained. Comparing to several reported studies, the continuously operated multistage packed-bed BAC reactor showed similar or superior results. In addition, the operation of large-packed-bed BAC reactors could be improved by using several shallow BAC bed stages, because the pressure drop caused by bed compaction of a support material constituted by small and fragile particles can be reduced.

Lactic acid bacteria production from whey.

The main purpose of this work was to isolate and characterize lactic acid bacteria (LAB) strains to be used for biomass production using a whey-based medium supplemented with an ammonium salt and with very low levels of yeast extract (0.25 g/L). Five strains of LAB were isolated from naturally soured milk after enrichment in whey-based medium. One bacterial isolate, designated MNM2, exhibited a remarkable capability to utilize whey lactose and give a high biomass yield on lactose. This strain was identified as Lactobacillus casei by its 16S rDNA sequence. A kinetic study of cell growth, lactose consumption, and titratable acidity production of this bacterial strain was performed in a bioreactor. The biomass yield on lactose, the percentage of lactose consumption, and the maximum increase in cell mass obtained in the bioreactor were 0.165 g of biomass/g of lactose, 100%, and 2.0 g/L, respectively, which were 1.44, 1.11, and 2.35 times higher than those found in flask cultures. The results suggest that it is possible to produce LAB biomass from a whey-based medium supplemented with minimal amounts of yeast extract.