Bioremediation, involving bioaugmentation and/or biostimulation, being an economical and eco-friendly approach, has emerged as the most advantageous soil and water clean-up technique for contaminated sites containing heavy metals and/or organic pollutants. Addition of pre-grown microbial cultures to enhance the degradation of unwanted compounds (bioaugmentation) and/or injection of nutrients and other supplementary components to the native microbial population to induce propagation at a hastened rate (biostimulation), are the most common approaches for in situ bioremediation of accidental spills and chronically contaminated sites worldwide. However, many factors like strain selection, microbial ecology, type of contaminant, environmental constraints, as well as procedures of culture introduction, may lead to their failure. These drawbacks, along with fragmented literature, have opened a gap between laboratory trials and on-field application. The present review discusses the effectiveness as well as the limitations of bioaugmentation and biostimulation processes. A summary of experimental studies both in confined systems under controlled conditions and of real case studies in the field is presented. A comparative account between the two techniques and also the current scenario worldwide for in situ biotreatment using bioaugmentation and biostimulation, are addressed.
Pharmaceuticals and personal care products (PPCPs) have been the focus of much recent research as concerns rise about their occurrence in bodies of water worldwide. In an effort to characterize the risk and determine the prevalence of these micropollutants in lakes and rivers, many researchers are examining PPCP removal from impaired water during wastewater treatment and water recycling (soil passage) processes. Biodegradation studies and projects considering combinations of biodegradation and other removal processes have been conducted over a wide range of compound categories and therapeutic classes, as well as across different systems and scales of study. This review summarizes the extent of PPCP removal observed in these various systems.
Increasing discharge and improper management of liquid and solid industrial wastes have created a great concern among industrialists and the scientific community over their economic treatment and safe disposal. White rot fungi (WRF) are versatile and robust organisms having enormous potential for oxidative bioremediation of a variety of toxic chemical pollutants due to high tolerance to toxic substances in the environment. WRF are capable of mineralizing a wide variety of toxic xenobiotics due to non-specific nature of their extracellular lignin mineralizing enzymes (LMEs). In recent years, a lot of work has been done on the development and optimization of bioremediation processes using WRF, with emphasis on the study of their enzyme systems involved in biodegradation of industrial pollutants. Many new strains have been identified and their LMEs isolated, purified and characterized. In this review, we have tried to cover the latest developments on enzyme systems of WRF, their low molecular mass mediators and their potential use for bioremediation of industrial pollutants.
Partial nitrification to nitrite (nitritation) can be achieved in a continuous process without sludge retention by wash out of nitrite oxidising bacteria (NOB) while retaining ammonia oxidising bacteria (AOB), at elevated temperatures (the SHARON process) and, as demonstrated in this paper, also at low dissolved oxygen (DO) concentrations. Enriched AOB was attained at a low DO concentration (0.4 mg l−1) and a dilution rate of 0.42 day−1 in a continuous process. A higher oxygen affinity of AOB compared to NOB seemed critical to achieving this. This was verified by determining the oxygen half saturation constant, K o, with similar oxygen mass transfer resistances for enriched AOB and NOB as 0.033 ± 0.003 mg l−1 and 0.43 ± 0.08 mg l−1, respectively. However, the extent of nitritation attained was found to be highly sensitive to process upsets.
The enrichment culture SL2 dechlorinating tetrachloroethene (PCE) to ethene with strong trichloroethene (TCE) accumulation prior to cis-1,2-dichloroethene (cis-DCE) formation was analyzed for the presence of organohalide respiring bacteria and reductive dehalogenase genes (rdhA). Sulfurospirillum-affiliated bacteria were identified to be involved in PCE dechlorination to cis-DCE whereas “Dehalococcoides”-affiliated bacteria mainly dechlorinated cis-DCE to ethene. Two rdhA genes highly similar to tetrachloroethene reductive dehalogenase genes (pceA) of S. multivorans and S. halorespirans were present as well as an rdhA gene very similar to the trichloroethene reductive dehalogenase gene (tceA) of “Dehalococcoides ethenogenes” strain 195. A single strand conformation polymorphism (SSCP) method was developed allowing the simultaneous detection of the three rdhA genes and the estimation of their abundance. SSCP analysis of different SL2 cultures showed that one pceA gene was expressed during PCE dechlorination whereas the second was expressed during TCE dechlorination. The tceA gene was involved in cis-DCE dechlorination to ethene. Analysis of the internal transcribed spacer region between the 16S and 23S rRNA genes revealed two distinct sequences originating from Sulfurospirillum suggesting that two Sulfurospirillum populations were present in SL2. Whether each Sulfurospirillum population was catalyzing a different dechlorination step could however not be elucidated.
Little is known of the attenuation of chemical mixtures created for hydraulic fracturing within the natural environment. A synthetic hydraulic fracturing fluid was developed from disclosed industry formulas and produced for laboratory experiments using commercial additives in use by Marcellus shale field crews. The experiments employed an internationally accepted standard method (OECD 301A) to evaluate aerobic biodegradation potential of the fluid mixture by monitoring the removal of dissolved organic carbon (DOC) from an aqueous solution by activated sludge and lake water microbial consortia for two substrate concentrations and four salinities. Microbial degradation removed from 57 % to more than 90 % of added DOC within 6.5 days, with higher removal efficiency at more dilute concentrations and little difference in overall removal extent between sludge and lake microbe treatments. The alcohols isopropanol and octanol were degraded to levels below detection limits while the solvent acetone accumulated in biological treatments through time. Salinity concentrations of 40 g/L or more completely inhibited degradation during the first 6.5 days of incubation with the synthetic hydraulic fracturing fluid even though communities were pre-acclimated to salt. Initially diverse microbial communities became dominated by 16S rRNA sequences affiliated with Pseudomonas and other Pseudomonadaceae after incubation with the synthetic fracturing fluid, taxa which may be involved in acetone production. These data expand our understanding of constraints on the biodegradation potential of organic compounds in hydraulic fracturing fluids under aerobic conditions in the event that they are accidentally released to surface waters and shallow soils.
Due to their amphipathic nature, biosurfactants are multifunctional molecules that have considerable potential in several industries, especially the petroleum industry. In this study, the commercial production of a biosurfactant from Pseudomonas cepacia CCT6659 grown on industrial waste was investigated in a semi-industrial 50-L bioreactor for use in the removal of hydrocarbons from oily effluents. A concentration of 40.5 g/L was achieved in the scale up and the surface tension was reduced to 29 mN/m. The biosurfactant was formulated with an added preservative, tyndallization and the combination of fluent vaporization plus the preservative. Formulated biosurfactant samples were stored for 120 days. Tensioactive properties and stability were evaluated with different pH values, temperatures and salt concentrations. The commercial biosurfactant obtained with all formulation methods demonstrated good stability, with tolerance to a wide range of pH values as well as high temperature and high salinity, enabling application in extreme environmental conditions, as it occurs in industrial plants. The biosurfactant proved to be economically viable for large-scale application, as demonstrated by the cost of the product, estimated at around US$ 0.14–0.15/L and US$ 0.02/g for the formulated and the isolated biosurfactant, respectively. Both products were applied in an oil-fired thermoelectric plant for the treatment of oily effluents and removed up to 100% of the oil. Therefore, this biosurfactant is suitable for application under extreme conditions, such as in the petroleum industry, and can be produced at a more attractive price compared to other commercially available products on the market.
The 16S rRNA sequence and biochemical characteristics revealed the isolated organism as Pseudomonas sp. SU-EBT. This strain showed 97 and 90% decolorization of a recalcitrant dye, Congo red (100 mg l−1) and textile industry effluent with 50% reduction in COD within 12 and 60 h, respectively. The optimum pH and temperature for the decolorization was 8.0 and 40°C, respectively. Pseudomonas sp. SU-EBT was found to tolerate the dye concentration up to 1.0 g l−1. Significant induction in the activity of intracellular laccase suggested its involvement in the decolorization of Congo red. The metabolites formed after decolorization of Congo red, such as p-dihydroxy biphenyl, 8-amino naphthol 3-sulfonic acid and 3-hydroperoxy 8-nitrosonaphthol were characterized using FTIR and GC–MS. Phytotoxicity study revealed nontoxic nature of the degradation metabolites to Sorghum bicolor, Vigna radiata, Lens culinaris and Oryza sativa plants as compared to Congo red and textile industry effluent. Pseudomonas sp. SU-EBT decolorized several individual textile dyes, dye mixtures and textile industry effluent, thus it is a useful strain for the development of effluent treatment methods in textile processing industries.
Laccase from Myceliophthora thermophila was covalently immobilised on Eupergit C and Eupergit C 250L yielding specific activities of up to 17 and 80 U/g, respectively. Due to its superior activity, Eupergit C 250L was chosen for further research. The somewhat lower catalytic efficiency (based on the ratio between the turnover number and the Michaelis constant, kcat/KM) of the immobilised enzyme in comparison with that of the free enzyme was balanced by its increased stability and broader operational window related to temperature and pH. The feasibility of the immobilised laccase was tested by using a packed bed reactor (PBR) operating in consecutive cycles for the removal of Acid Green 27 dye as model substrate. High degrees of elimination were achieved (88, 79, 69 and 57% in 4 consecutive cycles), while the levels of adsorption on the support varied from 18 to 6%, proving that dye removal took place mainly due to the action of the enzyme. Finally, a continuous PBR with the solid biocatalyst was applied for the treatment of a solution containing the following endocrine disrupting chemicals: estrone (E1), 17β-estradiol (E2) and 17α-ethinylestradiol (EE2). At steady-state operation, E1 was degraded by 65% and E2 and EE2 were removed up to 80% and only limited adsorption of these compounds on the support, between 12 and 22%, was detected. In addition, a 79% decrease in estrogenic activity was detected in the effluent of the enzymatic reactor while only 14% was attained by inactivated laccase.
The biodegradation of waters polluted by some bisphenols, endowed with endocrine activity, has been studied by means of laccase or tyrosinase immobilized on polyacrylonitrile (PAN) beads. Bisphenol A (BPA), Bisphenol B (BPB), Bisphenol F (BPF) and Tetrachlorobisphenol A (TCBPA) have been used. The laccase-PAN beads system has been characterized as a function of pH, temperature and substrate concentration. The biochemical parameters so obtained have been compared with those of the free enzyme to evidence the modification induced by the immobilization process. Once characterized, the laccase-PAN beads have been employed in a fluidized bed reactor to determine for each of the four bisphenols the degradation rate constant (k); the τ50, i.e., the time to obtain the 50% of degradation, and the removal efficiency (RE90) after 90 min of enzyme treatment. The same parameters have been measured for each of the four pollutants with the same fluidized bed bioreactor loaded with tyrosinase-PAN beads. The internal comparison, i.e., in each of the two catalytic systems, has shown that both enzymes exhibit a removal efficiency in the following order BPF>BPA>BPB>TCBPA. The external comparison, i.e., the comparison between the two catalytic system, has shown that the catalytic power of laccase were higher than that of tyrosinase. The operational stability of both catalytic systems resulted excellent, since they maintained more than 80% of the initial activity after 30 days of work.
A bacterial consortium (consortium GR) consisting of Proteus vulgaris NCIM-2027 and Micrococcus glutamicus NCIM-2168 could rapidly decolorize and degrade commonly-used sulfonated reactive dye Green HE4BD and many other reactive dyes. Consortium GR shows markedly higher decolorization activity than that of the individual strains. The preferable physicochemical parameters were identified to achieve higher dye degradation and decolorization efficiency. The supplementation of cheap co-substrates (e.g., extracts of agricultural wastes) could enhance the decolorization performance of consortium GR. Extent of mineralization was determined with TOC and COD measurements, showing nearly complete mineralization of Green HE4BD by consortium GR (up to 90% TOC and COD reduction) within 24 h. Oxidoreductive enzymes seemed to be involved in fast decolorization/degradation process with the evidence of enzymes induction in the bacterial consortium. Phytotoxicity and microbial toxicity studies confirm that the biodegraded products of Green HE4BD by consortium GR are non-toxic. Consortium GR also shows significant biodegradation and decolorization activities for mixture of reactive dyes as well as the effluent from actual dye manufacturing industry. This confers the possibility of applying consortium GR for the treatment of industrial wastewaters containing dye pollutants.
The bacterial strain F4, isolated from olive oil-contaminated soil, has been found to produce biosurfactants as confirmed by oil displacement test and the emulsification index results. The identification of the strain F4, by 16S ribosomal RNA gene, showed a close similarity to Bacillus safensis, therefore the strain has been termed Bacillus safensis F4. The Thin Layer Chromatography (TLC) and the High Pressure Liquid Chromatography-Mass Spectrometry (HPLC–MS/MS) demonstrated that the biosurfactant had a lipopeptide structure and was classified as surfactin. The present study showed also that the produced biosurfactant has an important antibacterial activity against several pathogen strains as monitored with minimum inhibitory concentration (MIC) micro-assays. In particular, it presented an interesting anti-planktonic activity with a MIC of 6.25 mg mL−1 and anti-adhesive activity which exceeded 80% against the biofilm-forming Staphylococcus epidermidis S61 strain. Moreover, the produced lipopeptide showed an antitumor activity against T47D breast cancer cells and B16F10 mouse melanoma cells with IC50 of 0.66 mg mL−1 and 1.17 mg mL−1, respectively. Thus, our results demonstrated that Bacillus safensis F4 biosurfactant exhibited a polyvalent activity via a considerable antibiofilm and antitumoral potencies.
A new arsenite-oxidizing bacterium was isolated from a low arsenic-containing (8.8 mg kg−1) soil. Phylogenetic analysis based on 16S rRNA gene sequencing indicated that the strain was closely related to Stenotrophomonas panacihumi. Batch experiment results showed that the strain completely oxidized 500 μM of arsenite to arsenate within 12 h of incubation in a minimal salts medium. The optimum initial pH range for arsenite oxidation was 5–7. The strain was found to tolerate as high as 60 mM arsenite in culture media. The arsenite oxidase gene was amplified by PCR with degenerate primers. The deduced amino acid sequence showed the highest identity (69.1 %) with the molybdenum containing large subunit of arsenite oxidase derived from Bosea sp. Furthermore the amino acids involved in binding the substrate arsenite, were conserved with the arsenite oxidases of other arsenite oxidizing bacteria such as Alcaligenes feacalis and Herminnimonas arsenicoxydans. To our knowledge, this study constitutes the first report on arsenite oxidation using Stenotrophomonas sp. and the strain has great potential for application in arsenic remediation of contaminated water.
Ionic liquids (ILs) are novel organic salts that have enormous potential for industrial use as green replacements for harmful volatile organic solvents. Varying the cationic components can alter the chemical and physical properties of ILs, including solubility, to suit a variety of industrial processes. However, to complement designer engineering, it is crucial to proactively characterize the biological impacts of new chemicals, in order to fully define them as environmentally friendly. Before introduction of ILs into the environment, we performed an analysis of the biodegradability of six ILs by activated sludge microorganisms collected from the South Bend, Indiana wastewater treatment plant. We examined biodegradability of 1-butyl, 1-hexyl and 1-octyl derivatives of 3-methyl-imidazolium and 3-methyl-pyridinium bromide compounds using the standard Organisation for Economic Cooperation and Development dissolved organic carbon Die-Away Test, changes in total dissolved nitrogen concentrations, and 1H-nuclear magnetic resonance analysis of initial and final chemical structures. Further, we examined microbial community profiles throughout the incubation period using denaturing gradient gel electrophoresis (DNA-PCR-DGGE). Our results suggest that hexyl and octyl substituted pyridinium-based ILs can be fully mineralized, but that imidazolium-based ILs are only partially mineralized. Butyl substituted ILs with either cation, were not biodegradable. Biodegradation rates also increase with longer alkyl chain length, which may be related to enhanced selection of a microbial community. Finally, DGGE analysis suggests that certain microorganisms are enriched by ILs used as a carbon source. Based on these results, we suggest that further IL design and synthesis include pyridinium cations and longer alkyl substitutions for rapid biodegradability.
Response surface methodology was applied to optimize the production of biosurfactants from Bacillus mojavensis I4 using Box–Behnken design with four variables. The optimal variable combination was 3% of glucose as carbon source, 0.6% of glutamic acid as nitrogen source, temperature of 35 °C and 10 g/l of NaCl which yielded to an optimal production of 4.12 g/l. Compositional analysis and FTIR spectrum revealed that the extracted biosurfactants was a lipopeptides. The biosurfactants achieved a critical micelle concentration value of 100 mg/l. Moreover, the extracted biosurfactants were effective at recovering up to 89.2% of motor oil from sand beach and achieved a dispersion rate of 78% of the initial diameter of the oil. These findings suggested the potential use of I4 biosurfactants in the oil industry.
Biological properties of ionic liquids (ILs) have been usually tested with the help of standard biodegradation or ecotoxicity tests. So far, several articles on the identification of intermediate metabolites of microbiological decay of ILs have been published. Simultaneously, the number of novel ILs with unrecognized characteristics regarding biodegradability and effect on organisms and environment is still increasing. In this work, seven imidazolium ionic liquids of different chemical structure were studied. Three of them are 1-alkyl-3-methyl-imidazolium bromides, while the other four are tetra- or completely substituted imidazolium iodides. This study focused on the identification of intermediate metabolites of the aforementioned ionic liquids subjected to biodegradation in a laboratory activated sludge system. Both fully substituted ionic liquids and 1-ethyl-3-methyl-imidazolium bromide were barely biodegradable. In the case of two of them, no biotransformation products were detected. The elongation of the alkyl side chain made the IL more susceptible for microbiological decomposition. 1-Decyl-3-methyl-imidazolium bromide was biotransformed most easily. Its primary biodegradation up to 100 % could be achieved. Nevertheless, the cleavage of the imidazolium ring has not been observed.
The indigenous microorganisms responsible for degrading phenanthrene (PHE) in activated biosludge were identified using DNA-based stable isotope probing. Besides the well-known PHE degraders Burkholderia, Ralstonia, Sinobacteraceae and Arthrobacter, we for the first time linked the taxa Paraburkholderia and Kaistobacter with in situ PHE biodegradation. Analysis of PAH-RHD alpha gene detected in the heavy DNA fraction of C-13-PHE treatment suggested the mechanisms of horizontal gene transfer or inter-species hybridisation in PAH-RHD gene spread within the microbial community. Additionally, three cultivable PHE degraders, Microbacterium sp. PHE-1, Rhodanobacter sp. PHE-2 and Rhodococcus sp. PHE-3, were isolated from the same activated biosludge. Among them, Rhodanobacter sp. PHE-2 is the first identified strain in its genus with PHE-degrading ability. However, the involvement of these strains in PHE degradation in situ was questionable, due to their limited enrichment in the heavy DNA fraction of C-13-PHE treatment and lack of PAH-RHD alpha gene found in these isolates. Collectively, our findings provide a deeper understanding of the diversity and functions of indigenous microbes in PHE degradation.
To obtain a restoring and protective calcite layer on degraded limestone, five different strains of the Bacillus sphaericus group and one strain of Bacillus lentus were tested for their ureolytic driven calcium carbonate precipitation. Although all the Bacillus strains were capable of depositing calcium carbonate, differences occurred in the amount of precipitated calcium carbonate on agar plate colonies. Seven parameters involved in the process were examined: calcite deposition on limestone cubes, pH increase, urea degrading capacity, extracellular polymeric substances (EPS)-production, biofilm formation, ζ-potential and deposition of dense crystal layers. The strain selection for optimal deposition of a dense CaCO3 layer on limestone, was based on decrease in water absorption rate by treated limestone. Not all of the bacterial strains were effective in the restoration of deteriorated Euville limestone. The best calcite precipitating strains were characterised by high ureolytic efficiency, homogeneous calcite deposition on limestone cubes and a very negative ζ-potential.
The current research focuses on the production and characterization of glycolipid biosurfactant (GB) from Pseudomonas plecoglossicida and its anthelmintic activity against Caenorhabditis elegans. The GB was purified and characterized by Fourier Transform Infrared Spectroscopy (FTIR) and Gas Chromatography and Mass Spectrometry (GC–MS) analysis. Anthelmintic activity of GB was studied at six different pharmacological doses from 10 to 320 µg/mL on C. elegans. Exposure of different developmental stages (L1, L2, L3, L4 and adult) of C. elegans to the GB reduced the survivability of worms in a dose and time-dependent manner. Adult and L4 worms were least susceptible, while L1, L2 and L3 were more susceptible to GB when compared to the untreated control. An increased exposure period drastically reduced the survival rate of worms and reduction in LC50 value. The GB significantly inhibited the development of C. elegans with an IC50 value of 53.14 µg/mL and even reduced the adult body length and egg hatching. Fecundity rate of the worms treated with GB at 20, 40 and 80 µg/mL decreased from 261.90 ± 3.21 to 239.70 ± 5.58, 164.20 ± 5.94 and 44.80 ± 6.22 eggs per worm, respectively. Besides the toxicological effects, prolonged exposure to GB significantly decreased (p ≤ 0.0001) the lifespan of wild type worms under standard laboratory conditions. Additionally, GB was found to be lethal towards ivermectin and albendazole resistant C. elegans strains. Overall, the data indicated that the GB extracted from P. plecoglossicida could be utilized for the control of non-susceptible and resistant gastrointestinal nematodes towards broad spectrum anthelmintic drugs, ivermectin and albendazole.
Pseudomonas strains isolated from oil contaminated soils were screened for biosurfactant production. Three out of eleven Pseudomonas isolates were selected for their high emulsifying activity (E24 value on n-hexadecane ~ 78%). These isolates (E39, E311 and E313) were identified as members of the P. putida group using phenotypical methods and a molecular approach. To identify the chemical nature of produced biosurfactants, thin layer chromatography and MALDI-ToF mass spectrometry analysis were carried out and revealed lipopeptides belonging to the syringafactin family. The activity of the produced biosurfactants was stable over a pH range of 6–12, at high salinity (10%) and after heating at 80 °C. Tests in contaminated sand micro-bioreactors showed that the three strains were able to degrade diesel. These results suggest the potential of these syringafactin producing strains for application in hydrocarbon bioremediation.