Effect of Rhodococcus opacus PD630 on selenium phytoremediation by Brassica oleracea.

The purpose of this study was to evaluate the potential of microbial-enhanced Brassica oleracea for the phytoremediation of seleniferous soils. The effect of selenite (Se(IV)) and selenate (Se(VI)) on B. oleracea (1-100 mg.L-1) was examined through germination (7 d) and pot (30 d) trials. Microbial analysis was conducted to verify the toxic effect of various Se concentrations (1-500 mg.L-1) on Rhodococcus opacus PD360, and to determine if it exhibits plant growth promoter traits. R. opacus PD630 was found to tolerate high concentrations of both Se(IV) and Se(VI), above 100 mg.L-1. R. opacus PD630 reduced Se(IV) and Se(VI) over 7 days, with a Se conversion efficiency between 60 and 80%. Germination results indicated lower concentrations (0-10 mg.L-1) of Se(IV) and Se(VI) gave a higher shoot length (> 4 cm). B. oleracea accumulated 600-1,000 mg.kg-1 dry weight (DW) of Se(IV) and Se(VI), making it a secondary accumulator of Se. Moreover, seeds inoculated with R. opacus PD360 showed increased Se uptake (up to 1,200 mg Se.kg-1 DW). In addition, bioconcentration and translocation factors were greater than one. The results indicate a synergistic effect between R. opacus PD630 and B. oleracea for Se phytoextraction from polluted soils.

This article examines how Brassica oleracea may be used to improve seleniferous soils and how Rhodococcus opacus can be added to increase biofortification. The research shows great potential for combining Brassica species with bacterial isolates to remove selenium from heavily contaminated soils.

Selenite (IV) and selenate (VI) uptake and accumulation capacity of Lemna minor L. from an aquatic medium.

The uptake of sodium selenite (Se(IV)) and sodium selenate (Se(VI)) from aqueous medium by Lemna minor L. and the influence of different Se concentrations on its growth, morphological and ultrastructural characteristics were studied. L. minor was grown at different concentrations (1, 3, 5 and 10 mg L-1) of Se(IV) and Se(IV). The Se(IV) concentration in the plant tissue ranged between 77.7 (± 4.3) to 453 (± 0) mg kg-1 DW. The Se(VI) concentration in plant tissues ranged between 117 (± 11) to 417 (± 2) mg kg-1 DW. The highest bioconcentration factor for Se(VI) was 127 (± 7) at 3 mg/L, with a Se removal efficiency of 44%. For Se(IV), the highest bioconcentration factor was 77.7 (± 4.3) at 1 mg L-1, which had a Se removal efficiency of 23%. Growth of L. minor was suppressed at 10 mg L-1 Se in both forms. The addition of Se promoted the formation of starch granules in L. minor which occupied a chloroplast area of 74% for Se(IV) and 77% for Se(VI). The efficient uptake of both Se forms by L. minor indicates the potential application of this species for phytoremediation of Se laden wastewaters and its use as an alternative feedstock in biofuel production.

Effect of copper, arsenic and nickel on pyrite-based autotrophic denitrification.

Pyritic minerals generally occur in nature together with other trace metals as impurities, that can be released during the ore oxidation. To investigate the role of such impurities, the presence of copper (Cu(II)), arsenic (As(III)) and nickel (Ni(II)) during pyrite mediated autotrophic denitrification has been explored in this study at 30 °C with a specialized microbial community of denitrifiers as inoculum. The three metal(loid)s were supplemented at an initial concentration of 2, 5, and 7.5 ppm and only Cu(II) had an inhibitory effect on the autotrophic denitrification. The presence of As(III) and Ni(II) enhanced the nitrate removal efficiency with autotrophic denitrification rates between 3.3 [7.5 ppm As(III)] and 1.6 [7.5 ppm Ni(II)] times faster than the experiment without any metal(loid) supplementation. The Cu(II) batches, instead, decreased the denitrification kinetics with 16, 40 and 28% compared to the no-metal(loid) control for the 2, 5 and 7.5 ppm incubations, respectively. The kinetic study revealed that autotrophic denitrification with pyrite as electron donor, also with Cu(II) and Ni(II) additions, fits better a zero-order model, while the As(III) incubation followed first-order kinetic. The investigation of the extracellular polymeric substances content and composition showed more abundance of proteins, fulvic and humic acids in the metal(loid) exposed biomass.

Deciphering anaerobic ethanol metabolic pathways shaped by operational modes.

Efficient anaerobic digestion requires the syntrophic cooperation among diverse microorganisms with various metabolic pathways. In this study, two operational modes, i.e., the sequencing batch reactor (SBR) and the continuous-flow reactor (CFR), were adopted in ethanol-fed systems with or without the supplement of powdered activated carbon (PAC) to examine their effects on ethanol metabolic pathways. Notably, the operational mode of SBR and the presence of CO2 facilitated ethanol metabolism towards propionate production. This was further evidenced by the dominance of Desulfobulbus, and the increased relative abundances of enzymes (EC: 1.2.7.1 and 1.2.7.11) involved in CO2 metabolism in SBRs. Moreover, SBRs exhibited superior biomass-based rates of ethanol degradation and methanogenesis, surpassing those in CFRs by 53.1% and 22.3%, respectively. Remarkably, CFRs with the extended solids retention time enriched high relative abundances of Geobacter of 71.7% and 70.4% under conditions with and without the addition of PAC, respectively. Although both long-term and short-term PAC additions led to the increased sludge conductivity and a reduced methanogenic lag phase, only the long-term PAC addition resulted in enhanced rates of ethanol degradation and propionate production/degradation. The strategies by adjusting operational mode and PAC addition could be adopted for modulating the anaerobic ethanol metabolic pathway and enriching Geobacter.

New insights into syntrophic ethanol oxidation: Effects of operational modes and solids retention times.

Anaerobic ethanol oxidation relies on syntrophic interactions among functional microorganisms to become thermodynamically feasible. Different operational modes (sequencing batch reactors, SBRs, and continuous flow reactors, CFRs) and solids retention times (SRT, 25 days and 10 days) were employed in four ethanol-fed reactors, named as SBR25d, SBR10d, CFR25d, and CFR10d, respectively. System performance, syntrophic relationships, microbial communities, and metabolic pathways were examined. During the long-term operation, 2002.7 ± 56.0 mg COD/L acetate was accumulated in CFR10d due to the washout of acetotrophic methanogens. Microorganisms with high half-saturation constants were enriched in reactors of 25-day SRT. Moreover, ethanol oxidizing bacteria and acetotrophic methanogens with high half-saturation constants could be acclimated in SBRs. In SBRs, Syner-01 and Methanothrix dominated, and the low SRT of 10 days increased the relative abundance of Geobacter to 38.0%. In CFRs, the low SRT of 10 days resulted in an increase of Desulfovibrio among syntrophic bacteria, and CFR10d could be employed in enriching hydrogenotrophic methanogens like Methanobrevibacter.

Influence of pH, Heat Treatment of Inoculum, and Selenium Oxyanions on Concomitant Selenium Bioremediation and Volatile Fatty Acid Production from Food Waste.

Developing novel strategies to enhance volatile fatty acid (VFA) yield from abundant waste resources is imperative to improve the competitiveness of biobased VFAs over petrochemical-based VFAs. This study hypothesized to improve the VFA yield from food waste via three strategies, viz., pH adjustment (5 and 10), supplementation of selenium (Se) oxyanions, and heat treatment of the inoculum (at 85 °C for 1 h). The highest VFA yield of 0.516 g COD/g VS was achieved at alkaline pH, which was 45% higher than the maximum VFA production at acidic pH. Heat treatment resulted in VFA accumulation after day 10 upon alkaline pretreatment. Se oxyanions acted as chemical inhibitors to improve the VFA yield at pH 10 with non-heat-treated inoculum (NHT). Acetic and propionic acid production was dominant at alkaline pH (NHT); however, the VFA composition diversified under the other tested conditions. More than 95% Se removal was achieved on day 1 under all the conditions tested. However, the heat treatment was detrimental for selenate reduction, with less than 15% Se removal after 20 days. Biosynthesized Se nanoparticles were confirmed by transmission and scanning electron microscopy and and energy dispersive X-ray analyses. The heat treatment inhibited the presence of nonsporulating bacteria and methanogenic archaea (Methanobacteriaceae). High-throughput sequencing also revealed higher relative abundances of the bacterial families (such as Clostridiaceae, Bacteroidaceae, and Prevotellaceae) that are capable of VFA production and/or selenium reduction.

Biofilm metagenomic characteristics behind high coulombic efficiency for propanethiol deodorization in two-phase partitioning microbial fuel cell.

Hydrophobic volatile organic sulfur compounds (VOSCs) are frequently found during sewage treatment, and their effective management is crucial for reducing malodorous complaints. Microbial fuel cells (MFC) are effective for both VOSCs abatement and energy recovery. However, the performance of MFC on VOSCs remains limited by the mass transfer efficiency of MFC in aqueous media. Inspired by two-phase partitioning biotechnology, silicone oil was introduced for the first time into MFC as a non-aqueous phase (NAP) medium to construct two-phase partitioning microbial fuel cell (TPPMFC) and augment the mass transfer of target VOSCs of propanethiol (PT) in the liquid phase. The PT removal efficiency within 32 h increased by 11-20% compared with that of single-phase MFC, and the coulombic efficiency of TPPMFC (11.01%) was 4.32-2.68 times that of single-phase MFC owing to the fact that highly active desulfurization and thiol-degrading bacteria (e.g., Pseudomonas, Achromobacter) were attached to the silicone oil surface, whereas sulfur-oxidizing bacteria (e.g., Thiobacillus, Commonas, Ottowia) were dominant on the anodic biofilm. The outer membrane cytochrome-c content and NADH dehydrogenase activity improved by 4.15 and 3.36 times in the TPPMFC, respectively. The results of metagenomics by KEGG and COG confirmed that the metabolism of PT in TPPMFC was comprehensive, and that the addition of a NAP upregulates the expression of genes related to sulfur metabolism, energy generation, and amino acid synthesis. This finding indicates that the NAP assisted bioelectrochemical systems would be promising to solve mass-transfer restrictions in low solubility contaminates removal.

Effect of different inocula on the granulation process, reactor performance and biodiesel production of algal-bacterial granular sludge (ABGS) under low aeration conditions.

The algal-bacterial granular sludge (ABGS) system is a prospective wastewater treatment technology, but few studies focused on the effects of different inoculum types on the establishment of the ABGS system under low aeration conditions (step-decrease superficial gas velocity from 1.4 to 0.5 cm/s). Results from this study indicated that compared with other inocula, the ABGS formed by co-inoculating aerobic granular sludge (AGS) and targeted algae (Chlorella) exhibited a shorter granulation period (shortened by 15 days), higher total nitrogen (89.4%) and PO43--P (95.0%) removal efficiencies, and a greater yield of fatty acid methyl esters (FAMEs) (9.04 mg/g MLSS). This was possibly attributed to that the functional bacteria (e.g. Thauera, Gemmobacter and Rhodobacter) in the inoculated AGS facilitated the ABGS granulation. The inoculated algae promoted their effective enrichment under illumination conditions and enhanced the production of extracellular polymeric substances, thus improving the stability of ABGS. The enriched algae were attached to the outer layer of the granules, which could provide sufficient oxygen for bacterial metabolism, revealing the inherent mechanisms for the good stability of ABGS under low aeration intensity. Overall, the rapid granulation of ABGS can be achieved by inoculating optimal inocula under low aeration conditions, which is convenient and economically feasible, and motivates the application of algal-bacterial consortia.

Simultaneous removal of sulfide and bicarbonate from synthetic wastewater using an algae-assisted microbial fuel cell.

The anaerobic digestion (AD) process is one of the most practiced technologies for the remediation of organic waste and maximization of energy recovery in terms of biogas or biomethane. The presence of other gaseous components in biogas, e.g. CO2 and H2S, often makes its direct application in engines and electricity production unsuitable. This work aimed to develop and utilize an algae-assisted microbial fuel cell (AMFC) for the purification of biogas by removing both CO2 and H2S and simultaneous bioelectricity generation. In addition to biogas clean-up, elemental sulfur recovery and CO2 utilization for algae cultivation add value to the proposed AMFC process. Experiments were performed with both sulfide and bicarbonate in their dissolved form, in the respective anodic and cathodic chambers of the AMFC. The sulfide concentration was varied from 100 to 800 mg/l and the AMFC exhibited a sulfide removal efficiency exceeding 97% at all concentrations tested. The process efficiency dropped, however, at sulfide concentrations above 300 mg/l in terms of both sulfide removal and power output. The AMFC performed best at 400 mg/l sulfide by exhibiting a power density of 24.99 mW/m3 and sulfide removal efficiency of 98.87%. The system exhibited columbic efficiency (CE %) in the range of 7.85-80%. The total alkalinity representing CO2, carbonate and bicarbonate levels in the algae-based system was reduced by 49.54%. The electrical energy recovered from the AMFC was 0.1 kWh/m3 and the total energy recovery, which is the sum of the electrical and algal lipid energy, amounted to 7.25 kWh/m3.

Integrated bioprocess for Se(VI) remediation using duckweed: Coupling selenate removal to biogas production.

The use of phytoremediation as a method for wastewater treatment or removal of pollutants is garnering significant interest and duckweed (DW), a free floating macrophyte, depicts significant potential for the removal of nutrients and toxic compounds from contaminated waters. The present work aimed to develop an integrated process for remediating selenate (Se(VI)) using DW biomass and subsequent use of Se(VI) enriched DW for biogas production. The main objective is to extend the application of selenium (Se) enriched DW biomass for biogas production. Se(VI) enriched DW biomass (Se-DW) gave higher methane production (48.38 ± 3.6 mL gCOD-1) than control DW biomass (C-DW) (24.46 ± 3.6 mL gCOD-1). To further enhance methane production, three pre-treatment approaches (acid, alkali and hydrothermal) were assessed and the solid and liquid fractions obtained after pre-treatment were used as a substrate. Pre-treatments increased biogas production in both Se-DW and C-DW than untreated conditions. Liquid fractions gave higher biogas production than solid fractions. In Se-DW, highest biogas production was observed in hydrothermal pre-treated Se-DW, while in C-DW, acid pre-treatment gave higher biogas production. Methane production was shown to be enhanced up to a Se(VI) concentration of 1.7 mg L-1, whereas a concentration beyond this lowered biogas production.

Sustainable biorefining and bioprocessing of green seaweed (Ulva spp.) for the production of edible (ulvan) and non-edible (polyhydroxyalkanoate) biopolymeric films.

A sustainable biorefining and bioprocessing strategy was developed to produce edible-ulvan films and non-edible polyhydroxybutyrate films. The preparation of edible-ulvan films by crosslinking and plasticisation of ulvan with citric acid and xylitol was investigated using Fourier transform infrared (FTIR) spectroscopy and differential scanning calorimetry (DSC) analysis. The edible ulvan film was tested for its gut-friendliness using Lactobacillus and Bifidobacterium spp. (yoghurt) and was shown to improve these gut-friendly microbiome's growth and simultaneously retarding the activity of pathogens like Escherchia coli and Staphylococcus aureus. Green macroalgal biomass refused after the extraction of ulvan was biologically processed by dark fermentation to produce a maximum of 3.48 (± 0.14) g/L of volatile fatty acids (VFAs). Aerobic processing of these VFAs using Cupriavidus necator cells produced 1.59 (± 0.12) g/L of biomass with 18.2 wt% polyhydroxybutyrate. The present study demonstrated the possibility of producing edible and non-edible packaging films using green macroalgal biomass as the sustainable feedstock.

Pilot and full scale applications of floating treatment wetlands for treating diffuse pollution.

Floating treatment wetlands (FTW) are nature-based solutions for the purification of open water systems such as rivers, ponds, and lakes polluted by diffuse sources as untreated or partially treated domestic wastewater and agricultural run-off. Compared with other physicochemical and biological technologies, FTW is a technology with low-cost, simple configuration, easy to operate; has a relatively high efficiency, and is energy-saving, and aesthetic. Water remediation in FTWs is supported by plant uptake and the growth of a biofilm on the water plant roots, so the selection of the macrophyte species is critical, not only to pollutant removal but also to the local ecosystem integrity, especially for full-scale implementation. The key factors such as buoyant frame/raft, plant growth support media, water depth, seasonal variation, and temperature have a considerable role in the design, operation, maintenance, and pollutant treatment performance of FTW. Harvesting is a necessary process to maintain efficient operation by limiting the re-pollution of plants in the decay phase. Furthermore, the harvested plant biomass can serve as a green source for the recovery of energy and value-added products.

Lactic acid fermentation of food waste in a semicontinuous SBR system: influence of the influent composition and hydraulic retention time.

Fermentation processes have been shown to be a good approach to food waste (FW) management. Among the commodities that can be bioproduced by using FW as an organic substrate and exploiting its biodegradability, there is lactic acid (LA). LA has gained the interest of research because of its role in the production of polylactic acid plastics. In this study, the influence of the HRT (2-5 days) used during the fermentation of the liquid fraction (∼12-13 g COD/L) of FW on LA yield and concentration was investigated. Moreover, the changes in the chemical composition (in terms of carbohydrates and organic metabolites concentration) of the influent occurring in the feeding tank were monitored and its influence on the downstream fermentation process was examined. High instability characterized the reactor run with the optimal production yield obtained on day 129 at an HRT 2 days with 0.81 g COD/g COD. This study shows the importance of the fluctuating composition of FW, a very heterogeneous and biologically active substrate, for the LA fermentation process. The non-steady state fermentation process was directly impacted by the unstable influent and shows that a good FW storage strategy has to be planned to achieve high and constant LA production.

Metagenomic analysis reveals the responses of microbial communities and nitrogen metabolic pathways to polystyrene micro(nano)plastics in activated sludge systems.

Microplastics (MPs) and nanoplastics (NPs) are prevalent in sewage and pose a potential threat to nitrogen biotransformation in wastewater treatment systems. However, investigations on how MPs and NPs affect the microbial nitrogen conversion and metabolism of the activated sludge are still scanty. Herein, the responses of microbiomes and functional genes to polystyrene MPs and NPs in activated sludge systems were investigated by metagenomic analysis. Results indicated that 1 mg/L MPs and NPs had marginal impacts on the nitrogen removal performance of the activated sludge systems, whereas high concentrations of MPs and NPs (20 and 100 mg/L) decreased the total nitrogen removal efficiency (13.4%-30.6%) by suppressing the nitrogen transformation processes. Excessive reactive oxygen species induced by MPs and NPs caused cytotoxicity, as evidenced by impaired cytomembranes and decreased bioactivity. Metagenomic analysis revealed that MPs and NPs diminished the abundance of denitrifiers (e.g. Mesorhizobium, Rhodobacter and Thauera), and concurrently reduced the abundance of functional genes (e.g. napA, napB and nirS) encoding for key enzymes involved in the nitrogen transformations, as well as the genes (e.g. mdh) related to the electron donor production, thereby declining the nitrogen removal efficiency. Network analysis further clarified the attenuate association between denitrifiers and denitrification-related genes in the plastic-exposed systems, elucidating that MPs and NPs restrained the nitrogen removal by inhibiting the contributions of microorganisms to nitrogen transformation processes. This study provides vital insights into the responses of the microbial community structure and nitrogen conversion processes to micro(nano)plastics disturbance in activated sludge systems.

Process stability in expanded granular sludge bed bioreactors enhances resistance to organic load shocks.

Environmental perturbations such as changes in organic loading rate (OLR) can have deleterious effects on the anaerobic digestion process, leading to VFA accumulation and process failure. However, the operational history of a reactor, such as prior exposure to VFA build up, can impact a reactor's resistance to shock loads. In the present study, the effects of long term (>100 days) bioreactor (un)stability on OLR shock resistance were assessed. Three 4 L EGSB bioreactors were subjected to varying levels of process stability. Operational conditions such as OLR, temperature and pH were maintained stable in R1; R2 was subjected to a series of minor OLR perturbations and R3 was subjected to a series of non-OLR perturbations, including ammonium, temperature, pH and sulfide. The effect of these different operational histories on each reactor's resistance to a sudden 8-fold increase in OLR were assessed by monitoring COD removal efficiency and biogas production. The microbial communities of each reactor were monitored using 16S rRNA gene sequencing to understand the relationship between microbial diversity and reactor stability. It was determined that the stable (un-perturbed) reactor performed best in terms of its resistance to a large OLR shock, despite its lower microbial community diversity.

Temperature dependence of nitrification in a membrane-aerated biofilm reactor.

The membrane-aerated biofilm reactor (MABR) is a novel method for the biological treatment of wastewaters and has been successfully applied for nitrification. To improve the design and adaptation of MABR processes for colder climates and varying temperatures, the temperature dependence of a counter-diffusional biofilm's nitrification performance was investigated. A lab-scale MABR system with silicone hollow fibre membranes was operated at various temperatures between 8 and 30°C, and batch tests were performed to determine the ammonia oxidation kinetics. Biofilm samples were taken at 8 and 24°C and analysed with 16S rRNA sequencing to monitor changes in the microbial community composition, and a mathematical model was used to study the temperature dependence of mass transfer. A high nitrification rate (3.08 g N m-2 d-1) was achieved at 8°C, and temperature dependence was found to be low (θ = 1.024-1.026) compared to suspended growth processes. Changes in the community composition were moderate, Nitrospira defluvii remaining the most dominant species. Mass transfer limitations were shown to be largely responsible for the observed trends, consistent with other biofilm processes. The results show that the MABR is a promising technology for low temperature nitrification, and appropriate management of the mass transfer resistance can optimise the process for both low and high temperature operation.

Microbial protein production from sulfide-rich biogas through an enrichment of methane- and sulfur-oxidizing bacteria.

This study evaluated the possibility of combining methane oxidizing bacteria (MOB) with sulfur oxidizing bacteria (SOB) to enable the utilization of sulfide-rich biogas for microbial protein production. For this purpose, a MOB-SOB mixed-culture enriched by feeding both methane and sulfide was benchmarked against an enrichment of solely MOB. Different CH4:O2 ratios, starting pH values, sulfide levels and nitrogen sources were tested and evaluated for the two enrichments. The MOB-SOB culture gave promising results in terms of both biomass yield (up to 0.07 ± 0.01 g VSS/g CH4-COD) and protein content (up to 73 ± 5% of VSS) at 1500 ppm of equivalent H2S. The latter enrichment was able to grow also under acidic pH (5.8-7.0), but as inhibited outside the optimal CH4:O2 ratio of 2:3. The obtained results show the capability of MOB-SOB mixed-cultures to directly upcycle sulfide-rich biogas into microbial protein potentially suited for feed, food or biobased product applications.

In vivo characterisation of the Vibrio vulnificus stressosome: A complex involved in reshaping glucose metabolism and motility regulation, in nutrient- and iron-limited growth conditions.

Stressosomes are signal-sensing and integration hubs identified in many bacteria. At present, the role of the stressosome has only been investigated in Gram-positive bacteria. This work represents the first in vivo characterisation of the stressosome in a Gram-negative bacterium, Vibrio vulnificus. Previous in vitro characterisation of the complex has led to the hypothesis of a complex involved in iron metabolism and control of c-di-GMP levels. We demonstrate that the stressosome is probably involved in reshaping the glucose metabolism in Fe- and nutrient-limited conditions and mutations of the locus affect the activation of the glyoxylate shunt. Moreover, we show that the stressosome is needed for the transcription of fleQ and to promote motility, consistent with the hypothesis that the stressosome is involved in regulating c-di-GMP. This report highlights the potential role of the stressosome in a Gram-negative bacterium, with implications for the metabolism and motility of this pathogen.

Generation of bio-energy after optimization and controlling fluctuations using various sludge activated microbial fuel cell.

An aerobic microbial fuel cell (MFC) was designed to produce bio-electricity using cow manure-pretreated slurry (CM) and sewage sludge (SS). A comparative study of parametric effects on power generation for various parameters like feed ratio of wastes, pH of anode media, and electrode depth was conducted. This experiment aimed to identify the most important system parameters and optimize them to develop a suitable controller for a stable output. Power production reached its maximum at an electrode depth of 7 cm, a pH of 6, and a feed ratio of 2:1 in the CM + SS system before applying the controller. Response surface methodology (RSM) was practiced to explore the relationships between various parameters and the response using MINITAB software. The regression equation of the most productive system deduced from the RSM result has an R2 value of 85.3%. The results show that an ON/OFF controller works satisfactorily in this study. The highest energy-generating setup has a chemical oxygen demand (COD) removal efficiency of 45%. The morphology and content of the used wastes indicate that they can be recycled in other applications.

Enhanced removal of mixed VOCs with different hydrophobicities by Tween 20 in a biotrickling filter: Kinetic analysis and biofilm characteristics.

Mass transfer limitation usually causes the poor performance of biotrickling filters (BTFs) for the treatment of hydrophobic volatile organic compounds (VOCs) during long-term operation. In this study, two identical lab-scale BTFs were established to remove a mixture of n-hexane and dichloromethane (DCM) gases using non-ionic surfactant Tween 20 by Pseudomonas mendocina NX-1 and Methylobacterium rhodesianum H13. A low pressure drop (≤110 Pa) and a rapid biomass accumulation (17.1 mg g-1) were observed in the presence of Tween 20 during the startup period (30 d). The removal efficiency (RE) of n-hexane was enhanced by 15.0%- 20.5% while DCM was completely removed with the inlet concentration (IC) of 300 mg·m-3 at different empty bed residence times in the Tween 20 added BTF. The viable cells and the relative hydrophobicity of the biofilm were increased under the action of Tween 20, which facilitated the mass transfer and enhanced the metabolic utilization of pollutants by microbes. Besides, Tween 20 addition enhanced the biofilm formation processes including the increased extracellular polymeric substance (EPS) secretion, biofilm roughness and biofilm adhesion. The kinetic model simulated the removal performance of the BTF with Tween 20 for the mixed hydrophobic VOCs, and the goodness-of-fit was above 0.9.