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The objective of this research was to evaluate the biodegradation of chloroform by using biotrickling filter BTF and determining the dominant bacteria responsible for the degradation. The research was conducted in three phases under anaerobic condition, namely, in the presence of co-metabolite Phase I , in the presence of co-metabolite and surfactant Phase II and in the presence of surfactant but no co-metabolite Phase III.

The equivalent elimination capacity EC was 0. This research also investigated the overall microbial ecology of the BTF utilizing culture-independent gene sequencing alignment of the 16S rRNA allowing identification of isolated species. Disinfection byproducts DBPs are formed as the results of the reaction of free chlorine and a series of complex organic precursors Li and Blatchley Many DBPs are carcinogens or have been known to cause other health risks Durmishi et al.

The precursors forming DBPs range from natural occurring humic and fulvic material to anthropological contaminants that persist in treated water Richardson et al. The highest concentrations of DBPs detected in drinking water constitute trihalomethanes THMs where chloroform is the major component Durmishi et al. Several physical and chemical removal methods such as adsorption, advanced oxidation Shemer and Narkis , and air stripping Clark et al.

Chloroform is a volatile organic compound VOC that readily evaporates to the atmosphere from treated water surfaces through air stripping Houel et al. Hence, the need for innovative control technology is becoming more enviable. In this research, biological treatment was used to treat the off-gas chloroform by bio-trickling filters BTF.

Most of the research on the biological treatment of chloroform has been limited to batch liquid phase processes at wastewater treatment plants or hazardous waste disposal sites McCulloch Yoon and Park studied the effect of gas residence time on the aerobic biodegradation of nine VOCs including chloroform. In their study they observed that chloroform showed lower removal efficiency compared to other compounds for longer empty bed residence time of 3 minutes Yoon and Park However, under anaerobic conditions, chloroform could undergo a reductive biotransformation by pure cultures of methanogens Egli et al.

Highly chlorinated atoms are good electron targets for anaerobes since chlorine atoms block the activity of oxygenase. Thus, biological techniques have resulted in either partial dechlorination of chloroform to dichloromethane Egli et al. Although most studies show successful biodegradation of chloroform in the liquid phase, there has been limited reported work on the use of biofiltration for the removal of chloroform from gaseous streams.

Biofiltration is one of the proven technologies for removing VOCs form high volume stream as it is environmentally friendly, cost effective and releases fewer byproducts Yoon et al. The use of aerobic biofiltration technique has been reported for the biotreatment of chloroform with other mixtures of different VOCs Yoon et al. Yoon et al. Similarly, Balasubramanian et al. Their study showed that increasing the rate of chloroform loading significantly reduced the degradation efficiency of the reactor for the mixture of VOCs.

The increase in chloroform loading rate from However, there has been a limited research work reported on the use of anaerobic BTF for the removal of chloroform from the gas phase at low concentration. Multiple investigations have been performed on co-metabolism, specifically for the treatment of trichloroethylene TCE contaminated air using BTF.

Chlorinated compounds can be degraded by using a more easily biodegradable non-chlorinated hydrocarbon serving as a co-metabolite Balasubramanian et al. Biofilters using methane, butane, propane, propylene, phenol, toluene, or ammonia as a co-metabolite have shown success in the treatment of trichloroethylene TCE Mahon Mahon et al.

Chloroform is a halogenated compound and recalcitrant to biodegrade. Hence, the biodegradation of higher order chlorinated volatile compounds like chloroform occur mostly under co-metabolic conditions in the presence of primary substrates such as methane, propane, phenol and toluene Kim et al.

A nonionic surfactant can be introduced in the biofiltration system as means for enhancing solubility. Hence, surfactant could increase the apparent solubility of chloroform by micellar formation, which commences at the critical micelle concentration and the apparent solubility is proportional to surfactant concentration Edwards et al. The use of surfactants in enhancing the bioavailability of hydrophobic compounds by facilitating their biotransformation under aerobic conditions has been studied by several researchers Yeh et al.

The utilization of surfactant increases the solubility which overcomes the rate limiting step Hassan and Sorial Researchers have found that addition of surfactants stimulated polycyclic aromatic hydrocarbon PAH biodegradation Tiehm ; F. Volkering et al. However, there are limited studies where surfactant was used for the dehalogenation in biofiltration system.

Yuan et al. In the present work, an effort was made to evaluate the performance of anaerobic BTF for biodegradation of chloroform. The experimental plan was designed to operate the BTF in the presence and absence of co-metabolite and surfactant at different phases. The research also included the application of molecular tools for characterization of the microbial community diversity throughout the BTF.

The BTF was used for the continuous removal of gaseous chloroform at three different phases and to gain an insight into the effects of halorespiration for dehalogenation of chloroform. The change in the biodiversity of the bacterial community during the three phases helps to identify the most abundant bacterial populations responsible for the dehalogenation of chloroform during the operation of the BTF.

Chloroform was used as a model THM compound; ethanol laden nitrogen gas stream was supplemented as co-metabolite and Tomadol 25 — 7 was used as a surfactant for the biodegradation. Chloroform with The measuring sensors for pH, nitrate, dissolved oxygen DO , and ammonia were acquired from Accumate Instruments. The experimental set-up for BTF consisted of a cylindrical glass column with multiple sections providing a total length of cm and an internal diameter of 7. A schematic set up of the experimental system is shown in Fig.

The BTF was operated in a co-current mode where chloroform and co-metabolite laden gases, and liquid nutrient solution were introduced into the column from the top. The gas flow was controlled with two mass flow controllers Sierra Instruments. The composition of the nutrient solution was similar to the one used by Atikovic et al. Atikovic et al. Wu et al. Initially, these bacteria were obtained from nutrient enriched solution kept under a blanket of nitrogen gas that was acclimated in our lab to chloroform in a 4 liter amber batch reactor for two months.

The chloroform feed was step-wise increased from 5 to 50 ppmv within the two-month period. This inoculum was mixed in the ratio of with other methanogenic bacteria acquired from another bioreactor that was treating food waste prior to seeding the BTF. A mixture of chloroform and ethanol co-metabolite was continuously fed into the BTF in a flowing nitrogen carrier gas at a flow rate of 0. The nutrient solution buffered at pH 7 was fed intermittently to the BTF bed by a solenoid valve at a rate of 2.

The presence of co-metabolite enabled treatment strategies that stimulated biodegradation of chloroform, which alone was not enough to provide the carbon or energy benefit to the microorganism. Measurements of the effluent liquid pH, and organic matter, the gas flow pressure drop across the bed, and operating temperature were taken.

Gas phase samples were taken on-line from different points along the BTF column using an electrically controlled low-bleed eight-port Valco valve and analyzed by gas chromatograph. The samples were analyzed for chloroform, ethanol, and by-products such as methane and carbon dioxide. The carrier gas He flow rate was set at 3. Retention time for chloroform was 3. The samples were filtered through a 0. The concentration of ammonia was determined using an ammonia electrode sensor.

Dissolved total carbon and dissolved inorganic carbon content of the liquid samples were determined with a Shimadzu total organic carbon analyzer model TOC - L Shimadzu Corp. Biofilm samples were collected from the BTF within the media as shown in Fig. The samples were taken from port 2 first port from the top within the media at the end of each phase before proceeding to the next phase.

This method was previously done by other researchers Zehraoui et al. The samples consisted of about five media pellets covered with biomass and placed in O 2 free sampling tubes. Sequencing was carried out at Molecular Research LP www. Sequence data were processed using a proprietary analysis pipeline. Sequences were first depleted of barcodes and primers, and those under bp or with ambiguous base calls or with homopolymer runs exceeding 6bp were removed. In this research, the effects of co-metabolite and surfactant on the performance of BTF were evaluated.

Even though chloroform is a recalcitrant compound to biological transformation under conventional aerobic conditions, it can be transformed in the presence of a co-metabolite under aerobic and anaerobic environments Zitomer and Speece The co-metabolite was mixed with chloroform stream to achieve higher removal efficiency by providing additional electron donor to the micro-organisms.

Anaerobic dehalogenation of chloroform has been observed by different researchers by using methanogenic microbes with electron donating co-metabolites in reductive chloroform biotransformation Mikesell and Boyd ; Bagley and Gossett ; Bouwer et al.

Ethanol was used as a co-metabolite since it readily mixes with chloroform and water, and is non-toxic to microbial community. In Phase I, the BTF started up with chloroform influent concentration of 5 ppmv and ethanol concentration of 25 ppmv providing a corresponding chloroform loading rate of 0. The operating conditions and different phases of operation are summarized in Table 1.

Therefore, emphasis is placed on the performance of the BTF on chloroform removal. The lower boundary of the box denotes the lower quartile, a line within the box marks the median, and the boundary of the box furthest from zero indicates the upper quartile.

Whiskers error bars above and below the box indicate the 90 th and 10 th percentiles. Chloroform is used as an electron acceptor by the reducing bacteria and dehalogenation was enhanced by adding electron donor supply.

In previously reported studies, co-metabolite assisted degradation of chloroform were all conducted in batch liquid phase reactions. Thus, there are limited studies on the use of co-metabolite for a continuous gas phase studies.

It was also observed that lower fractional degradation of chloroform was obtained as the initial concentration increased. Gupta et al. Performance of the BTF in the three phases. Phase I 44 days presence of cometabolite, phase II 97 days presence of cometabolite and surfactant, phase III 95 days presence of surfactant with no cometabolite.

To further improve the dehalogenation of chloroform, a nonionic surfactant was mixed with the nutrient feed solution. Hence, in Phase II, a surfactant was added along with the co-metabolite. It is worthwhile to note that the behavior of this BTF was very similar to the reported performance of n-hexane trickle bed air biofilter using Tomadol 25 — 7 Hassan and Sorial The microorganisms utilize surfactants as substrates for energy and nutrients during biodegradation process Ying Phase II test was run for 97 days to give the microbes sufficient time to acclimate and attain a stable condition.

The increased run time allowed the BTF more cell production and increase of biomass throughout the bed. As shown in Fig. In a previous study, the use of Tomadol 25 — 7 improved the performance of BTF by doubling the elimination capacity for n-hexane, allowing a robust operation, and decreasing the fluctuation of the effluent stream n-hexane concentrations Hassan and Sorial The amount of methane produced in our research, during Phases I and II, were 0.

This phase has the same operating conditions as Phase II and was run for 95 days. The removal efficiency was higher than that of Phase I and the overall dehalogenation of chloroform was high in the absence of ethanol. The methane production for phase III was 0. The drop in the performance compared to the previous Phase II could be due to the limitation of biomass growth as was depicted by measurements of VSS.

The reductive dehalogenation of chloroform along the BTF was measured weekly by collecting gas samples from ports that are located at 7. During the reductive dehalogination process see Supplementary data, Appendix A , the hydrodehalogenation and hydrogenation of chloroform can be hypothesized as in Fig S1 Supplementary data, Appendix B.

The kinetic analysis was conducted using the data from sampling ports within the media as there is a possibility of biodegradation on the top portion of the BTF above the media or at the bottom disengagement chamber used for separation of liquid and gas effluents. The chloroform concentrations in these samples along with the influent stream concentration were used to develop the transformation kinetics as a pseudo first order reaction rate based on a plug flow reactor model.

The kinetics reaction rate constants were obtained from the slopes of the regression lines, from the following equation:. The error bars represent the standard deviations of three replicas. The results in the figure show higher reaction rate of 0. The addition of surfactant prompted higher reduction rate constant. This could be due to the co-metabolism and dissolution effects of the surfactants that resulted in excess biomass retention within the BTF bed. As discussed in the performance section, these values correlated with efficiencies of the BTF for the different phases.

The higher reaction rate was obtained due to the enhancement and improvement gained from the co-metabolite and surfactant added to the system, which increased microbial activity within the biofilter bed. Reaction rate constants for chloroform in three phases. Three data sets have been collected per phase and the error bars present the standard deviations for these replicas.

The cumulative CO 2 equivalent of for this research is presented in Fig. To study the carbon cycle within the bed for both the liquid and gaseous phases, all the carbon sources and products were measured. The influent consisted of gaseous concentrations of chloroform and ethanol, plus aqueous inorganic and organic carbon. The effluent included aqueous inorganic and organic carbon, a carbon equivalence of volatile suspended solids, gaseous carbon dioxide, gaseous methane and a carbon equivalence of chloroform and ethanol concentrations.

The CO 2 equivalence of all the carbon components was calculated in moles and a cumulative input and output CO 2 equivalence of carbon was plotted on sequential time Fig. To avoid toxicity to the microbes, chloroform was supplied to the BTF at a relatively lower flow rate, which resulted in the lower recovery.

The loss of influent and effluent carbon was produced as biomass within the BTF Hassan and Sorial This hypothesis is justified by comparing the loss of carbon to the amount of biomass accumulated within the bed. The cellular composition for typical heterogeneous anaerobic microorganisms is represented as C 4. These compositions were used as the basis for relating the ammonia consumed in building up new biomass to estimate the amount of biomass retained within the BTF.

A two-tailed t-test was performed to compare the results of the carbon consumed and the biomass produced. It is worthwhile to note that the main carbon contributors to the carbon balance of BTF are the gas phase concentrations of the influent and effluent chloroform and ethanol concentration, and effluent gaseous carbon dioxide and methane. As a microbial degradation process, biofiltration is based on the ability of microorganisms to degrade organic and inorganic compounds.

Webster et al. Although, gram negative bacteria has been reported as the dominant bacterial community of some biofilters, few studies have been done on the microbial diversity of BTF Bruce and Perry ; Webster et al. Similarly, Amann et al. Hence, in order to understand and analyze the microbial community structure and the diversity of the BTF during the dechlorination processes, molecular methods have been proven to be powerful tools for analyzing the diversity and structure of the microbiome Fakruddin and Mannan In this research, Ion Torrent system was used to examine the impact of the presence and absence of cometabolite and surfactant on the bacterial community structure in the BTF.

To get a high diversity of microbes, inocula usually come from digested activated sludge or previously cultivated microflora Wagner et al. Initially, in this research microbes were acclimated from chloroform based culture and methanogenic bacteria from food waste. The community dominant compositions were A. Especially, A. The prevalence of these species has also been reported previously from various microbial utilization and studies related to anaerobic biodegradation.

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