A comparison between biostimulation and bioaugmentation in a solid treatment of anaerobic sludge: Drug content and microbial evaluation.

Emerging pollutants can reach the environment through the sludge of Wastewater Treatment Plants. In this work, the use of Trametes versicolor in biopiles at lab-scale was studied, evaluating its capacity to remove the most hydrophobic Pharmaceuticals and assessing the evolution of the biopiles microbial communities. The total removal of drugs at real concentrations from sewage sludge was assessed for non-inoculated and fungal inoculated biopiles, testing if the re-inoculation of the biopiles after 22 days of treatment would improve the removal yields. It was found that 2 out of the 15 initially detected pharmaceuticals were totally degraded after 22 days, and re-inoculated fungal biopiles achieved higher removal rates than non-re-inoculated fungal biopiles for single compounds and for all the drugs simultaneously: 66.45% and 49.18% re-inoculated and non-re-inoculated biopiles, respectively. Finally, the study of the bacterial and fungal communities revealed that fungal inoculated and non-inoculated biopiles evolved to similar communities adapted to the presence of those drugs.

The role of sorption processes in the removal of pharmaceuticals by fungal treatment of wastewater.

The contribution of the sorption processes in the elimination of pharmaceuticals (PhACs) during the fungal treatment of wastewater has been evaluated in this work. The sorption of four PhACs (carbamazepine, diclofenac, iopromide and venlafaxine) by 6 different fungi was first evaluated in batch experiments. Concentrations of PhACs in both liquid and solid (biomass) matrices from the fungal treatment were measured. Contribution of the sorption to the total removal of pollutants ranged between 3% and 13% in relation to the initial amount. The sorption of 47 PhACs in fungi was also evaluated in a fungal treatment performed in 26days in a continuous bioreactor treating wastewater from a veterinary hospital. PhACs levels measured in the fungal biomass were similar to those detected in conventional wastewater treatment (WWTP) sludge. This may suggest the necessity of manage fungal biomass as waste in the same manner that the WWTP sludge is managed.

Comparison between several reactors with Trametes versicolor immobilized on lignocellulosic support for the continuous treatments of hospital wastewater.

Hospital wastewater is a major source of pharmaceutically active compounds (PhACs), which are not all removed in conventional wastewater treatment plants. White rot fungi can degrade PhACs, but their application has been limited to non-sterile conditions due to the competition with other microorganisms for growth. In this study, immobilization of Trametes versicolor on different lignocellulosic supports was studied as strategy to ensure fungal survival under continuous treatment conditions. A fluidized bed reactor and a trickling packed-bed reactor with T. versicolor immobilized on pallet wood were employed for the removal of ibuprofen, ketoprofen and naproxen. Best results were obtained with the trickling packed-bed reactor, which operated for 49days with high removal values in real hospital wastewater.

Pharmaceuticals removal and microbial community assessment in a continuous fungal treatment of non-sterile real hospital wastewater after a coagulation-flocculation pretreatment.

Hospital wastewaters are a main source of pharmaceutical active compounds, which are usually highly recalcitrant and can accumulate in surface and groundwater bodies. Fungal treatments can remove these contaminants prior to discharge, but real wastewater poses a problem to fungal survival due to bacterial competition. This study successfully treated real non-spiked, non-sterile wastewater in a continuous fungal fluidized bed bioreactor coupled to a coagulation-flocculation pretreatment for 56 days. A control bioreactor without the fungus was also operated and the results were compared. A denaturing gradient gel electrophoresis (DGGE) and sequencing approach was used to study the microbial community arisen in both reactors and as a result some bacterial degraders are proposed. The fungal operation successfully removed analgesics and anti-inflammatories, and even the most recalcitrant pharmaceutical families such as antibiotics and psychiatric drugs.

Continuous treatment of non-sterile hospital wastewater by Trametes versicolor: How to increase fungal viability by means of operational strategies and pretreatments.

Hospital wastewaters have a high load of pharmaceutical active compounds (PhACs). Fungal treatments could be appropriate for source treatment of such effluents but the transition to non-sterile conditions proved to be difficult due to competition with indigenous microorganisms, resulting in very short-duration operations. In this article, coagulation-flocculation and UV-radiation processes were studied as pretreatments to a fungal reactor treating non-sterile hospital wastewater in sequential batch operation and continuous operation modes. The influent was spiked with ibuprofen and ketoprofen, and both compounds were successfully degraded by over 80%. UV pretreatment did not extent the fungal activity after coagulation-flocculation measured as laccase production and pellet integrity. Sequential batch operation did not reduce bacteria competition during fungal treatment. The best strategy was the addition of a coagulation-flocculation pretreatment to a continuous reactor, which led to an operation of 28days without biomass renovation.

Fungal treatment for the removal of antibiotics and antibiotic resistance genes in veterinary hospital wastewater.

The emergence and spread of antibiotic resistance represents one of the most important public health concerns and has been linked to the widespread use of antibiotics in veterinary and human medicine. The overall elimination of antibiotics in conventional wastewater treatment plants is quite low; therefore, residual amounts of these compounds are continuously discharged to receiving surface waters, which may promote the emergence of antibiotic resistance. In this study, the ability of a fungal treatment as an alternative wastewater treatment for the elimination of forty-seven antibiotics belonging to seven different groups (ß-lactams, fluoroquinolones, macrolides, metronidazoles, sulfonamides, tetracyclines, and trimethoprim) was evaluated. 77% of antibiotics were removed after the fungal treatment, which is higher than removal obtained in conventional treatment plants. Moreover, the effect of fungal treatment on the removal of some antibiotic resistance genes (ARGs) was evaluated. The fungal treatment was also efficient in removing ARGs, such as ermB (resistance to macrolides), tetW (resistance to tetracyclines), blaTEM (resistance to ß-lactams), sulI (resistance to sulfonamides) and qnrS (reduced susceptibility to fluoroquinolones). However, it was not possible to establish a clear link between concentrations of antibiotics and corresponding ARGs in wastewater, which leads to the conclusion that there are other factors that should be taken into consideration besides the antibiotic concentrations that reach aquatic ecosystems in order to explain the emergence and spread of antibiotic resistance.

Stable Carbon Isotope Fractionation During 1,2-Dichloropropane-to-Propene Transformation by an Enrichment Culture Containing Dehalogenimonas Strains and a dcpA Gene.

A stable enrichment culture derived from Besòs river estuary sediments stoichiometrically dechlorinated 1,2-dichloropropane (1,2-DCP) to propene. Sequential transfers in defined anaerobic medium with the inhibitor bromoethanesulfonate produced a sediment-free culture dechlorinating 1,2-DCP in the absence of methanogenesis. Application of previously published genus-specific primers targeting 16S rRNA gene sequences revealed the presence of a Dehalogenimonas strain, and no amplification was obtained with Dehalococcoides-specific primers. The partial sequence of the 16S rRNA amplicon was 100% identical with Dehalogenimonas alkenigignens strain IP3-3. Also, dcpA, a gene described to encode a corrinoid-containing 1,2-DCP reductive dehalogenase was detected. Resistance of the dehalogenating activity to vancomycin, exclusive conversion of vicinally chlorinated alkanes, and tolerance to short-term oxygen exposure is consistent with the hypothesis that a Dehalogenimonas strain is responsible for 1,2-DCP conversion in the culture. Quantitative PCR showed a positive correlation between the number of Dehalogenimonas 16S rRNA genes copies in the culture and consumption of 1,2-DCP. Compound specific isotope analysis revealed that the Dehalogenimonas-catalyzed carbon isotopic fractionation (εC(bulk)) of the 1,2-DCP-to-propene reaction was -15.0 ± 0.7‰ under both methanogenic and nonmethanogenic conditions. This study demonstrates that carbon isotope fractionation is a valuable approach for monitoring in situ 1,2-DCP reductive dechlorination by Dehalogenimonas strains.

Degradation of Orange G by laccase: fungal versus enzymatic process.

Biodegradation of the Orange G azo dye by pellets of Trametes versicolor in a fluidized bioreactor operating under conditions of laccase production was studied. The percentage of decolorization obtained was 97% in batch mode and both the biomass and the broth, were colorless. In vitro degradations carried out with purified commercial laccase from Trametes versicolor demonstrated that laccase is able to degrade the dye. In spite of the high level of decolorization reached in both processes, an important difference between the fungal and enzymatic treatments was detected. At the end of the experiments carried out in vitro, a final residual color appears (different to the initial one). Consequently, measuring the yield of decolorization as a percentage of absorbance lambdamax variation is not the best indicator of the treated wastewater quality, but the analysis of the visible color spectrum makes it possible to detect changes in color. The results demonstrate that better results are obtained with fungal Orange G biodegradation because a further breakdown of the enzymatic products is achieved with the fungus.

Treatment of toxic industrial wastewater in fluidized and fixed-bed batch reactors with Trametes versicolor: influence of immobilisation.

This work presents the results obtained in the treatment of industrial pulp mill wastewater (black liquor) with the white-rot fungus Trametes versicolor immobilised in nylon and polyurethane foam cubes. Reductions in colour (36%), aromatic compounds (54%) and toxicity (3.15 times reduction of the initial value) were obtained when the fungus was immobilised in nylon and good toxicity reduction (5.7-fold reduction of the initial value) when polyurethane foam was used. These results were compared with those obtained with Trametes versicolor in the form of pellets for colour and aromatic compounds (84.8% and 70.2% respectively). Correlations among different parameters have been studied. Relationships between colour and changes in the molecular weight distribution profiles, as well as a correlation between laccase production and toxicity reduction have been found. For laccase production vs. toxicity reduction a different behaviour has been observed depending on the bioreactor configuration (fixed-bed reactor with immobilised Trametes versicolor or fluidised bed reactor with pellets of Trametes versicolor).

Recombinant production of serine hydroxymethyl transferase from Streptococcus thermophilus and its preliminary evaluation as a biocatalyst.

The glyA gene encoding a serine hydroxymethyl transferase (SHMT) with threonine aldolase activity was isolated from Streptococcus thermophilus YKA-184 chromosomal DNA. This aldolase is a pyridoxal 5'-phosphate-dependent enzyme that stereospecifically catalyzes the interconversion of L-threonine to glycine and acetaldehyde. The enzyme was overexpressed in Escherichia coli M15 as a recombinant protein of 45 kDa with a His6-tag at its N-terminus. The recombinant enzyme was purified to homogeneity by a single chromatographic step using Ni-nitrilotriacetic acid affinity, obtaining a high activity-recovery yield (83%). Lyophilized and precipitated enzymes were stable at least for 10 weeks when stored at -20 degrees C and 4 degrees C. It was observed that the Km for L-allo-threonine was 38-fold higher than that for L-threonine, suggesting this enzyme can be classified as a specific L-allo-threonine aldolase. The optimum pH range of threonine aldolase activity for the recombinant SHMT was pH 6-7. When tested for aldol addition reactions with non-natural aldehydes, such as benzyloxyacetaldehyde and (R)-N-Cbz-alaninal, two possible beta-hydroxy-alpha-amino acid diastereoisomers were produced, but with moderate stereospecificity. The enzyme showed potential as a biocatalyst for the stereoselective synthesis of beta-hydroxy-alpha-amino acids.

Mechanism of textile metal dye biotransformation by Trametes versicolor.

The biodegradation of Grey Lanaset G, which consists of a mixture of metal complexed dye, was studied. Experiments were carried out in a bioreactor with retained pellets of the fungus Trametes versicolor that was operated under conditions of laccase production. Although decolorization was highly efficient (90%), no direct relationship to extracellular enzyme was apparent. Moreover, the extracellular enzyme was found to be unable to degrade the dye in vitro. The process involves several steps. Thus, the initial adsorption of the dye and its transfer into cells is followed by breaking of the metal complex bond in the cells release of the components. The metal (Cr and Co) contents of the biomass and treated solutions, and their closer relationship to intracellular enzyme and degradation of the dye, confirm the initial hypothesis.

Olive oil mill waste waters decoloration and detoxification in a bioreactor by the white rot fungus Phanerochaete flavido-alba.

Olive oil mill wastewater (OMW) is produced as waste in olive oil extraction. With the purpose of treating this highly polluting waste, a number of experiments were conducted in a laboratory-scale bioreactor with the white rot fungus Phanerochaete flavido-alba (P. flavido-alba). It is known that this fungus is capable of decolorizing OMW in static or semistatic cultures at Erlenmeyer scale and at 30 degrees C. The objective of this work was to prove that P. flavido-alba could decolorize OMW in submerged cultures and that it is capable of reducing OMW toxicity at room temperature (25 degrees C) and in a laboratory-scale bioreactor. In the experiments conducted, manganese peroxidase (MnP) and laccase enzymes were detected; however, unlike other studies, lignin peroxidase was not found to be present. Decoloration obtained after treatment was 70%. The reduction of aromatic compounds obtained was 51%, and the toxicity of the culture medium was reduced by up to 70%. We can therefore state that P. flavido-alba is capable of reducing important environmental parameters of industrial effluents and that prospects are positive for the use of this process at a larger scale, even when working at room temperature.

Influence of phosphate on rhamnose-containing exopolysaccharide rheology and production by Klebsiella I-714.

Physiological conditions enhancing rhamnose-containing polysaccharide synthesis by Klebsiella I-714 were studied in batch culture (0.3-l and 2-l bioreactors). The four carbon sources tested, sucrose, sorbitol, Neosorb and Cerelose, allowed exopolysaccharide production. Larger amounts of polymer were produced when high carbon/nitrogen ratios and complex nitrogen sources were used. Exopolysaccharide synthesis was greatest at 30 degrees C, which was a suboptimal growth temperature. A reduction in the phosphate content of the medium enhanced rhamnose-containing polysaccharide production. When the initial carbon source concentration was augmented, byproducts other than exopolysaccharide were formed. Rhamnose-containing polysaccharide rheology can be modulated by changing the phosphate content of the medium.

Reaction engineering for consecutive enzymatic reactions in peptide synthesis: application to the synthesis of a pentapeptide.

A single-pot enzymatic synthesis of Z-CCK5 (4-8) is presented in this work, employing Z-Gly-Trp-OBzl as acyl donor, under kinetic control. The first goal of the work is the development of a synthetic strategy allowing the use of the same medium for two reactions catalyzed by immobilized alpha-chymotrypsin, discriminating between simultaneous and consecutive addition systems. The second goal is the maximization of the pentapeptide yield as a function of the molar excess of both nucleophiles employed. A maximum yield of 36% was obtained, and the addition strategy as well as the optimal initial concentrations of substrates have been determined.

Enzymatic condensation of cholecystokinin CCK-8 (4-6) and CCK-8 (7-8) peptide fragments in organic media.

The kinetically controlled condensation reaction of Z-Gly-Trp-Met-OR(1) (R(1): Et, Al, Cam) and H-Asp-(OR(2))-Phe-NH(2) (R(2): H, Bu(t)) catalyzed by alpha-chymotrypsin deposited onto polyamide in organic media was studied. The effect of the drying process of the enzyme-support preparation, substrate concentrations, reaction medium, acyl donor, and nucleophile structure on both enzymatic activity and pentapeptide yield was investigated. The immobilized preparation directly equilibrated at a(w) = 0.113, gave higher enzymatic activities than dried with vacuum first, and then equilibrated at a(w) = 0.113. The addition of triethylamine to the reaction medium increased dramatically the enzymatic activity. However, the pentapeptide yield was affected neither by the drying procedure nor by the addition of triethylamine. The donor ester Z-Gly-Trp-Met-OAl gave initial reaction rates 2.6 times higher than the conventional ethyl ester derivative but rendered similar yields. The best results were obtained using Z-Gly-Trp-Met-OCam as acyl-donor ester; 80% yield and initial reaction rates 4 times higher than the ethyl ester derivative. In all cases, acetonitrile containing Tris-HCl 50 mM pH 9 buffer (0.5% v/v) and triethylamine (0.5% v/v) was found to be the best reaction system. Under these conditions, it was possible to use the nucleophile H-Asp-Phe-NH(2) with beta-unprotected aspartic acid residue. In this case, 50% yield was obtained, but economic considerations could lead to select it as nucleophile. Finally, the fragment condensation reaction was carried out at gram scale, obtaining a 39% yield which included the reaction, removal of protecting groups and purification steps. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 456-463, 1997.

Enzymatic synthesis of a CCK-8 tripeptide fragment in organic media.

The enzymatic synthesis of the tripeptide derivative Z-Gly-Trp-Met-OEt is reported. This tripeptide is a fragment of the cholecystokinin C-terminal octapeptide CCK-8. Studies on the alpha-chymotrypsin catalyzed coupling reaction between Z-Gly-Trp-R(1) and Met-R(2) have focused on low water content media, using deposited enzyme on inert supports such as Celite and polyamide. The effect of additives (polar organic solvents), the acyl-donor ester structure, the C-alpha protecting group of the nucleophile, enzyme loading, and substrate concentration were tested. The best reaction medium found was acetonitrile containing buffer (0.5%, v/v) and triethylamine (0.5%, v/v) using the enzyme deposited on Celite as catalyst (8 mg of alpha-chymotrypsin/g of Celite). A reaction yield of 81% was obtained with Z-Gly-Trp-OCam as acyl donor, at an initial concentration of 80 mM. The tripeptide synthesis was scaled up to the production of 2 g of pure tripeptide with an overall yield of 71%, including reaction and purification steps.

Biomass production by a thermotolerant yeast: Hansenula polymorpha.

Biomass production at high temperature by Hansenula polymorpha as part of a lignocellulosic utilizing process was studied. Compromise growth conditions (45 degrees C and pH = 4.8) with an eventual saccharification step were established. The effects of stirring rate and initial glucose concentration on biomass yield coefficient, volumetric productivity and maximal cell density were determined. Process optimization led to a fed-batch fermentation process: high yield (0.63 g dry cell g-1 glucose), volumetric productivity (1.3 g dry cell dm-3 h-1) and cell concentration (60 g dry cell dm-3) were obtained. At these conditions, significant arabitol excretion (18 g dm-3) as a unique by-product associated with cell mass production was obtained, making more interesting a high temperature operating process.

An approach in mathematical modeling of an upflow packed-bed reactor for enzymatic hydrolysis of wheat straw.

The behavior of a packed-bed reactor for enzymatic hydrolysis of pretreated wheat straw has been described by means of a mathematical model. The flow pattern has been evaluated by residence time distribution experiments. Small deviations from ideal plug flow behavior were found using the dispersion model. The kinetic model proposed for enzymatic hydrolysis of cellulosic fraction of pretreated wheat straw has been derived from batch experimental data. Variations of enzyme concentration throughout the straw bed have been approximately described using a ramp variation of adsorbed enzyme. The final explains qualitatively the experimental results.

Application of extended Kalman filter to identification of enzymatic deactivation.

A recursive estimation scheme, the Extended Kalman Filter (EKF) technique, was applied to study enzymatic deactivation in the enzymatic hydrolysis of pretreated cellulose using a model previously developed by the authors. When no deactivation model was assumed, the results showed no variation with time for all the model parameters except for the maximum rate of cellobiose-to-glucose conversion (r'(m)).The r'(m) variation occurred in two zones with a grace period. A new model of enzymatic hydrolysis of pretreated cellulose deactivation was proposed and validated showing better behavior than the old deactivation model. This approach allows one to study enzyme deactivation without additional experiments and within operational conditions.

Dilute acid hydrolysis of wheat straw hemicellulose at moderate temperature: a simplified kinetic model.

Wheat straw has been hydrolized with sulfuric acid at 34 and 90 degrees C. The treatment at 90 degrees C yields complete solubilization of hemicellulose to xylose and arabinose without significant amounts of furfural. The influence of acid concentration was studied and the kinetics of the acid-catalyzed hydrolysis has been modeled suggesting a two-consecutive reactions mechanism. This model is useful to explain the different behavior of the concentration of the two main sugars produced. The enhanced cellulose accessibility to enzymatic attack is also reported.