Effects of potential inducers to enhance laccase production and evaluating concomitant enzyme immobilisation.

This work investigated non-polar solvent hexane and polar solvents methanol and ethanol as inducers besides a well-known inducer, copper, for laccase production with and without mesoporous silica-covered plastic packing under sterilised and unsterilised conditions. The potential of waste-hexane water, which is generated during the mesoporous silica production process, was also investigated as a laccase inducer. During the study, the free and immobilised laccase activity on the packing was measured. The results showed that the highest total laccase activity, approximately 10,000 Units, was obtained under sterilised conditions with 0.5 mM copper concentration. However, no immobilised laccase activity was detected except in the copper and ethanol sets under unsterilised conditions. The maximum immobilised laccase activity of the sets that used waste hexane as an inducer was 1.25 U/mg packing. According to its significant performance, waste hexane can be an alternative inducer under sterilised conditions. Concomitant immobilised packing showed satisfactory laccase activities and could be a promising method to reduce operation costs and improve the cost-efficiency of enzymatic processes in wastewater treatment plants.

Occurrence of the Persistent Antimicrobial Triclosan in Microwave Pretreated and Anaerobically Digested Municipal Sludges under Various Process Conditions.

Treatment of emerging contaminants, such as antimicrobials, has become a priority topic for environmental protection. As a persistent, toxic, and bioaccumulative antimicrobial, the accumulation of triclosan (TCS) in wastewater sludge is creating a potential risk to human and ecosystem health via the agricultural use of biosolids. The impact of microwave (MW) pretreatment on TCS levels in municipal sludge is unknown. This study, for the first time, evaluated how MW pretreatment (80 and 160 °C) itself and together with anaerobic digestion (AD) under various sludge retention times (SRTs: 20, 12, and 6 days) and temperatures (35 and 55 °C) can affect the levels of TCS in municipal sludge. TCS and its potential transformation products were analyzed with ultra-high-performance liquid chromatography and tandem mass spectrometry. Significantly higher TCS concentrations were detected in sludge sampled from the plant in colder compared to those in warmer temperatures. MW temperature did not have a discernible impact on TCS reduction from undigested sludge. However, AD studies indicated that compared to controls (no pretreatment), MW irradiation could make TCS more amenable to biodegradation (up to 46%), especially at the elevated pretreatment and digester temperatures. At different SRTs studied, TCS levels in the thermophilic digesters were considerably lower than that of in the mesophilic digesters.

Occurrence and fate of antimicrobial triclocarban and its transformation products in municipal sludge during advanced anaerobic digestion using microwave pretreatment.

This study, for the first time, investigated the impact of microwave pretreatment on the fate of the pervasive antimicrobial triclocarban (TCC) that was already present in municipal sludge, before and during advanced anaerobic digestion (AD) under thermophilic and mesophilic conditions. A range of microwave temperature (80 and 160 °C) and exposure duration (1 and 30 min) configurations were studied by employing ten bench-scale anaerobic digesters fed with mixed sludge at three different solids retention times (SRTs) including 20, 12, and 6 days. Seasonal changes influenced the levels of TCC in municipal sludge sampled from a plant employing the biological nutrient removal. Initial batch pretreatment studies showed that microwave irradiation itself can achieve TCC removal efficiencies up to 30 ± 4 and 64 ± 5% at 80 and 160 °C, respectively. The control digesters utilizing un-pretreated mixed sludge showed limited TCC removals, between 18 and 32% and 11-26% respectively, under thermophilic and mesophilic temperatures. On the other hand, the highest TCC elimination (78 ± 2%) was obtained from the thermophilic digester utilizing microwaved sludge at 160 °C for 30 min at SRT of 12 days. The non-chlorinated carbanilide (a transformation product of TCC) was detected and quantified for the first time during conventional and microwave-pretreated anaerobic sludge digestion. The formation of carbanilide in biosolids through reductive dechlorination could be an indicator of efficient and complete TCC transformation. This research demonstrated that AD coupled with microwave pretreatment can be used to reduce environmental concentrations of TCC in municipal sludge and biosolids.

Kinetic and microbial response of activated sludge community to acute and chronic exposure to tetracycline.

Current study aimed to discover both kinetic and microbial response of activated sludge biomass to continuous exposure to tetracycline, one of the most frequently detected antibiotics in wastewaters. Respirometric analysis and model evaluation of the oxygen utilization rate profiles generated at critical phases of the experimental period showed that, continuous exposure to tetracycline caused complete suppression of substrate storage aside from mild inhibition on the growth kinetics and it exerted a significant binding action with available organic carbon, leading to less oxygen consumption. Additionally, increase in endogenous decay rates by 1.5 fold was associated with maintenance energy dictated by the presence and production of antibiotic resistance genes, as demonstrated by resistance gene profile. High-throughput sequencing results showed that continuously exposure to tetracycline caused a significant shift in the community structure at species level so that tetracycline resistant bacteria like Arthrobacter sp and Diaphorobacter sp dominated the bacterial community.

Chronic impact of sulfamethoxazole on the metabolic activity and composition of enriched nitrifying microbial culture.

This study investigated the chronic impact of sulfamethoxazole (SMX) on activated sludge sustaining an enriched nitrifying biomass. For this purpose, a laboratory scale fill and draw reactor was operated with 100 mg COD/L of peptone mixture and 50 mg N/L of ammonia at a sludge age of 15 days. Additionally, the biomass was exposed to a daily SMX dose of 50 mg/L once the reactor reached steady-state conditions. The reactor performance and microbial composition were monitored for 37 days with conventional parameters and molecular techniques based on the gene for ammonia monooxygenase subunit A (amoA) and the prokaryotic 16S rRNA gene. Denaturing gradient gel electrophoresis (DGGE) and 16S rRNA gene cloning analyses suggested a microbial community change concurrent with the addition of SMX. Specifically, quantitative polymerase chain reaction analyses (qPCR/RT-qPCR) revealed a significant reduction in the levels and activity of ammonia oxidizing bacteria (AOB). However, the acclimation period ended with high amoA mRNA levels and improved nitrification efficiency. Partial degradation of SMX by heterotrophic bacteria was also observed.

Is the chronic impact of sulfamethoxazole different for slow growing culture? The effect of culture history.

The study evaluated impact of sulfamethoxazole on acetate utilization kinetics and microbial community structure using respirometric analysis and pyrosequencing. A fill and draw reactor fed with acetate was sustained at a sludge age of 10 days. Acute impact was assessed by modeling of respirometric data in batch reactors started with sulfamethoxazole doses in the range of 25-200 mg/L. Fill and draw operation resumed with continuous sulfamethoxazole dosing of 50 mg/L and the chronic impact was evaluated with acclimated biomass after 20 days. Acute impact revealed higher maintenance energy requirements, activity reduction and slight substrate binding. Chronic impact resulted in retardation of substrate storage. A fraction of acetate was utilized at a much lower rate with partial biodegradation of sulfamethoxazole by the acclimated biomass. Pyrosequencing indicated that Amaricoccus sp. and an unclassified Bacteroidetes sp., possibly with the ability to co-metabolize sulfamethoxazole, dominated the community.

Chronic impact of tetracycline on nitrification kinetics and the activity of enriched nitrifying microbial culture.

This study evaluated the chronic impact of tetracycline on biomass with enriched nitrifying community sustained in a lab-scale activated sludge system. For this purpose, a fill and draw reactor fed with 100 mg COD/L of peptone mixture and 50 mg N/L of ammonia was sustained at a sludge age of 15 days. At steady-state, the reactor operation was continued with a daily tetracycline dosing of 50 mg/L for more than 40 days, with periodic monitoring of the microbial composition, the nitrifying bacteria abundance, as well as the amoA and 16S rRNA gene activity, using molecular techniques. Changes in the kinetics of nitrification were quantified by modelling concentration profiles of major nitrogen fractions and oxygen uptake rate profiles derived from parallel batch experiments. Activated sludge modeling results indicated inhibitory impact of tetracycline on the growth of nitrifiers with a significant increase of the half saturation coefficients in corresponding rate equations. Tetracycline also inactivated biomass components of the enriched culture at a gradually increasing rate with time of exposure, leading to total collapse of nitrification. Molecular analyses revealed significant changes in the composition of the microbial community throughout the observation period. They also showed that continuous exposure to tetracycline inflicted significant reduction in amoA mRNA and 16S rRNA levels directly affecting nitrification. The chronic impact was much more pronounced on the ammonia oxidizing bacteria (AOB) community. These observations explained the basis of numerical changes identified in the growth kinetics of nitrifiers under stress conditions.

Acute impact of erythromycin and tetracycline on the kinetics of nitrification and organic carbon removal in mixed microbial culture.

The study evaluated acute impact of erythromycin and tetracycline on nitrification and organic carbon removal kinetics in mixed microbial culture. Acclimated biomass was obtained from a fill and draw reactor fed with peptone mixture selected as synthetic substrate and operated at a sludge age of 10 days. Acute inhibition was tested in batch reactors involving a control unit started solely with substrate and the others with additional doses of each antibiotic. Modeling indicated that both steps of nitrification were totally blocked by erythromycin. Tetracycline inhibited and retarded nitrification kinetics at 50 mg/L and stopped nitrite oxidation at 200 mg/L, leading to nitrite accumulation. Both antibiotics also affected organic carbon removal by inducing partial inactivation of the heterotrophic community in the culture, increased substrate storage and accelerated endogenous respiration, with a relatively slight impact on heterotrophic growth. Major inhibitory effect was on process stoichiometry, leading to partial utilization of organic substrate.

Biodegradation characteristics and size fractionation of landfill leachate for integrated membrane treatment.

The fate of organics and nitrogen during the biological treatment with MBR and subsequent membrane filtration processes (nano filtration, NF; reverse osmosis, RO) were investigated for a landfill leachate. The chemical oxygen demand (COD) and total Kjeldahl nitrogen (TKN) removal performances of membrane bioreactor (MBR) were obtained to be around 89% and 85%, respectively. The effluent COD of MBR was measured to be 1935 mg/L (30 kDa) which is much lower than experimentally determined soluble inert COD of 3200 mg/L using 0.45 µm filter. The readily and slowly biodegradable COD fractions were estimated to be 17% and 52% of raw influent COD, respectively. The respirometry based modeling test performed on raw leachate exhibited much slower degradation kinetics compared to municipal wastewater. A unique subset of model parameters was extracted from batch respirometry by using acclimated MBR sludge. The sequential ultrafiltration (UF) experiments (particle size distribution, PSD) revealed that most of the organics was below 2 nm filter mesh size. In addition, NF/RO post treatment after MBR system was required to increase COD and total nitrogen (TN) removal performances up to 99%. Relatively lower salt rejection rates around 94% was obtained for RO system as a post treatment of MBR system.

Effect of sludge age on population dynamics and acetate utilization kinetics under aerobic conditions.

The study addressed acetate utilization by an acclimated mixed microbial culture under different growth conditions. It explored changes in the composition of the microbial community and variable process kinetics induced by different culture history. Sequencing batch reactors were operated at steady-state at different sludge ages of two and ten days. Microbial population structure was determined using high-throughput sequencing of 16S rRNA genes. Parallel batch experiments were conducted with acclimated biomass for respirometric analyses. A lower sludge age sustained a different community, which also reflected as variable kinetics for microbial growth and biopolymer storage. The maximum growth rate was observed to change from 3.9/d to 8.5/d and the substrate storage rate from 3.5/d to 5.9/d when the sludge age was decreased from 10 d to 2.0 d. Results challenge the basic definition of heterotrophic biomass in activated sludge models, at least by means of variable kinetics under different growth conditions.

Characteristics of mixed microbial culture at different sludge ages: effect on variable kinetics for substrate utilization.

The study focused on variable kinetics for substrate utilization, primarily addressing the following issue: Is variable process kinetics observed under different operating conditions and culture history (sludge ages), the result of changes inflicted on the metabolic machinery of the same microbial culture? Or, is this the result of a different microbial population selected under different operating conditions? For this purpose, the study mainly emphasized to assess the microbial population composition sustained at different sludge ages. It explored the relationship between observed process kinetics and microbial population structure using respirometric modeling and high-throughput sequencing of 16S rRNA gene. Experimental results indicated a significant change in the composition of the microbial community fed with the same organic substrate, when the culture history was changed, lower sludge age selecting a different and faster growing microbial community.

Is ammonification the rate limiting step for nitrification kinetics?

This study investigated relative magnitude of hydrolysis and ammonification by separate analysis of ammonia release and nitrification mechanisms. A peptone mixture was used as substrate in two parallel experiments seeded with nitrifying biomass conducted with and without nitrification inhibitor. Results were evaluated by means of model analysis of the ammonia and the oxygen uptake rate (OUR) profiles. A dual hydrolysis mechanism with maximum rate coefficients of 6.3 and 0.5/day characterized the peptone mixture and a kinetic balance was established for the ammonia release mechanism with a corresponding ammonification rate of 0.08 m(3)/g COD day. The experiments also showed a low soluble ammonia nitrogen generation that was rapidly depleted, confirming the existence of ammonification. These rate coefficients were verified using model calibration of the OUR profile related to simultaneous carbon removal and nitrification. Results indicated that ammonification would not be rate limiting for wastewaters such as domestic sewage, with lower hydrolysis kinetics.