Evaluation of scale-up effect on cold-active enzyme production and biodegradation tests using pilot-scale bioreactors and a 3D soil tank.

Despite recent attention being paid to the biodegradation of petroleum hydrocarbons in cold environments, scale-up studies of biodegradation are lacking. Herein, the effect of scale-up on the enzymatic biodegradation of highly contaminated soil at low temperatures was studied. A novel cold-adapted bacteria (Arthrobacter sp. S2TR-06) was isolated that could produce cold-active degradative enzymes (xylene monooxygenase (XMO) and catechol 2,3-dioxygenase (C2,3D)). Enzyme production was investigated on 4 different scales (lab to pilot scale). The results showed a shorter fermentation time, and the highest production of enzymes and biomass (107 g/L for biomass, 109 U/mL, and 203 U/mL for XMO and C2,3D after 24 h) was achieved in the 150-L bioreactor due to enhanced oxygenation. Multi-pulse injection of p-xylene into the production medium was needed every 6 h. The stability of membrane-bound enzymes can be increased up to 3-fold by adding FeSO4 at 0.1% (w/v) before extraction. Soil tests also showed that biodegradation is scale-dependent. The maximum biodegradation rate decreased from 100% at lab-scale to 36% in the 300-L sand tank tests due to limited access of enzymes to trapped p-xylene in soil pores, low dissolved oxygen in the water-saturated zone, soil heterogeneity, and the presence of the free phase of p-xylene. The result demonstrated that formulation of enzyme mixture with FeSO4 and direct injection of enzyme mixture (third scenario) can increase the efficiency of bioremediation in heterogeneous soil. In this study, it was demonstrated that cold-active degradative enzyme production can be scaled up to an industrial scale and enzymatic treatment can be used to effectively bioremediate p-xylene contaminated sites. This study could provide key scale-up guidance for the enzymatic bioremediation of mono-aromatic pollutants in water-saturated soil under cold conditions.

Pilot-scale production and in-situ application of petroleum-degrading enzyme cocktail from Alcanivorax borkumensis.

Petroleum degrading enzymes can be used as an alternative way to improve petroleum bioremediation approaches. Alcanivorax borkumensis is an alkane-degrading bacteria that can produce petroleum degrading enzymes such as alkane hydroxylase and lipase. In this study, pilot-scale Alcanivorax borkumensis fermentation was developed for producing large volumes of petroleum degrading enzymes cocktail (∼900 L). Different process conditions, such as inoculum age 72 h and size 4% v/v, temperature 30 ± 1 °C, agitation speed at 150 rpm and, fermentation period 3 days were determined as the optimum for producing alkane hydroxylase and lipase activity. The oxygen transfer capacity was studied for obtaining better bacterial growth and higher enzyme activities in bioreactor process optimization as well as scale-up. Results showed that the maximum values of oxygen mass transfer coefficient (kLa), oxygen uptake rate (OUR), oxygen transfer rate (OTR), alkane hydroxylase, lipase, and cell count were 196.95 h-1, 0.92 mmol O2/L/h, 1.8 mmol O2/L/h, 222.49 U/mL, 325 U/mL, and 8.6 × 1010 CFU/mL, respectively. Compared with the bench-scale bioreactors, the 150 L fermenter showed a better oxygen transfer rate which affected the cell growth that doubled the number and enzymes production that increased. Then, the enzyme cocktail was used for a field test in a diesel source zone using a 5-spot well pattern. The results showed a significant reduction in concentrations of C10 - C50 (from 36% to > 99%) after one injection of enzyme cocktail, mainly for the contaminated soils located in the saturated zone of the unconfined aquifer. This study confirmed the scaling-up ofalkane-degrading enzyme production to an industrial-scale and its application for effective bioremediation of petroleum contaminated sites.

Enzymatic biodegradation of highly p-xylene contaminated soil using cold-active enzymes: A soil column study.

Enzymatic bioremediation is a sustainable and environment-friendly method for the clean-up of contaminated soil and water. In the present study, enzymatic bioremediation was designed using cold-active enzymes (psychrozymes) which catalyze oxidation steps of p-xylene biodegradation in highly contaminated soil (initial concentration of 13,000 mg/kg). The enzymes were obtained via co-culture of two psychrophilic Pseudomonas strains and characterized by kinetic studies and tandem LC-MS/MS. To mimic in situ application of enzyme mixture, bioremediation of p-xylene contaminated soil was carried out in soil column (140 mL) tests with the injection (3 pore volume) of different concentrations of enzyme cocktails (X, X/5, and X/10). Enzyme cocktail in X concentration contained about 10 U/mL of xylene monooxygenase (XMO) and 20 U/mL of catechol 2, 3 dioxygenases (C2,3D). X/5 and X/10 correspond to 5x and 10x dilution of enzyme cocktail respectively. The results showed that around 92-94% p-xylene removal was achieved in the treated soil column with enzyme concentration X, X/5 after second enzyme injection. While the p-xylene removal rate obtained by X/10 concentration of enzyme was less than 30% and near to untreated soil column (22.2%). The analysis of microbial diversity and biotoxicity assay (root elongation and seed germination) confirmed the advantage of using enzymes as a green and environmentally friendly approach for decontamination of pollutants with minimal or even positive effects on microbial community and also enrichment of soil after treatment.

Biodegradation of p-xylene-a comparison of three psychrophilic Pseudomonas strains through the lens of gene expression.

p-Xylene is considered a recalcitrant compound despite showing a similar aromatic structure to other BTEXs (benzene, toluene, ethylbenzene, xylene isomers). This study evaluated the p-xylene biodegradation potential of three psychrophilic Pseudomonas strains (Pseudomonas putida S2TR-01, Pseudomonas synxantha S2TR-20, and Pseudomonas azotoformans S2TR-09). The p-xylene metabolism-related catabolic genes (xylM, xylA, and xylE) and the corresponding regulatory genes (xylR and xylS) of the selected strains were investigated. The biodegradation results showed that the P. azotoformans S2TR-09 strain was the only strain that was able to degrade 200 mg/L p-xylene after 60 h at 15 °C. The gene expression study indicated that the xylE (encoding catechol 2,3-dioxygenase) gene represents the bottleneck in p-xylene biodegradation. A lack of xylE expression leads to the accumulation of intermediates and the inhibition of biomass production and complete carbon recovery. The activity of xylene monooxygenase and catechol 2,3-dioxygenase was significantly increased in P. azotoformans S2TR-09 (0.5 and 0.08 U/mg, respectively) in the presence of p-xylene. The expression of the ring cleavage enzyme and its encoding gene (xylE) and activator (xylS) explained the differences in the p-xylene metabolism of the isolated bacteria and can be used as a novel biomarker of efficient p-xylene biodegradation at contaminated sites.

Column tests for evaluation of the enzymatic biodegradation capacity of hydrocarbons (C10-C50) contaminated soil.

Though many studies pertaining to soil bioremediation have been performed to study the microbial kinetics in shake flasks, the process efficiency in column tests is seldom. In the present study, soil columns tests were carried out to study the biodegradation of soil contaminated with a high concentration of diesel (≈19.5 g/kg) petroleum hydrocarbons expressed as C10-C50. Experiments were done with crude enzymatic cocktail produced by the hydrocarbonoclastic bacterium, Alcanivorax borkumensis. A. borkumensis was grown on a media with 3% (v/v) motor oil as the sole carbon and energy source. The effects of the enzyme concentration, treatment time and oxidant on the bioremediation efficiency of C10-C50 were investigated. A batch test was also carried out in parallel to investigate the stability of the enzymes and the effect of the biosurfactants on the desorption and the bioconversion of C10-C50. Batch tests indicated that the biosurfactants significantly affected the desorption and alkane hydroxylase and lipase enzymes, maintained their catalytic activity during the 20-day test, with a half-life of 7.44 days and 8.84 days, respectively. The crude enzyme cocktail, with 40 U/mL of lipase and 10 U/mL of alkane hydroxylase, showed the highest conversion of 57.36% after 12 weeks of treatment with a degradation rate of 0.0218 day-1. The results show that the soil column tests can be used to optimize operating conditions for hydrocarbon degradation and to assess the performance of the overall bioremediation process.

Sustainable production and co-immobilization of cold-active enzymes from Pseudomonas sp. for BTEX biodegradation.

Toluene/o-Xylene Monooxygenase (ToMO) is equipped with a broad spectrum of aromatic substrate specificity (such as BTEX; benzene, toluene, ethylbenzene, and isomers of xylenes). TOMO has can hydroxylate more than a single position of aromatic rings in two consecutive monooxygenation reactions. Catechol 1,2-dioxygenase (C1,2D) is an iron-containing enzyme able to cleave the ring of catechol (the converted product from ToMO) for complete detoxification of BTEX. In this study, cold-active ToMO and C1,2D were produced using newly isolated psychrophilic Pseudomonas S2TR-14 in the minimal salt medium supplemented with crustacean waste and different concentrations of used motor oil (0.2-2% (v/v)). Crude ToMO and C1,2D were immobilized into micro/nano biochar-chitosan matrices and used for BTEX biodegradation. The results showed that the highest enzyme production (12 U/mg for ToMO and 22 U/mg for C1,2D) was achieved at the presence of 0.5% v/v used motor oil compared to the control group without motor oil (0.07 and 0.06 U/mg). High immobilization yield was achieved due to covalent bonding of ToMO (92.26% for micro matrix and 77.20% for nano matrix) and C1,2D (87.57% for micro matrix and 74.79% for nano matrix) with matrices. FTIR spectra confirmed the immobilization of enzymes on the surface of microbiochar and nanobiochar-chitosan matrices as proper support. The immobilization increased the storage stability of the enzymes with more than 50% residual activity after 30 days at 4 ± 1 °C, while the free form of enzymes had less than 10% of its activity. Immobilized enzymes degraded more than 80% of BTEX (~200 mg/L in groundwater and ~10,000 mg/kg in soil) at 10 ± 1 °C in groundwater and soil. Therefore, integrated use of microbiochar and nanobiochar with chitosan for co-immobilization of ToMO and C1,2D can be a potential way to remove petroleum hydrocarbons with higher efficiency from contaminated groundwater and soil.

Critical insights into psychrophilic anaerobic digestion: Novel strategies for improving biogas production.

Anaerobic digestion (AD) under psychrophilic temperature has only recently garnered deserved attention. In major parts of Europe, USA, Canada and Australia, climatic conditions are more suited for psychrophilic (<20 ℃) rather than mesophilic (35 - 37 ℃) and thermophilic (55 - 60 ℃) AD. Low temperature has adverse effects on important cellular processes which may render the cell biology inactive. Moreover, cold climate can also alter the physical and chemical properties of wastewater, thereby reducing the availability of substrate to microbes. Hence, the use of low temperature acclimated microbial biomass could overcome thermodynamic constraints and carry out flexible structural and conformational changes to proteins, membrane lipid composition, expression of cold-adapted enzymes through genotypic and phenotypic variations. Reduction in organic loading rate is beneficial to methane production under low temperatures. Moreover, modification in the design of existing reactors and the use of hybrid reactors have already demonstrated improved methane generation in the lab-scale. This review also discusses some novel strategies such as direct interspecies electron transfer (DIET), co-digestion of substrate, bioaugmentation, and bioelectrochemical system assisted AD which present promising prospects. While DIET can facilitate syntrophic electron exchange in diverse microbes, the addition of organic-rich co-substrate can help in maintaining suitable C/N ratio in the anaerobic digester which subsequently can enhance methane generation. Bioaugmentation with psychrophilic strains could reduce start-up time and ensure daily stable performance for wastewater treatment facilities at low temperatures. In addition to the technical discussion, the economic assessment and future outlook on psychrophilic AD are also highlighted.

Fumaric acid production using alternate fermentation mode by immobilized Rhizopus oryzae-a greener production strategy.

The current work investigates the impact of using immobilized Rhizopus oryzae NRRL 1526 for bioproduction of fumaric acid using agro-industrial residues as feedstock. This use of agro-industrial residues, a renewable feedstock, for the production of bio-based platform chemical makes the process cost-competitive as well as greener by preventing the release of assimilable organic carbon to the environment, thereby reducing the generation of greenhouse gases. Immobilization of R. oryzae has been proposed previously to alleviate operational difficulties confronted during free mycelial fungal fermentation. To this effect, three synthetic refuse materials namely polystyrene foam, polyester sponge and polyurethane foam were investigated for their suitability towards fumaric acid bioproduction. Polystyrene foam was identified as the most suitable support material for immobilization as well as fumaric acid production. In addition to the considerable reduction in the lag-phase (from 48 to 24 h) the reduction in the size of the support material from cubes of 1 cm to beads of 0.1-0.3 cm led to a 42% improvement in fumaric acid production (27 g/L against 19 g/L). Growing the polystyrene foam bead immobilized R. oryzae on apple pomace ultrafiltration sludge as sole feedstock yielded a final fumaric acid titer of 7.9 g/L whereas free mycelial fermentation yielded 6.3 g/L. Moreover, upon operating the fermentation with intermittent feeding, a three-fold increase (1.7 g/L to 5.1 g/L) in fumaric acid production was obtained upon supplementation of the apple pomace sludge media with molasses, an agro-industrial residue, as feed.

Feasibility of the use of different types of enzymatically treated cellulosic fibres for polylactic acid (PLA) recycling.

In the present study, the potential use of cellulosic microfibers (CMFs) extracted from hemp fiber (HF) and pulp and paper solid waste (mixed sludge (MS), deinked sludge (DS)) as a reinforcing agent in novel bio composite materials produced from recycled Polylactic acid (rPLA) was investigated. CMFs were extracted and treated using physicochemical method followed by enzymatic treatment with laccase and cellulase. The effects of CMFs concentrations (1.5, 3 and 6% w/w) and fiber size (75 µm-1.7 mm) on the mechanical properties (impact and tensile) and biodegradability of the biocomposite samples were investigated. A modified interfacial adhesion between rPLA matrix and the three fibers used, was clearly observed through mechanical tests due to alkali and enzymatic treatments. The use of different types of enzymatically treated cellulosic fibers for polylactic acid (PLA) recycling was assessed by Scaning electron microscopy (SEM), X-ray diffraction (XRD) and Fourier transform infrared spectroscopy (FTIR). The combined physicochemical and enzymatic treatments led to a considerable size reduction of the cellulosic fibers (HF, MS and DS) resulting in the enhanced interfacial adhesion between rPLA matrix and fibers. The biocomposite obtained with rPLA with HF gave the most favorable values for Young's modulus (324.53 ± 3.10 MPa, p-value 0.03), impact strength (27.61 ± 2.94 kJ/m2, p-value 0.01) and biodegradation rate (1.97%).

Psychrozymes as novel tools to biodegrade p-xylene and potential use for contaminated groundwater in the cold climate.

Sites contaminated by petroleum hydrocarbons in cold-climate regions have recently received significant attention due to their sensitive ecosystem and human health impacts. Two cold-adapted pseudomonas strains were isolated from contaminated groundwater and soil. As xylene monooxygenase from Pseudomonas synxantha S2TR-26 and catechol 2,3-dioxygenase from Pseudomonas mandelii S2TR-08, have a matching end product, they acted in symphony to degrade p-xylene. Their unique thermodynamic and kinetic behavior permits them to achieve rapid degradation of p-xylene at low temperatures (<15 °C). The results showed that the sequential action led to the conversion of 200 mg/l of p-xylene within 72 h and complete degradation after 120 h. The cocktail of these enzymes with a ratio of 1:1.5 (xylene monooxygenase: catechol 2, 3-dioxygenase) confirmed the complete degradation of p-xylene within 48 h at 15 °C. This approach will allow efficient biodegradation of p-xylene to minimize the bioremediation duration in cold-climate regions.

Biodegradation of aflatoxin B1 with cell-free extracts of Trametes versicolor and Bacillus subtilis.

Aflatoxin B1 (AFB1) is one of the most common contaminants of poultry feed and has been linked to adverse effects on animal health and productivity. In this study, the degradation of AFB1 was studied with cell-free extracts (CFE) of Trametes versicolor and Bacillus subtilis using High-Performance Liquid chromatography (HPLC). CFE from B. subtilis and T. versicolor gave 60% and 34% of AFB1 degradation respectively, while heat-inactivated extracts showed no degradation. By-products obtained at the end of AFB1 degradation were analyzed by Liquid Chromatography with tandem mass spectrometry (LC-MS/MS). After 96 h of incubation, by-products with lower m/z values were obtained with CFE from B. subtilis as compared to that from T. versicolor, indicating a higher degradation efficiency of the former. Additionally, the detection of a by-product which could correspond to AFB1-8,9 dihydrodiol - a less toxic derivative of AFB1 - after 72 and 96 h of incubation with CFE from B. subtilis, could indicate the simultaneous detoxification along with degradation of AFB1 by B. subtilis CFE.

Evaluation of pyroligneous acid as a therapeutic agent against Salmonella in a simulated gastrointestinal tract of poultry.

Pyroligneous acid (PA) was evaluated as a potential alternative to therapeutic antibiotics in poultry. Antimicrobial activity of PA was studied at acidic pH (2.0) and neutral pH (7.0) of the liquid against Salmonella enterica and Lactobacillus acidophilus. Acidic PA gave a MIC value of 0.8% (v/v) and 1.6% (v/v), and neutralized PA gave a MIC value of 1.6% (v/v) and 3.2% (v/v) against S. enterica and L. acidophilus respectively. Acidic PA was evaluated at different concentrations in a simulated poultry digestive tract and cecal fermentation to study its effect on the cecal microflora and fermentation profile. PA at a concentration of 1.6% (v/v) completely inhibited S. enterica and was also found to have a similar effect on lactobacilli count as compared with the control (p = 0.17). Additionally, PA at this concentration was found not to have a significant effect on acetic acid production after 24 h of cecal fermentation (p = 0.20). Graphical abstract.

Ciprofloxacin removal via sequential electro-oxidation and enzymatic oxidation.

The combination of electro-oxidation and enzymatic oxidation was tested to evaluate the potency of this system to remove ciprofloxacin (CIP), a fluoroquinolone antibiotic, from water. For the electro-oxidation boron-doped diamond (BDD) and mixed metal oxides anodes were tested, at three current densities (4.42, 17.7 and 35.4 A/cm2). BDD anode at 35.4 A/cm2 exhibited the highest removal efficiency in the shortest time (>90 % removal in 6 min). For the enzymatic oxidation, laccase from Trametes versicolor was chosen. Laccase alone was not able to remove CIP; hence the influence of redox mediators was investigated. The addition of syringaldehyde (SA) and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) resulted in enhanced CIP transformation. About 48.9±4.0 % of CIP remained after 4 h of treatment when SA-mediated laccase was applied and 87.8±6.6 % in the case of ABTS-mediated laccase. The coupling of enzymatic oxidation followed by electro-oxidation led to 73 % removal of the antibiotic. Additionally, the antimicrobial activity increased up to its original efficiency after the treatment. The combination of electro-oxidation followed by enzymatic oxidation led to 97-99 % removal of CIP. There was no antimicrobial activity of the solution after the treatment. The tests with wastewater confirmed the efficacy of the system to remove CIP from the complex matrix.

Microwave-assisted extraction of chitosan from Rhizopus oryzae NRRL 1526 biomass.

Microwave-assisted extraction (MAE) of chitosan from dried fungal biomass of Rhizopus oryzae NRRL1526, obtained by culturing on potato dextrose broth (PDB), was performed and the optimal conditions required were identified using statistical analysis for the first time in this study. This microwave-assisted extraction (MAE) was compared against the conventional autoclave assisted method of chitosan extraction. The full factorial experimental design was used to investigate the impact of operating parameters of MAE, microwave power (100 W-500 W), and duration (10 min-30 min), on alkaline insoluble material (AIM) yield, chitosan yield, and degree of deacetylation (DDA). The effect of operating conditions was then evaluated using full factorial data analysis and optimum condition for MAE of chitosan was identified using response surface methodology to be 300 W and 22 min. This optimum condition identified was then further evaluated and the chitosan obtained characterized. Higher chitosan yield of 13.43 ± 0.3% (w/w) of fungal biomass was obtained when compared to that obtained, 6.67% ± 0.3% (w/w) of dry biomass, for the conventional extraction process. MAE yielded chitosan of higher degree of deacetylation, 94.6 ± 0.9% against 90.6 ± 0.5% (conventional heating), but the molecular weight was observed to be similar to that obtained by using conventional autoclave heating. MAE of chitosan was observed to yield a higher quantity of chitosan when compared to conventional extraction process and obtained chitosan exhibited a higher degree of deacetylation as well as molecular weight. The lower energy consumption of 0.11 kW h for MAE (5 kW h for conventional process) and the concomitant reduction in the energy bill to 1.1 cents from 50 cents, in addition to the above results, show that microwave irradiation is a more efficient and environment-friendly means to obtain chitosan from fungal biomass.

Bioproduction of fumaric acid: an insight into microbial strain improvement strategies.

Fumaric acid (FA), a metabolic intermediate, has been identified as an important carbohydrate derived platform chemical. Currently, it is commercially sourced from petrochemicals by chemical conversion. The shift to biochemical synthesis has become essential for sustainable development and for the transition to a biobased economy from a petroleum-based economy. The main limitation is that the concentrations of FA achieved during bioproduction are lower than that from a chemical process. Moreover, the high cost associated with bioproduction necessitates a higher yield to improve the feasibility of the process. To this effect, genetic modification of microorganism can be considered as an important tool to improve FA yield. This review discusses various genetic modifications strategies that have been studied in order to improve FA production. These strategies include the development of recombinant strains of Rhizopus oryzae, Escherichia coli, Saccharomyces cerevisiae, and Torulopsis glabrata as well as their mutants. The transformed strains were able to accumulate fumaric acid at a higher concentration than the corresponding wild strains but the fumaric acid titers obtained were lower than that reported with native fumaric acid producing R. oryzae strains. Moreover, one plausible adoption of gene editing tools, such as Agrobacterium-mediated transformation (AMT), CRISPR CAS-9 and RNA interference (RNAi) mediated knockout and silencing, have been proposed in order to improve fumaric acid yield. Additionally, the introduction of the glyoxylate pathway in R. oryzae to improve fumaric acid yield as well as the biosynthesis of fumarate esters have been proposed to improve the economic feasibility of the bioprocess. The adoption of some of these genetic engineering strategies may be essential to enable the development of a feasible bioproduction process.

Bench-scale production of enzymes from the hydrocarbonoclastic bacteria Alcanivorax borkumensis and biodegradation tests.

This study investigates motor oil (3, 5, 7.5 and 10% (v v-1)) as a sole carbon source for the production of Alcanivorax borkumensis in shake flasks and a 5 L bench-scale fermenter in comparison to the standard media. Shake flask studies showed a significant and higher cell growth (p=0.000038), lipase (p = 0.006900) and alkane hydroxylase production (p = 0.000921) by Alcanivorax borkumensis when motor oil was used as the substrate. Based on Tukey post-hoc tests, 5% motor oil concentration was selected as the optimal substrate concentration. The 5 L fermenter experiments conducted using motor oil at 5% (v v-1) concentration, under controlled conditions exhibited significant and higher alkane hydroxylase and lipase activities (55.6 U mL-1 (p = 0.018418) and 208.30 U mL-1 (p = 0.020087), respectively) as compared with those of motor oil at 3% (v v-1) and n-hexadecane at 3% (v v-1) concentration which was used as control. Cell growth was significantly higher when motor oil (3 or 5%) was used as a substrate (p = 0.024705). Enzymatic degradation tested on two different polycyclic aromatic hydrocarbons (PAHs) contaminated groundwaters showed 37.4% removal after 5 days with a degradation rate of 196.6 ppb day-1 and 82.8% removal after 10 days with a degradation rate of 217.54 ppb day-1 for the 1st site and an almost complete biodegradation with 95% removal and 499.02 ppb day-1 removal rate after only 5 days for the 2nd site.

Use of antibiotics in broiler production: Global impacts and alternatives.

Antibiotics are used to fight bacterial infections. However, a selective pressure gave rise to bacteria resistant to antibiotics. This leaves scientists worried about the danger to human and animal health. Some strategies can be borrowed to reduce the use of antibiotics in chicken farms. Much research has been carried out to look for natural agents with similar beneficial effects of growth promoters. The aim of these alternatives is to maintain a low mortality rate, a good level of animal yield while preserving environment and consumer health. Among these, the most popular are probiotics, prebiotics, enzymes, organic acids, immunostimulants, bacteriocins, bacteriophages, phytogenic feed additives, phytoncides, nanoparticles and essential oils.

Adsorptive immobilization of agro-industrially produced crude laccase on various micro-biochars and degradation of diclofenac.

Although enzymes are gifted with unique and unprecedented catalytic activity and selectivity over a wide range of pollutants, still their stability related issues often hinder their application in real environmental conditions. In this study, agro-industrially produced crude laccase was concentrated using ultrafiltration. Crude laccase was immobilized on pine wood (BC-PW), pig manure (BC-PM) and almond shell (BC-AS) biochar microparticles. Immobilization of laccase was investigated at various laccase activities on micro-biochars and the release (desorption) of the enzyme has been studied. It was observed that for all the biochars, as the initial concentration of laccase increased in the crude solution, the binding capacity and as result immobilization efficiency also increased. BC-PM was found to be the most effective (31.4 ±â€¯3.1 U g-1) at 10 U mL-1 of enzyme activity followed by BC-AS (24.3 ±â€¯4.8 U g-1) and BC-PW (14.58 ±â€¯3.3 U g-1). In addition, the biochars were functionalized with citric acid for possible surface modifications and the effect of biochars for the adsorption of enzymes has been investigated. Isotherm studies of enzyme loading onto biochar established homogeneous monolayer adsorption as the major mechanism. The desorption of laccase from all biochars followed pseudo-second-order model. Immobilized laccase exhibited superior storage ability and shelf-life which were three times higher than free laccase. Finally, the immobilized laccase was used for the degradation of micropollutant, DCF and near 100% removal was obtained within 5 h at an environmentally relevant concentration (500 µg L-1).

Production and characterization of novel hydrocarbon degrading enzymes from Alcanivorax borkumensis.

This study investigates the production of alkane hydroxylase, lipase and esterase by the marine hydrocarbon degrading bacteria Alcanivorax borkumensis. The focus of this study is the remediation of petroleum hydrocarbons, hexane, hexadecane and motor oil as model substrates. A. borkumensis showed an incremental growth on these substrates with a high cell count. Growth on motor oil showed highest alkane hydroxylase and lipase production of 2.62 U/ml and 71 U/ml, respectively, while growth on hexadecane showed the highest esterase production of 57.5 U/ml. The percentage of hexane, hexadecane, and motor oil degradation during A. borkumensis growth after 72 h, was around 80%, 81.5% and 75%, respectively. Zymogram showed two different bands with a molecular weight of approx. 52 and 40 kDa, respectively with lipase and esterase activity. Alkane hydroxylase reached optimum activity at pH 8.0 and 70 ±â€¯1 °C for hexane and hexadecane and 75 ±â€¯1 °C for motor oil. Lipase and esterase showed optimum activity at 35 ±â€¯1 °C and 40 ±â€¯1 °C, respectively and pH 7.0. The crude enzymes showed higher stability in a wide range of pH, but they were not thermostable at higher temperatures.