Trickle-bed Bioreactor Research Articles

Ex-situ single-culture biomethanation operated in trickle-bed configuration: Microbial H2 kinetics and stoichiometry for biogas conversion into renewable natural gas

Biomethanation converts carbon dioxide (CO2) emissions into renewable natural gas (RNG) using mixed microbial cultures enriched with hydrogenotrophic archaea. This study examines the performance of a single methanogenic archaeon converting biogas with added hydrogen (H2) into methane (CH4) using a trickle-bed bioreactor with enhanced gas–liquid mass transport. The process in continuous operation followed the theoretical reaction of hydrogenotrophic methanogenesis (CO2 + 4 H2 → CH4 + 2 H2O), producing RNG with over 99 % CH4 and more than 0.9 H2 conversion efficiency. The Monod constants of H2 uptake were experimentally determined using kinetic modelling. Also, a dimensionless parameter was used to quantify the ratio between the H2 mass transfer rate and the maximum attainable H2 consumption rate. Single-culture biomethanation averts the formation of secondary metabolites and bicarbonate buffer interferences, resulting in lower demands for H2 than mixed-culture biomethanation.

Continuous biogas production and ex-situ biomethanation in a trickling bed bioreactor under mesophilic and thermophilic conditions

The utilization of biogas as a renewable energy source necessitates a reduction in its CO2 content. "Ex-situ biomethanation" is employed to convert CO2 in biogas to CH4 through hydrogenotrophic methanogens. In this study, a biogas stream (32–36 % CO2) originating from cow manure digestion was subjected to treatment in a trickle bed bioreactor (4 liters). The presence of microorganisms on the packing material was confirmed by using a 16 S rRNA test and SEM images. This study stands out by considering all four combinations of thermophilic and mesophilic conditions, monitoring the transition and steady-state phases in a two-reactor series setup. The mesophilic-thermophilic mode yielded the highest purity (92 %) with an 86 % CO2 conversion rate and an energy density of 13,870 kJ/m3. Additionally, the performance consistency was also assessed using a larger TBB (8 liters). Extending the residence time, the thermophilic-thermophilic mode yielded the highest CH4 concentration at 98 %. The 16 S rRNA results revealed that, influenced by the designated growth conditions encompassing culture media, pH, temperature, and reactor retention time, an enrichment of hydrogenotrophic methanogens occurred. Furthermore, promising results in terms of CH4 concentration and process efficiency were demonstrated by the trickled bed bioreactor employing liquid-in-gas dispersion.

Application of a generalized hybrid machine learning model for the prediction of H2S and VOCs removal in a compact trickle bed bioreactor (CTBB)

This study presents a generalized hybrid model for predicting H2S and VOCs removal efficiency using a machine learning model: K–NN (K - nearest neighbors) and RF (random forest). The approach adopted in this study enabled the (i) identification of odor removal efficiency (K) using a classification model, and (ii) prediction of K <100%, based on inlet concentration, time of day, pH and retention time. Global sensitivity analysis (GSA) was used to test the relationships between the inputs and outputs of the K-NN model. The results from classification model simulation showed high goodness of fit for the classification models to predict the removal of H2S and VOCs (SPEC = 0.94–0.99, SENS = 0.96–0.99). It was shown that the hybrid K-NN model applied for the “Klimzowiec” WWTP, including the pilot plant, can also be applied to the “Urbanowice” WWTP. The hybrid machine learning model enables the development of a universal system for monitoring the removal of H2S and VOCs from WWTP facilities.

Assessment of Potential Use of a Composite Based on Polyester Textile Waste as Packing Elements of a Trickle Bed Bioreactor.

Biological wastewater treatment using trickle bed reactors is a commonly known and used solution. One of the key elements of the proper operation of the trickle bed bioreactor is the appropriate selection of biofilm support elements. The respective properties of the bioreactor packing media used can influence, among other things, the efficiency of the treatment process. In this study, the possibility of polyester waste material usage for the preparation of the biofilm support elements was tested. The following properties were checked: adsorption capacity, swelling, surface morphology, microbicidal properties, as well as the possibility of their use in biological wastewater treatment. The tested elements did not adsorb copper nor showed microbicidal properties for bacterial strains Escherichia coli and Staphylococcus aureus as well as fungal strains Aspergillus niger and Chaetomium globosum. The hydrophilic and rough nature of the element surface was found to provide a friendly support for biofilm formation. The durability of the elements before and after their application in the biological treatment process was confirmed by performing tests such as compressive strength, FTIR analysis, hardness analysis and specific surface area measurement. The research confirmed the applicability of the packing elements based on polyester textile waste to the treatment of textile wastewater. The treatment efficiency of the model wastewater stream was above 90%, while in the case of a stream containing 60% actual industrial wastewater it was above 80%. The proposed solution enables the simultaneous management of textile waste and wastewater treatment, which is consistent with the principles of a circular economy. The selected waste raw material is a cheap and easily available material, and the use of the developed packing elements will reduce the amount of polyester materials ending up in landfills.

Characterization of the metabolism of the yeast Yarrowia lipolytica growing as a biofilm.

Yarrowia lipolytica is a well-characterized yeast with remarkable metabolic adaptability. It is capable of producing various products from different carbon sources and easily switching between planktonic and biofilm states. A biofilm represents a natural means of cell immobilization that could support continuous cultivation and production processes, such as perfusion cultivation. However, the metabolic activities of Y. lipolytica in biofilms have not yet been studied in detail. Therefore, this study aimed to compare the metabolic activities of Y. lipolytica in biofilm and planktonic states. Conventionally, a stirred tank bioreactor was used to cultivate Y. lipolytica in a planktonic state. On the other hand, a trickle bed bioreactor system was used for biofilm cultivation. The low pH at 3 was maintained to favor polyol production. The accumulation of citric acid was observed over time only in the biofilm state, which significantly differed from the planktonic state. Although the biofilm cultivation process has lower productivity, it has been observed that the production rate remains constant and the total product yield is comparable to the planktonic state when supplied with 42% oxygen-enriched air. This finding indicates that the biofilm state has the potential for continuous bioprocessing applications and is possibly a feasible option.

Odor and volatile organic compounds biotreatment using compact trickle bed bioreactors (CTBB) in a wastewater treatment plant

The main aim of this study was to optimize and maximize the impacts of odor and volatile organic compounds (VOCs) biodegradation in a wastewater treatment plant utilizing a pilot-scale compact trickle bed bioreactor (CTBB). A CTBB was built and tested for its long-term performance during which gases were supplied from the tank containing semi-liquid fats, oils, and fat waste. The concentrations of pollutants ranged from 0 to 140.75 mg/m3 H2S, 0 to 2500 mg/m3 VOCs, and 0 to 21.5 mg/m3 NH3. The CTBB was tested at different gas flow rates and at two pH values for the liquid phase: pH = 7.0 and 5.0. In the liquid phase, the pollutant removal efficiency was higher at pH = 7.0 than at pH = 5.0. Overall, the removal efficiency was between 81.5 % and 99.5 % for the VOCs and 87.5 % and 98.9 % for H2S, while NH3 removals were >99 %.

Removal of volatile organic compounds and hydrogen sulfide in biological wastewater treatment plant using the compact trickle-bed bioreactor

Removal of volatile organic compounds and hydrogen sulfide in biological wastewater treatment plant using the compact trickle-bed bioreactor

Trickle-Bed Bioreactors for Acetogenic H2/CO2 Conversion

Acetic acid is an essential industrial building block and can be produced by acetogenic bacteria from molecular hydrogen (H2) and carbon dioxide (CO2). When gasses are supplied as substrates, bioreactor design plays an important role for their availability. Trickle-bed bioreactors (TBs) have an enhanced gas-to-liquid mass transfer and cells remain in the system by forming a biofilm on the carriers. So far, TBs have been investigated extensively for bio-methanation processes, whereas studies for their use in acetic acid production are rare. In this study, we evaluated the reproducibility of two parallel TBs for acetic acid production from H2:CO2(= 70:30) by a mixed culture with a gas flow rate of 3.8 mL min−1and a medium flow rate of 10 mL min−1. Additionally, the effect of glucose addition during the starting phase on the resulting products and microbial composition was investigated by setting up a third TB2. Partial medium exchanges to decrease the internal acetic acid concentration (AAC) combined with recycling of withdrawn cells had a positive impact on acetic acid production rates with maxima of around 1 g L−1d−1even at high AACs of 19–25 g L−1. Initial glucose addition resulted in the accumulation of unwanted butyric acid up to concentrations of 2.60 ± 0.64 g L−1. The maximum AAC of 40.84 g L−1was obtained without initial glucose addition. The main families identified in the acetogenic TBs were Peptococcaceae, Ruminococcaceae, Planococcaceae, Enterobacteriaceae, Clostridiaceae, Lachnospiraceae, Dysgonomonadaceae and Tannerellaceae. We conclude that a TB is a viable solution for conversion of H2/CO2to acetate using an anaerobic enrichment culture.

Development and adaptation of the technology of air biotreatment in trickle-bed bioreactor to the automotive painting industry

The automotive painting industry is a source of environmental pollution caused by Volatile Organic Compounds (VOCs) present in the discharged ventilation air. To contribute to the mitigation of this type of air pollution, Ekoinwentyka Ltd. developed – from pilot scale to full scale – and adapted the technology of Compact Trickle Bed Bioreactor (CTBB) whose operating principle builds upon co-current downflow of the gas phase (polluted air) and liquid phase (solution of mineral salts) through a packed bed where active microorganisms are immobilized in the biofilm on the surfaces of packing elements. Pilot-scale bioreactor 0.32 m in diameter and 1.50 m total height had its packed bed inoculated with a consortium of microorganisms dominated by Pseudomonas fluorescens bacteria. During the experimental programme that lasted several months, the flow rate of air drawn from the ventilation system of the painting shop was changing between 1.0 and 10.0 m3/h and the inlet concentration of VOCs ranged from 10 to 200 ppm. By measuring VOC concentration in the purified air, the factor of VOC biodegradation was found to range between 85 and 99%. Based on pilot-scale experiments, full-scale CTBB has been developed 2.8 m in diameter and 10 m total height and installed as an add-on component of the ventilation system of the painting shop. Test operation at gas flow rates up to 6000 m3/h, confirmed VOC biodegradation factor at the level of 85–99% thus proving a positive result of CTBB technology adaptation to the conditions of the automotive painting industry.

Scale up study of a thermophilic trickle bed reactor performing syngas biomethanation

Biomethane constitutes an important biofuel for the transition towards a biobased circular economy with a sustainable energy sector. An environmentally friendly pathway for its production is through the gasification of 2nd generation biomass that generates syngas (H2, CO, and CO2), followed by syngas biomethanation. A novel design thermophilic trickle bed bioreactor was successfully tested at semi-pilot scale. It was operated continuously performing biomethanation of an artificial syngas mixture (45% H2, 20% CO, 25% CO2 and 10% N2). Its effectiveness was compared to a primitive design lab scale trickle bed reactor with a 28 times lower bed volume under identical operating conditions. At an empty bed residence time of 0.6 h, the novel semi-pilot scale trickle bed reactor converted 100% and 98% of the influent H2 and CO, respectively and produced CH4 at a rate of 10.6 ± 0.2 mmol·lbed−1·h−1, whereas the lab scale reactor converted 89% of the H2 and 73% of the CO and achieved a CH4 productivity of 8.5 mmol·lbed−1·h−1. The maximum CH4 productivity achieved in the semi-pilot scale reactor was 17.6 ± 0.6 mmol·lbed−1·h−1 at an empty bed residence time of 0.33 h with a 99.2 ± 0.1% product selectivity. Furthermore, the semi-pilot scale reactor was connected in series with a fluidized bed gasifier fed with wood pellets to assess the biomethanation potential of the system when supplied with real syngas. The obtained results showed no process inhibition in the semi-pilot scale reactor, which accomplished 100% H2 conversion efficiency and 92.4 ± 0.6% CO conversion efficiency at an empty bed residence time of 0.6 h.

Removal of pharmaceuticals and ecotoxicological changes in wastewater using Trametes versicolor: A comparison of fungal stirred tank and trickle-bed bioreactors

Hospital wastewater is loaded with pharmaceutically active compounds (PhACs), which sometimes end up in the environment unaltered due to the lack of effective and specific treatments. The use of white rot fungi, specifically Trametes versicolor, as a biological treatment has proven to be an effective and environmentally friendly technology to tackle this problem. However, only a few studies have dealt with the removal of pharmaceuticals from wastewater by immobilizing white rot fungi in a lignocellulosic substrate. In this work, two bioreactor configurations are utilized to treat both synthetic and real hospital wastewater by the use of T. versicolor. The stirred tank bioreactor (STB) was able to remove to a great extent 16 pharmaceuticals spiked in the synthetic wastewater (95.7%) and those naturally present in the hospital wastewater (85.0%). Nonetheless, acute toxicity tests with Daphnia magna and germination index with Lactuca sativa showed an increase in the general toxicity of both wastewater matrices after the treatment. On the other hand, a trickle-bed bioreactor (TBB), using fungal biomass immobilized on rice husks, achieved an elimination of 88.6% and 89.8% in synthetic and real wastewater, respectively. However, 73.3% of the removal was ascribed to adsorption to the bed’s biomass. Additionally, toxicological tests showed a decrease in the hospital wastewater’s toxicity after the treatment in the TBB. These findings suggest that the fungal fixed-bed reactor could be more suitable than the stirred tank reactor to remove pharmaceutical compounds from wastewater, since it was able to remove PhACs to a great extent and simultaneously detoxify real wastewater.

Implementation to Industry and Municipal Sector the Compact Trickle Bed Bioreactors Technology to Odor and Vocs Removal

Abstract Biotrickling filters are one of the most effective methods of air bio-purification, this bioremediation process if of high efficiency in pollution reduction. It is an eco-friendly process and economically viable. The technology of biotrickling filters includes Compact Trickle Bed Bioreactors (CTBB), which are currently used in an increasingly wide range. The aim of this work will be an objective assessment of the implementation potential of the CTBB technology to various industries, including the municipal sector. The paper briefly discusses the characteristics and operating parameters of biotrickling filters, a review of their applications as an effective method of VOC and odor removal including sources of their emissions, as well as the characteristics of CTBB and implementation possibilities to various industries. It was concluded that CTBB are promising solution for the future, as it combines the high degradation efficiency of a wide range of pollutants with cost-effectiveness and ecology. According to the analyzed data and results, this technology can be successfully used to remove VOCs and odors from various industries.

Fungal bioremediation of diuron-contaminated waters: Evaluation of its degradation and the effect of amendable factors on its removal in a trickle-bed reactor under non-sterile conditions

The occurrence of the extensively used herbicide diuron in the environment poses a severe threat to the ecosystem and human health. Four different ligninolytic fungi were studied as biodegradation candidates for the removal of diuron. Among them, T. versicolor was the most effective species, degrading rapidly not only diuron (83%) but also the major metabolite 3,4-dichloroaniline (100%), after 7-day incubation. During diuron degradation, five transformation products (TPs) were found to be formed and the structures for three of them are tentatively proposed. According to the identified TPs, a hydroxylated intermediate 3-(3,4-dichlorophenyl)-1-hydroxymethyl-1-methylurea (DCPHMU) was further metabolized into the N-dealkylated compounds 3-(3,4-dichlorophenyl)-1-methylurea (DCPMU) and 3,4-dichlorophenylurea (DCPU). The discovery of DCPHMU suggests a relevant role of hydroxylation for subsequent N-demethylation, helping to better understand the main reaction mechanisms of diuron detoxification. Experiments also evidenced that degradation reactions may occur intracellularly and be catalyzed by the cytochrome P450 system. A response surface method, established by central composite design, assisted in evaluating the effect of operational variables in a trickle-bed bioreactor immobilized with T. versicolor on diuron removal. The best performance was obtained at low recycling ratios and influent flow rates. Furthermore, results indicate that the contact time between the contaminant and immobilized fungi plays a crucial role in diuron removal. This study represents a pioneering step forward amid techniques for bioremediation of pesticides-contaminated waters using fungal reactors at a real scale.

Biodegradation of PCBs in contaminated water using spent oyster mushroom substrate and a trickle-bed bioreactor

Due to their persistence, polychlorinated biphenyls (PCBs) represent a group of important environmental pollutants, but conventional physicochemical decontamination techniques for their removal are usually expensive. The main aim of this work was to develop a cost-effective method for PCB bioremediation, focusing on contaminated water and utilizing the well-known degradation capability of Pleurotus ostreatus (the oyster mushroom). For this purpose, the conditions of several laboratory-scale reactors (working volume 1 L) were optimized. Spent oyster mushroom substrate obtained from a commercial farm was used as a fungal inoculum and growth substrate. The highest degradation efficiency (87%) was recorded with a continuous low-flow setup, which was subsequently scaled up (working volume 500 L) and used for the treatment of 4000 L of real contaminated groundwater containing 0.1–1 μg/L of PCBs. This trickle-bed pilot-scale bioreactor was able to remove 82, 80, 65, and 30–50% of di-, tri-, tetra- and pentachlorinated PCB congeners, respectively. No degradation was observed for hexa- or heptachlorinated congeners. Multiple mono- and dichlorobenzoic acids (CBAs) were identified as transformation products by mass spectrometry, confirming the role of biodegradation in PCB removal. A Vibrio fischeri bioluminescence inhibition test revealed slight ecotoxicity of the primary reactor effluent (sampling after 24 h), which was quickly suppressed once the effluent passed through the reactor for the second time. Moreover, no other effluent exhibited toxicity for the rest of the experiment (71 days in total). Microbial analyses (phospholipid fatty acid analysis and next-generation sequencing) showed that P. ostreatus was able to degrade PCBs in the presence of an abundance of other fungal species as well as aerobic and anaerobic bacteria. Overall, this study proved the suitability of the use of spent oyster mushroom substrate in a bioremediation practice, even for pollutants as recalcitrant as PCBs.

Biological Treatment Processes for the Removal of Organic Micropollutants from Wastewater: a Review

Micropollutants or contaminants of emerging concern (CECs) are released into the environment from a wide variety of sources. Due to the adverse effect on human health, micropollutant-containing wastewater needs to be treated before its discharge. A number of conventional physicochemical methods have been extensively studied for micropollutant degradation. However, owing to their one or more disadvantages, biological treatment using suitable microorganisms is of recent interest. Numerous bacteria and fungi are capable of degrading these micropollutants even at high concentrations. However, in order for the biological treatment to be commercially viable and industrially scalable, bioprocess development with efficient bioreactor systems is highly essential. This paper reviews state-of-the-art techniques for the removal of micropollutants by conventional biological systems such as activated sludge process, biofilm-based reactor, and trickling bed bioreactor. However, compared with conventional systems, advanced biological systems, namely two-phase partitioning bioreactor, membrane-based reactor, and cell-immobilized bioreactor systems, have not been examined and, hence, need detailed exploration. Such advanced treatment systems are capable of tolerating high pollutant load and are also able to treat highly water insoluble pollutants. Furthermore, hybrid systems comprising of a combination of different physicochemical and biological processes are discussed in this paper, which are not only capable of improving the treatment efficiency but also eliminate any accumulation of the toxic by-product produced during the treatment. Among the different hybrid systems, a combination of different biological systems is found to be highly efficient in treating micropollutant-containing wastewater. Finally, scope for future research prospects in the field are derived and addressed in details.

Production of Fungal Phytase in an Innovative Trickle Bed Bioreactor

Excessive temperature rise and drying of substrates are two common problems in solid state fermentation. In order to overcome these problems, an innovative trickle bed bioreactor (TBBR) was designed and tested for the production of fungal phytase by the cultivation of Aspergillus ficuum on wheat straw. In this reactor, a helical perforated bed acts as a support for the substrate. Rotation of this bed in a baffled column produces gentle mixing and separates agglomerated substrates. Intermittent water trickling during fermentation removes product and heat from the fermentation medium partially. For phytase production in the TBBR, the bioreactor was filled with moistened sterilized wheat straw, inoculated with the spores of the fungus. After 3 days, water was trickled on the substrate for 1 min every 24 h. The enzyme in the trickling liquid, and the extracted enzyme from the residual straw after fermentation, were quantified. An equal size traditional packed bed bioreactor (PBBR) was also operated as a control. Phytase production in this TBBR was 54.95% higher than the PBBR. Monitoring the temperature in both bioreactors, moreover, showed lower temperature increase in the TBBR compared to the PBBR.

Application of a compact trickle-bed bioreactor for the removal of odor and volatile organic compounds emitted from a wastewater treatment plant

A compact trickle-bed bioreactor (CTBB) was tested for the removal of volatile organic compounds (VOCs) and hydrogen sulphide (H2S) present in the exhaust air of a wastewater treatment plant. At gas-flow rates varying between 2.0 and 30.0 m3/h and for specific pollutant loads up to 20 g/(m3·h), removal efficiencies for H2S and VOC were >95%. The CTBB was designed for a maximum H2S concentration of ∼200 ppm and removal efficiencies >97% were noticed. VOC concentrations were in the range of 25–240 ppmv and the removal efficiency was in the range of 85–99%. Possible consequences of an excessive pollutant overload and the time required for regenerating the microbial activity and reviving stable process conditions in the CTBB were also investigated. An increase in the H2S concentration from 400 to 600 ppmv for a few hours caused bioreactor poisoning; however, when original H2S concentrations were restored, stable CTBB operation was ascertained within 3 h.

Biodegradation of vegetable residues by polygalacturonase-agar using a trickle-bed bioreactor

Bacterial pectinases degrade the pectic substances present in plant tissues and particularly, polygalacturonases catalyze the hydrolysis of α-(1,4) glycosidic bonds linking d-galacturonic acid units. In this study, polygalacturonase from Streptomyces halstedii ATCC 10897 was immobilized by the matrix entrapment technique using different thermogels. Bacteriological agar added with magnesium cation produced beads with a more stabilized microstructure for enzyme retention, monitored by oscillatory measurements of storage and loss modulus. Agar concentration and protein content were optimized to maximize protein entrapment, product conversion, and reaction yield. Results showed that the mixture at 10:90% (v/v) of protein (2mg/mL) and agar (4% w/v) was the best immobilization condition to retain 91% of protein and hydrolyze 38% of pectin to allow the highest reaction yield (9.279g/g) and increase stability up to 48h of successive reactions. Agarose bead biocatalysts were used in a trickle-bed column operated with recirculation, and this bioreactor allowed the degradation of pear and cucumber residues by enzymatic liquefaction to enhance sugar content up to 15.33 and 9.35mg/mL, respectively, and decrease viscosity by 92.3%. The scale-up of this process adds value to vegetable residues such as fructooligosaccharides or fermentable sugars, which become a sustainable source of fuels and chemicals.

Development of the mathematical model of the biotreatment process of water-soluble gaseous emissions

In the experimental research, the kinetic characteristics of hydrogen sulfide, sulfur dioxide and ammonia biooxidation were determined. It was found that the hydrogen sulfide and sulfur dioxide oxidation rates varied from 12 mg/g of biomass per hour in the region of the minimum pollution concentrations to the maximum value of 40 mg/g·h. As for ammonia, the variation range was 1.5–5 mg/g·h, respectively. The analytical description of the dependence of the specific degradation rate of pollutants on their concentration was proposed. The obtained quantitative values and variation nature of the specific oxidation rate prove the technological possibility of using the trickle-bed bioreactor for treatment of water-solvable gaseous emissions. Based on the experimental research, the mathematical description of the non-stationary biooxidation process of water-soluble gaseous hydrogen sulfide, sulfur dioxide and ammonia was developed. The developed mathematical model is based on the mass balance in the trickling layer of the bioreactor in the course of absorption and biodegradation processes. The analytical dependencies consider the emergence of dynamic balance between the harmful matter arrival intensity and oxidation. The state of dynamic balance determines the boundary of the bioreactor efficiency. The results allow evidence-based calculations of the hydrogen sulfide, sulfur dioxide and ammonia biotreatment process in the trickle-bed bioreactor.

Development and evaluation of a trickle bed bioreactor for enhanced mass transfer and methanol production from biogas

Biological conversion of the biogas produced by landfills and anaerobic digestion systems (60–70% methane (CH4), 30–40% carbon dioxide (CO2)) to methanol using methanotrophs (aerobic CH4-oxidizing bacteria) is an emerging approach to convert waste-derived biogas to liquid chemicals and fuels. The purpose of this work was to develop a trickle-bed reactor (TBR) to improve mass transfer of CH4 and oxygen (O2) to methanotroph growth media for enhanced CH4 oxidation and methanol production. Mass transport of O2 in a TBR packed with ceramic balls was nearly two-fold higher than an unpacked TBR. CH4 oxidation in the TBR (0.4–0.6mmol/h) was about four times higher than that in shake flasks that used similar inoculum and headspace:volume and biogas:air ratios. Using optimal operating parameters (biogas:air=1:2.5, 12mmol formate addition, 3.6mmol phosphate), methanol productivity (0.9g/L/d) from the non-sterile TBR was among the highest reported in the literature. Operation under non-sterile conditions caused differences in the microbial community composition between experiments, and the most predominant methanotrophs appeared to be members of the genus in which the inoculum is classified (Methylocaldum sp. 14B).