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.

Chitinase induction in Trichoderma harzianum: a solid-state fermentation approach using shrimp waste and wheat bran/commercial chitin for chitooligosaccharides synthesis.

This study innovatively employed solid-state fermentation (SSF) to evaluate chitinase induction in Trichoderma harzianum. Solid-state fermentation minimizes water usage, a crucial global resource, and was applied using shrimp waste chitin and a mixture of commercial chitin with wheat bran as substrates. Shrimp waste and wheat bran were pretreated and characterized for SSF, and the fungus's utilization of the substrates was assessed using spectrophotometric and microscopic methods. The resulting enzymes' ability to produce chitooligosaccharides (COS) mixtures was studied. Wheat bran/commercial chitin demonstrated superior performance, with a 1.8-fold increase in chitinase activity (76.3 U/mg protein) compared to shrimp waste chitin (41.8 U/mg protein). Additionally, the COS mixture obtained from wheat bran/commercial chitin showed a higher concentration of reducing sugars, reaching 87.85 mM, compared to shrimp waste chitin (14.87 mM). The COS profile from wheat bran/commercial chitin included monomers to heptamers, while the profile from shrimp waste chitin was predominantly composed of monomers. These results highlight the advantages of SSF for chitinase induction and COS production in T. harzianum, offering potential applications as dietary fiber, antioxidants, and antimicrobial agents. The findings contribute to by-product valorization, waste reduction, and the sustainable generation of valuable products through SSF-based enzyme production.

RP-HPLC Separation and 1H NMR Identification of a Yellow Fluorescent Compound-Riboflavin (Vitamin B2)-Produced by the Yeast Hyphopichia wangnamkhiaoensis.

The yeast Hyphopichia wangnamkhiaoensis excretes a brilliant yellow fluorescent compound into its growth culture. In this study, we isolated and identified this compound using reverse-phase high-performance liquid chromatography-diode array detector (RP-HPLC-DAD) as well as 1H NMR and UV-Vis spectroscopy. Two of the three RP-HPLC-DAD methods used successfully separated the fluorescent compound and involved (1) a double separation step with isocratic flow elution, first on a C18 column and later on a cyano column, and (2) a separation with a linear gradient elution on a phenyl column. The wavelengths of maximum absorption of the fluorescent compound-containing HPLC fractions (~224, 268, 372, and 446 nm) are in good agreement with those exhibited by flavins. The 1H NMR spectra revealed methyl (δ 2.30 and 2.40) and aromatic proton (δ 7.79 and 7.77) signals of riboflavin. The 1H NMR spectra of the samples spiked with riboflavin confirmed that the brilliant yellow fluorescent compound is riboflavin. The maximum excitation and emission wavelengths of the fluorescent compound were 448 and 528 nm, respectively, which are identical to those of riboflavin.

Fundaments and Concepts on Screening of Microorganisms for Biotechnological Applications. Mini Review.

Microbial biotechnology uses microorganisms and their derivatives to generate industrial and/or environmental products that impact daily life. Modern biotechnology uses proteomics, metabolomics, quantum processors, and massive sequencing methods to yield promising results with microorganisms. However, the fundamental concepts of microbial biotechnology focus on the specific search for microorganisms from natural sources and their correct analysis to implement large-scale processes. This mini-review focuses on the methods used for the isolation and selection of microorganisms with biotechnological potential to empathize the importance of these concepts in microbial biotechnology. In this work, a review of the state of the art in recent years on the selection and characterization of microorganisms with a basic approach to understanding the importance of fundamental concepts in the field of biotechnology was carried out. The proper selection of isolation sources and the design of suitable selection criteria according to the desired activity have generated substantial changes in the development of biotechnology for more than three decades. Some examples include Taq polymerase in the PCR method and CRISPR technology. The objective of this mini review is to establish general ideas for the screening of microorganisms based on basic concepts of biotechnology that are left aside in several articles and maintain the importance of the basic concepts that this implies in the development of modern biotechnology.

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.

Evaluation and comparison of advanced oxidation processes for the degradation of 2,4-dichlorophenoxyacetic acid (2,4-D): a review.

Organochlorine pesticides have generated public concern worldwide because of their toxicity to human health and the environment, even at low concentrations, and their persistence, being mostly nonbiodegradable. The use of 2,4-dichlorophenoxyacetic acid (2,4-D) has increased in recent decades, causing severe water contamination. Several treatments have been developed to degrade 2,4-D. This manuscript presents an overview of the physicochemical characteristics, uses, regulations, environmental and human health impacts of 2,4-D, and different advanced oxidation processes (AOPs) to degrade this organic compound, evaluating and comparing operation conditions, efficiencies, and intermediaries. Based on this review, 2,4-D degradation is highly efficient in ozonation (system O3/plasma, 99.8% in 30 min). Photocatalytic, photo-Fenton, and electrochemical processes have the optimal efficiencies of degradation and mineralization: 97%/79.67% (blue TiO2 nanotube arrays//UV), 100%/98% (Fe2+/H2O2/UV), and 100%/84.3% (MI-meso SnO2), respectively. The ozonation and electrochemical processes show high degradation efficiencies, but energy costs are also high, and photocatalysis is more expensive with a separation treatment used to recover the catalyst in the solution. The Fenton process is a viable economic-environmental option, but degradation efficiencies are often low (50-70%); however, they are increased when solar UV radiation is used (90-100%). AOPs are promising technologies for the degradation of organic pollutants in real wastewater, so evaluating their strengths and weaknesses is expected to help select viable operational conditions and obtain optimal efficiencies.

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.

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.

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.