The combination of enzymes and nanomaterials represents an innovative technology that can be used in the field of environmental bioremediation. Various biologically active molecules can be effectively immobilized onto nanomaterials, which are considered very interesting matrices because of their unique physicochemical properties. Nanoparticles are carriers that have been intensively investigated because of their potential to produce nanobiocatalysts. Currently, the development of novel hybrid nanocomposites such as nanographene, nanotubes, nanofibers, and nanogels is significantly increasing. In the first part of this chapter, importance and advantages of enzyme immobilization, properties of free enzymes, and parameters influencing the immobilization processes are discussed. Description of different types of nanomaterials and the corresponding immobilization techniques such as adsorption, covalent binding, entrapment, encapsulation, and cross-linking is also involved. The second part of this chapter is about degradation of various pollutants including phenolic compounds, synthetic dyes, pharmaceuticals, and PAHs by nanobiocatalysts. Many studies have been published over the past 5 years, which indicates an increasing interest in this application. The most important parameters and results of the published papers have been reviewed and organized in a comprehensive table. It is obvious that the utilization of nanobiocatalysts for remediating contaminated wastewater and soil is still under development. However, enzyme-based nanomaterials found an interesting application in the field of biosensors, which is described at the end of this chapter. Nanobiosensors are rapidly developing and more and more often used for detecting and monitoring various pollutants in the environment.

The consumption of methylphenidate, a nootropic drug used to improve mental performance, is becoming increasingly serious. Methylphenidate is metabolized in human liver to ritalinic acid, which has been commonly detected in sewage and surface waters. Additionally, ritalinic acid serves as a biomarker in sewage epidemiology studies. Thus knowledge of the stability and microbial degradation pathways of ritalinic acid is essential for proper estimation of methylphenidate consumption. In the study reported here, we describe the fast formation of a previously unknown, dead-end metabolite of ritalinic acid by Nocardioides sp. strain MW5. HRMS and 2D NMR analyses allowed precisely identification of the compound as an imidazole-based alkaloid cation with chemical formula 11-[3-(formylamino)propyl]-1,2,3,4,6,7,8,9-octahydrodipyrido [1,2-a:1',2'-dimidazole-5-ium. In experiments, Nocardioides sp. strain MW5 transformed 34% of ritalinic acid into this metabolite, while 52% was mineralized into CO2. Alkaloid was not biodegraded during the OECD 301 F test. This study provides new insight into the environmental fate of methylphenidate and its metabolites. The data collected are essential for assessing nootropic drug consumption by sewage epidemiology and should lead to a better understanding of microbial degradation of ritalinic acid.

Piracetam (2-oxo-1-pyrrolidine acetamide) is a popular cognitive enhancer, which has recently been detected in waste and drinking water. Nootropic drugs are designed to affect human metabolism and act on the nervous system, but their environmental effects have yet to be the subject of detailed studies. In this report, we present the efficient biodegradation of the cognitive enhancer, piracetam. Two bacterial strains capable of using this compound as the sole carbon source were isolated and later identified as Ochrobactrum anthropi strain MW6 and Ochrobactrum intermedium strain MW7. The compound's mineralization and the cleavage of the heterocyclic ring were shown in the experiments with 14C-labeled piracetam. This is also the first report of a pharmaceutical's degradation by the Ochrobactrum genus. This study presents model microorganisms that can be used in further investigation of piracetam's degradation pathways as well as enzymes and genes involved in the process.

Methylphenidate is widely used as a medication for the treatment of attention deficit hyperactivity disorder (ADHD) in children. Less than 1% of methylphenidate is excreted unchanged in urine, while 80% of an oral dose is excreted as ritalinic acid (which is reportedly poorly degradable). This study aims to investigate the biotransformation of ritalinic acid by free and immobilized enzymes. The influence of various laccase mediators on biotransformation efficiency has been tested. Formation of the main transformation products has been monitored and their potential structures suggested. The effective transformation of ritalinic acid was observed only in the presence of 2,2,6,6-tetramethylpiperidine 1-oxyl mediator (TEMPO). The most effective enzyme was the laccase of T. versicolor 159. The main transformation product was an N-methyl derivative of ritalinic acid. Ritalinic acid was also reduced to aldehyde and alcohol, and a broad spectrum of intermediate complexes with oxoammonium ion of TEMPO were detected. This is the first time the biotransformation of ritalinic acid has been investigated in detail.

In the last decade, biological degradation and mineralization of antibiotics have been increasingly reported feats of environmental bacteria. The most extensively described example is that of sulfonamides that can be degraded by several members of Actinobacteria and Proteobacteria. Previously, we reported sulfamethoxazole (SMX) degradation and partial mineralization by Achromobacter denitrificans strain PR1, isolated from activated sludge. However, further studies revealed an apparent instability of this metabolic trait in this strain. Here, we investigated this instability and describe the finding of a low-abundance and slow-growing actinobacterium, thriving only in co-culture with strain PR1. This organism, named GP, shared highest 16S rRNA gene sequence similarity (94.6–96.9%) with the type strains of validly described species of the genus Leucobacter. This microbial consortium was found to harbor a homolog to the sulfonamide monooxygenase gene (sadA) also found in other sulfonamide-degrading bacteria. This gene is overexpressed in the presence of the antibiotic, and evidence suggests that it codes for a group D flavin monooxygenase responsible for the ipso-hydroxylation of SMX. Additional side reactions were also detected comprising an NIH shift and a Baeyer–Villiger rearrangement, which indicate an inefficient biological transformation of these antibiotics in the environment. This work contributes to further our knowledge in the degradation of this ubiquitous micropollutant by environmental bacteria.

Nano-scale zero-valent iron (nZVI) began attracting research attention in remediation practice in recent decades as a prospective nanomaterial applicable to various contaminated matrices. Despite concerns about the negative effects of nanomaterials on ecosystems, the number of reliable toxicity tests is limited. We have developed a test based on the evaluation of oxidative stress (OS). The test employed the analysis of a typical OS marker (malondialdehyde, MDA), after exposure of six bacterial strains to the tested nanomaterial. We also attempted to use other OS and cell membrane damage assays, including the determination of glutathione and lactate dehydrogenase, respectively. However, we found that the components of these assays interfered with nZVI; therefore, these tests were not applicable. The MDA assay was tested using nZVI and three newly engineered oxide shell nZVI materials with different oxide thicknesses. Six different bacterial species were employed, and the results showed that the test was fully applicable for the concentrations of nanomaterials used in remediation practice (0.1-10 g/L). MDA was produced in a dose-response manner, and the bacteria showed a similar response toward pure pyrophoric nZVI, reaching EC50 values of 0.3-1.1 g/L. We observed different responses in the absolute production of MDA; however, the MDA concentrations were correlated with the cell membrane surfaces of the individual strains (R > 0.75; P < 0.09). Additionally, the EC50 values correlated with the thickness of the oxide shells (except for Escherichia coli: R > 0.95; P < 0.05), documenting the reliability of the assay, where reactivity was confirmed to be an important factor for reactive oxygen species production.

The objective of this work was to test the PCB-degrading abilities of two white-rot fungi, namely Pleurotus ostreatus and Irpex lacteus, in real contaminated soils with different chemical properties and autochthonous microflora. In addition to the efficiency in PCB removal, attention was given to other important parameters, such as changes in the toxicity and formation of PCB transformation products. Moreover, structural shifts and dynamics of both bacterial and fungal communities were monitored using next-generation sequencing and phospholipid fatty acid analysis. The best results were obtained with P. ostreatus, which resulted in PCB removals of 18.5, 41.3 and 50.5% from the bulk, top (surface) and rhizosphere, respectively, of dumpsite soils after 12 weeks of treatment. Numerous transformation products were detected (hydoxylated and methoxylated PCBs, chlorobenzoates and chlorobenzyl alcohols), which indicates that both fungi were able to oxidize and decompose the aromatic moiety of PCBs in the soils. Microbial community analysis revealed that P. ostreatus efficiently colonized the soil samples and suppressed other fungal genera. However, the same fungus substantially stimulated bacterial taxa that encompass putative PCB degraders. The results of this study finally demonstrated the feasibility of using this fungus for possible scaled-up bioremediation applications.

The consumption of nootropic drugs has increased tremendously in the last decade, though the studies on their environmental fate are still scarce. Nootropics are bioactive compounds designed to alter human's physiology therefore the adverse effects towards wildlife can be expected. In order to understand their environmental impact, the knowledge on their transformation pathways is necessary. Methylphenidate belongs to the most prescribed neuro-enhancers and is among the most favored smart drugs used in non-medical situations. It is metabolized in human liver and excreted as ritalinic acid. Here, we showed for the first time that ritalinic acid can be biodegraded and used as a sole carbon and nitrogen source by various microbial strains originating from different environmental samples. Five axenic strains were isolated and identified as: Arthrobacter sp. strain MW1, MW2 and MW3, Phycicoccus sp. strain MW4 and Nocardioides sp. strain MW5. Our research provides the first insight into the metabolism of ritalinic acid and suggests that it may differ depending on the strain and growth conditions, especially on availability of nitrogen. The isolates obtained in this study can serve as model organisms in further studies on the catabolism of ritalinic acid and methylphenidate but potentially also other compounds with similar structures. Our findings have important implication for the sewage epidemiology. We demonstrated that ritalinic acid is subject to quick and efficient biodegradation thus its use as a stable biomarker should be reconsidered.

The feasibility of decontaminating creosote-treated wood (CTW) by co-composting with agricultural wastes was investigated using two bulking agents, grass cuttings (GC) and broiler litter (BL), each employed at a 1:1 ratio with the matrix. The initial concentration of total polycyclic aromatic hydrocarbons (PAHs) in CTW (26,500 mg kg(-1)) was reduced to 3 and 19% after 240 d in GC and BL compost, respectively. PAH degradation exceeded the predicted bioaccesible threshold, estimated through sequential supercritical CO2 extraction, together with significant detoxification, assessed by contact tests using Vibrio fisheri and Hordeum vulgare. GC composting was characterized by high microbial biomass growth in the early phases, as suggested by phospholipid fatty acid analyses. Based on the 454-pyrosequencing results, fungi (mostly Saccharomycetales) constituted an important portion of the microbial community, and bacteria were characterized by rapid shifts (from Firmicutes (Bacilli) and Actinobacteria to Proteobacteria). However, during BL composting, larger amounts of prokaryotic and eukaryotic PLFA markers were observed during the cooling and maturation phases, which were dominated by Proteobacteria and fungi belonging to the Ascomycota and those putatively related to the Glomeromycota. This work reports the first in-depth analysis of the chemical and microbiological processes that occur during the co-composting of a PAH-contaminated matrix.

The removal of aged hydrophobic contaminants from fine-textured soils is a challenging issue in remediation. The objective of this study was to compare the efficacy of augmentation treatments to that of biostimulation in terms of total aliphatic hydrocarbon (TAH) and toxicity removal from a historically contaminated clay soil and to assess their impact on the resident microbial community. To this aim, Pleurotus ostreatus, Botryosphaeria rhodina and a combination of both were used as the inoculants while the addition of a sterilized lignocellulose mixture to soil (1:5, w/w) was used as a biostimulation approach. As opposed to the non-amended control soil, where no changes in TAH concentration and residual toxicity were observed after 60days, the activation of specialized bacteria was found in the biostimulated microcosms resulting in significant TAH removal (79.8%). The bacterial community structure in B. rhodina-augmented microcosms did not differ from the biostimulated microcosms due to the inability of the fungus to be retained within the resident microbiota. Best TAH removals were observed in microcosms inoculated with P. ostreatus alone (Po) and in binary consortium with B. rhodina (BC) (86.8 and 88.2%, respectively). In these microcosms, contaminant degradation exceeded their bioavailability thresholds determined by sequential supercritical CO2 extraction. Illumina metabarcoding of 16S rRNA gene showed that the augmentation with Po and BC led to lower relative abundances of Gram(+) taxa, Actinobacteria in particular, than those in biostimulated microcosms. Best detoxification, with respect to the non-amended incubation control, was found in Po microcosms where a drop in collembola mortality (from 90 to 22%) occurred. At the end of incubation, in both Po and BC, the relative abundances of P. ostreatus sequences were higher than 60% thus showing the suitability of this fungus in bioaugmentation-based remediation applications.

The aim of this study was to evaluate the ability of bacterial cultures isolated from cattle, poultry or pig faeces and manure to produce rhamnolipids, as well as to investigate the influence of interspecies communication on possible quantitative differences in the production of rhamnolipid congeners. Initial screening methods (oil spreading, drop collapse, haemolytic activity and emulsification activity) showed that approximately 36% of the 51 isolated cultures exhibited the ability to produce biosurfactants. Subsequent studies using a selected culturable mixed culture (which included Enterococcus faecalis, Pseudomonas aeruginosa and Escherichia coli) revealed that only P. aeruginosa was able to produce this biosurfactant. HPLC–MS analysis showed that the surface active compounds were rhamnolipids. Further comparative studies confirmed that the total yield of rhamnolipids was notably higher in the bioreactor inoculated with the selected mixed culture (940.58 ± 1.10 mg/L) compared to the bioreactor inoculated with the axenic strain of P. aeruginosa (108.47 ± 0.41 mg/L). Twelve rhamnolipid congeners were identified during cultivation of the selected mixed culture, whereas six congeners were detected during cultivation of the sole axenic strain of P. aeruginosa. Furthermore, increased production of rhamnolipids was observed when the concentration of autoinducer molecules (AI-2) responsible for interspecies signaling increased, suggesting the influence of quorum-sensing communication on biosynthesis efficiency. This observation may be of importance for large-scale production of this biosurfactant, as it opens new possible solutions based on the use of mixed cultures or external addition of stimulating autoinducers.

Lignin, a complex three-dimensional amorphous polymer, is considered to be a potential natural renewable resource for the production of low-molecular-weight aromatic compounds. In the present study, a novel sequential lignin treatment method consisting of a biocatalytic oxidation step followed by a formic acid-induced lignin depolymerization step was developed and optimized using response surface methodology. The biocatalytic step employed a laccase mediator system using the redox mediator 1-hydroxybenzotriazole. Laccases were immobilized on superparamagnetic nanoparticles using a sorption-assisted surface conjugation method allowing easy separation and reuse of the biocatalysts after treatment. Under optimized conditions, as much as 45 wt% of lignin could be solubilized either in aqueous solution after the first treatment or in ethyl acetate after the second (chemical) treatment. The solubilized products were found to be mainly low-molecular-weight aromatic monomers and oligomers. The process might be used for the production of low-molecular-weight soluble aromatic products that can be purified and/or upgraded applying further downstream processes.

The degradation of polycyclic aromatic hydrocarbons (PAHs) including fluorene, phenanthrene, anthracene, fluoranthene and pyrene were investigated under thermophilic and mesophilic anaerobic-methanogenic conditions. By using central composite design, the impact of PAH concentration and soil to inocula ratio were systematically studied for their influence on PAH removal and biogas production. During the first 30 days, PAH concentration decreased in all samples indicating that the inocula used were able to biodegrade the contaminants; although an inhibition effect was initially observed in biogas production. Phospholipids fatty acids analysis was used to monitor the microbial communities present during the process. These communities were more complex in samples containing moderate to high PAH contamination concentrations. After 50 days, the concentration of PAHs increased in the majority of samples indicating possible reversed biotransformation of these compounds.

A group of white rot fungi (Irpex lacteus, Panus tigrinus, Dichomitus squalens, Trametes versicolor and Pleurotus ostreatus) was investigated for the biodegradation of norfloxacin (NOR), ofloxacin (OF) and ciprofloxacin (CIP). The selected fluoroquinolones were readily degraded almost completely by I. lacteus and T. versicolor within 10 and 14 d of incubation in liquid medium, respectively. The biodegradation products were identified by liquid chromatography-mass spectrometry. The analyses indicated that the fungi use similar mechanisms to degrade structurally related antibiotics. The piperazine ring of the molecules is preferably attacked via either substitution or/and decomposition. In addition to the degradation efficiency, attention was devoted to the residual antibiotic activities estimated using Gram-positive and Gram-negative bacteria. Only I. lacteus was able to remove the antibiotic activity during the course of the degradation of NOR and OF. The product-effect correlations evaluated by Principal Component Analysis (PCA) enabled elucidation of the participation of the individual metabolites in the residual antibacterial activity. Most of the metabolites correlated with the antibacterial activity, explaining the rather high residual activity remaining after the biodegradation. PCA of ligninolytic enzyme activities indicated that manganese peroxidase might participate in the degradation.

Sorption of hydrophobic pollutants on organics in soil causes a substantial decrease in pollutant availability for transport and transformation processes. The pollutants gradually become unavailable for organisms, which can result in a reduction of ecotoxicity but also in a decrease in biodegradation efficiency. This review describes various biological and chemical methods that can be used for determination of bioaccessible or bioavailable compounds. The biological methods make it possible to study bioaccumulation or to measure toxicity of the pollutants. The chemical methods are based on incomplete extraction of the pollutants from the contaminated soil. The bioaccessible portion can be estimated at the equilibrium stage. Other and probably more appropriate approaches are represented by sequential dynamic methods modelling two-site desorption kinetic curves of pollutants which can be used for relatively precise and reliable assessment of the bioaccessibility (bioavailability) parameter.

Polychlorinated biphenyl (PCB) organohalide-respiring communities from the efflux channel of a former Delor manufacturer in Eastern Slovakia were assessed using metagenomic, statistical and cultivation-adapted approaches. Multivariate analysis of environmental factors together with terminal restriction fragment length polymorphisms of the bacterial communities in the primary sediments revealed both temporal and spatial heterogeneity in the distribution of microbial populations, which reflects the dynamic pattern of contamination and altered conditions for biodegradation activity along the channel. Anaerobic microcosms were developed from eight sediments sampled along the channel, where high concentrations of PCBs – from 6.6 to 136mg/kg dry weight, were measured. PCB dehalorespiring activity, congruent with changes in the microbial composition in all microcosms, was detected. After 10 months of cultivation, the divergently evolved consortia achieved up to 35.9 percent reduction of the total PCB concentration. Phylogenetic-analysis of the active Chloroflexi-related organohalide-respiring bacteria by partial sequencing of 16S rRNA genes in cDNA from microcosms with the highest PCB dechlorination activity revealed diverse and unique complexity of the populations. The predominant organohalide respirers were either affiliated with Dehalococcoides sp. and Dehalococcoides-like group (DLG) organisms or were composed of currently unknown distant clades of DLG bacteria. The present study should encourage researchers to explore the full potential of the indigenous PCB dechlorinating populations to develop effective bioremediation approaches that can perform the complete mineralization of PCBs in polluted environments.

The present work was aimed at isolating and identifying the main members of the mycobiota of a clay soil historically contaminated by mid- and long-chain aliphatic hydrocarbons (AH) and to subsequently assess their hydrocarbon-degrading ability. All the isolates were Ascomycetes and, among them, the most interesting was Pseudoallescheria sp. 18A, which displayed both the ability to use AH as the sole carbon source and to profusely colonize a wheat straw:poplar wood chip (70:30, w/w) lignocellulosic mixture (LM) selected as the amendment for subsequent soil remediation microcosms. After a 60 d mycoaugmentation with Pseudoallescheria sp. of the aforementioned soil, mixed with the sterile LM (5:1 mass ratio), a 79.7% AH reduction and a significant detoxification, inferred by a drop in mortality of Folsomia candida from 90 to 24%, were observed. However, similar degradation and detoxification outcomes were found in the non-inoculated incubation control soil that had been amended with the sterile LM. This was due to the biostimulation exerted by the amendment on the resident microbiota, fungi in particular, the activity and density of which were low, instead, in the non-amended incubation control soil.