Biodegradation of high concentrations of halomethanes by a fermentative enrichment culture
© Shan et al.; licensee Springer 2014
Received: 8 May 2014
Accepted: 14 May 2014
Published: 14 June 2014
A fermentative enrichment culture (designated DHM-1) that grows on corn syrup was evaluated for its ability to cometabolically biodegrade high concentrations of chloroform (CF), carbon tetrachloride (CT), and trichlorofluoromethane (CFC-11). When provided with corn syrup and vitamin B12 (0.03 mol B12 per mol CF), DHM-1 grew and biodegraded up to 2,000 mg/L of CF in 180 days, with only minor transient accumulation of dichloromethane and chloromethane. CT (15 mg/L) and CFC-11 (25 mg/L) were also biodegraded without significant accumulation of halomethane daughter products. The rate of CF biodegradation followed a Michaelis-Menten-like pattern with respect to the B12 concentration; one-half the maximum rate (66 mg CF/L/d) occurred at 0.005 mol B12 per mol CF. DHM-1 was able to biodegrade 500 mg/L of CF at an inoculum level as low as 10−8 mg protein/L. The highest rate of CF biodegradation occurred at pH 7.7; activity decreased substantially below pH 6.0. DHM-1 biodegraded mixtures of CT, CFC-11, and CF, although CFC-11 inhibited CF biodegradation. Evidence for compete defluorination of CFC-11 was obtained based on a fluoride mass balance. Overall, the results suggest that DHM-1 may be effective for bioaugmentation in source zones contaminated with thousands of milligrams per liter of CF and tens of milligrams per liter of CT and CFC-11.
Over the past two decades, substantial progress has been made in use of bioremediation for treating halogenated solvents in groundwater. Nevertheless, in situ bioremediation strategies for groundwater with high concentrations of halogenated methanes such as carbon tetrachloride (CT), chloroform (CF), and trichlorofluoromethane (CFC-11) are still lacking. Among these compounds, CF is often the focal point for evaluating the feasibility of bioremediation because of its high toxicity to most microbes. For instance, inhibition of chlororespiration of chloroethenes by CF is a concern for sites co-contaminated with CF, and can only be overcome by removing the CF first (Bagley et al. ). CF and CT rank highly on the Agency for Toxic Substances and Disease Registry based on their frequency, toxicity, and potential for human exposure at National Priority List sites (ATSDR ). Although not ranked by ATSDR, CFC-11 is often a co-contaminant with CT and CF. For example, approximately 500 mg/L CF, 26 mg/L CFC-11 and 10 mg/L CT were detected in the source zone at a former industrial site (Shan et al. [2010b]). Bioremediation of mixtures of high concentrations of halomethanes is especially challenging.
Most previous research on bioremediation of CT, CF and CFC-11 at elevated concentrations focused on individual compounds. For example, in the presence of vitamin B12, a fermentative culture grown on dichloromethane (DCM) transformed up to 270 mg/L CF (Becker and Freedman ) and a sulfate reducing enrichment culture developed from anaerobic digester sludge transformed up to 350 mg/L CT (Freedman et al. ). The highest concentration of CFC-11 evaluated previously was 2.2 mg/L and resulted in accumulation of dichlorofluoromethane (HCFC-21), which is not an acceptable endpoint (Krone and Thauer ). Although bioaugmentation continues to mature as an option for treatment of chlorinated ethenes (ESTCP 2005), there has been less progress with halogenated methanes. SDC-9™ biodegrades CT and CF, but it has been evaluated at relatively low concentrations, i.e., 2.3 mg/L CT and 3.2 mg/L CF (ESTCP 2005). Recently, Dehalobacter spp. have been described that reductively dechlorinate up to approximately 60 mg/L of CF to DCM via organohalide respiration (Grostern et al. ; Lee et al. ). DCM is not an acceptable endpoint, but microbes that use DCM as a sole carbon and energy source have been reported (Freedman and Gossett ; Justicia-Leon et al. ; Mägli et al. ); Lee et al. () described a mixture of Dehalobacter spp. that reduced 50 mg/L of CF to DCM, which was subsequently fermented to acetate, CO2, and H2; evaluation of higher CF concentrations was not reported.
In a microcosm study of the abovementioned industrial site, we demonstrated that bioaugmentation is a potentially feasible remediation option for the highest concentration areas of the plume, containing CT, CFC-11, and CF (Shan et al. [2010b]). Cometabolic biodegradation of the halomethanes was accomplished via addition of a fermentative enrichment culture that grows on corn syrup, along with vitamin B12 at a dose of 0.03 mol B12 per mol of CT + CFC-11 + CF. Addition of only corn syrup + B12 was also effective, but took twice as long. Accumulation of DCM, chloromethane (CM), HCFC-21, and chlorofluoromethane (HCFC-31) was minor. Following numerous transfers of the culture (designated DHM-1) in mineral salts medium (MSM) amended with corn syrup, CF, and B12, we demonstrated that DHM-1 grows equally well in the presence or absence of 500 mg/L of CF (Shan et al. [2010a]). This was promising from the perspective that bioaugmentation cultures should be able to grow in the presence of the contaminants.
The objectives of this study were to further characterize the DHM-1 enrichment culture with respect to its ability to biodegrade CF at concentrations up to 4000 mg/L, CT (15 mg/L), and CFC-11 (25 mg/L), individually and in mixtures; to evaluate the effect of B12 dose and inoculum level on the maximum rate of CF biodegradation; to determine the effect of pH on the rate of CF biodegradation, and to evaluate the fate of CFC-11 using a fluoride mass balance.
Materials and methods
Inoculum, chemicals and MSM
Experiments were performed with the DHM-1 enrichment culture (ATCC no. PTA-120292) grown on corn syrup (regular type, Sweetener Products Company, Vernon, CA) and supplemented with cyanocobalamin (i.e., vitamin B12, USP grade; Research Organics, Inc., Cleveland, OH). The bicarbonate-buffered, sulfide-reduced MSM used to grow DHM-1 is described elsewhere (Shan et al. [2010a]).
Stock solutions of [14C]CT and [14C]CF were prepared in distilled deionized water at concentrations of approximately 4.5 μCi per mL, using neat [14C]CT (1.0 mCi/mmol) and [14C]CF (0.5 mCi/mmol) from American Radiolabeled Chemicals, Inc. (Saint Louis, MO). CT (99.9%, Sigma-Aldrich, Saint Louis, MO), CF (99.7%, Sigma-Aldrich), DCM (99.9%, AlliedSignal, Morristown, NJ), CFC-11 (99%, Sigma-Aldrich), and carbon disulfide (CS2, 100%, J.T. Baker, Center Valley, PA) were obtained as neat liquids. CM (99.9%, Praxair, Danbury, CT), HCFC-21 (dichlorofluoromethane, 99%, SynQuest Labs, Alachua, FL), HCFC-31 (chlorofluoromethane, 99%, SynQuest Labs), and methane (99.99%, Matheson, Longmont, CO) were obtained as neat gases. All other chemicals used were reagent grade.
Transformation of individual halomethanes
The ability of the DHM-1 enrichment culture to biodegrade individual halomethanes (~15 mg/L CT, 25 mg/L CFC-11, or 500–4000 mg/L CF) was evaluated with an initial inoculum of 2.5-5.0 mg/L protein (except in experiments that evaluated lower inoculum levels). Treatments were prepared with B12 (0.03 mol per mol of halomethane, except in experiments that evaluated lower inoculum levels), and without B12, in 160 mL serum glass bottles with 100 mL of MSM, in an anaerobic chamber (Coy Laboratory Products, Inc.) containing an atmosphere of approximately 98% N2 and 2% H2. The initial dose of corn syrup was 900 mg/L (~960 mg/L as chemical oxygen demand) (Shan et al. [2010a]). After purging the headspace with 30% CO2/70% N2 for 1 min, the bottles were sealed with 20-mm Teflon-faced red rubber septa and aluminum crimp caps. [14C]CT and [14C]CF were purified on a gas chromatograph prior to addition to the serum bottles to provide an initial 14C activity of 0.45 μCi/bottle, as previously described (Shan et al. [2010a]). The fate of CFC-11 was assessed based on release of fluoride instead of using [14C]CFC-11, which was prohibitively expensive.
Non-labeled CF, CT and CFC-11 were added using neat compounds. Media controls (no substrate, no culture) with and without B12 were prepared for each halomethane. The bottles were incubated quiescently in an inverted position at room temperature (22-24°C) in the anaerobic chamber. pH was monitored weekly and maintained between 6.7-7.7. Decreases in pH indicated that the corn syrup was undergoing fermentation; when decreases in pH stopped, a second dose of corn syrup was added.
The highest concentration of CF tested with DHM-1 in previous investigations was 500 mg/L (Shan et al. [2010a]). Experiments in this study evaluated CF concentrations of 1000, 2000, and 4000 mg/L. Serum bottles were prepared as described above, except that only unlabeled CF was added. Most experiments included water controls (WC), consisting of 100 mL of distilled deionized water and CF, CT, and CFC-11.
Effect of B12 concentration and DHM-1 inoculum
The effect of DHM-1 inoculum concentration on the CF biodegradation rate was evaluated using 10 treatments. Half of the treatments received varying inoculum levels of DHM-1; the others consisted of abiotic controls. Treatments were prepared in the same manner described in the section for transformation of individual halomethanes, with an initial CF addition of approximately 500 mg/L (without [14C]CF).
Effect of pH
Biodegradation rates for 500 mg/L of CF by DHM-1 were measured at pH levels from 5.0 to 7.7. B12 (0.03 mol B12 per mol of CF added) and corn syrup (900 mg/L) were added to all treatments. Serum bottles were prepared as described above for the B12 dose experiment, with the following modifications. The MSM was prepared at the target pH by varying the amounts of K2HPO4 and KH2PO4. After adding sodium sulfide, the final pH was adjusted using either H3PO4 (1 M) or NaOH (8 M); the MSM was incubated for six days to ensure equilibrium was reached at the target pH, before inoculating the DHM-1 enrichment culture (5% v/v). It was not necessary to sparge the headspace of the bottles with 30% CO2/70% N2. Each time CF was analyzed on the gas chromatograph, the pH was measured (0.2 mL sample) and, as needed, increased back to the target level using NaOH (8 M); decreases in pH were caused by fermentation of the corn syrup to organic acids. The highest CF biodegradation rate at a given pH was determined in the same manner described above for varying B12 doses. Lag times were based on the time from day zero to the first data point used to determine the highest biodegradation rate.
Transformation of mixtures of halomethanes
The ability of DHM-1 to biodegrade mixtures of CT, CF, and CFC-11 was evaluated in mixtures of two or three compounds. With two compounds, a single dose of B12 was provided at the start (0.03 mol B12 per mol of total halomethanes added). When all of the halomethanes were present, B12 was added in a stepwise manner, i.e., the first dose of B12 was made based on the initial moles of CT; when CT transformation was nearly complete, a second dose of B12 was made based on the initial moles of CFC-11, and when CFC-11 was nearly consumed, a third dose was added based on the initial moles of CF. Along with the second dose of B12, the bottles were reinoculated with DHM-1 (i.e., another 5 μg protein per mL), based on preliminary tests that indicated the culture’s activity on CFC-11 and CF diminished after completing transformation of CT, presumably because CT transformation yields inhibitory intermediates (Lewis et al. ). Controls with CT, CF and CFC-11 present included MSM + B12 added (but not inoculated), autoclaved (AC; inoculated with DHM-1 in MSM and then autoclaved for 1 h), and water only.
Analytical methods and 14C distribution
The amounts of CT, CFC-11, CF, DCM, CM, HCFC-21, HCFC-31, methane and CS2 present in serum bottles were determined by analysis of headspace samples using a gas chromatographic method (Shan et al. [2010a, b]). Aqueous phase concentrations were calculated using Henry’s Law constants (Shan et al. [2010a, b]). Fluoride was measured by ion chromatography (details in Additional file 1). The amount of 14C activity and its distribution in the gas phase (quantified using gas chromatography followed by combustion) and liquid phase were determined as previously described (Shan et al. [2010a, b]). Protein concentration was measured with a BCA™ protein assay kit (Pierce Chemical Company) by following the manufacturer’s enhanced protocol after lysing the cells (Coleman et al. ).
Transformation of individual halomethanes
Products from biodegradation of [ 14 C] CT and [ 14 C] CF by DHM-1
14C added as:
% of14C added recovered as a
Fluoride mass balance from degradation of CFC-11 a
No. of bottles
CFC-11 consumed (μmol/bottle)
CHCl2F formed (μmol/bottle)
CH2ClF formed (μmol/bottle)
F−recovery (%) b
DHM-1 + CS + B12 c
61.6 ± 2.1 d
57.4 ± 4.6
3.1 ± 1.0
0.8 ± 0.9
99.5 ± 8.0
DHM-1 + CS e
1.8 ± 1.2
0.9 ± 0.1
0.09 ± 0.04
MSM + B12
27.1 ± 2.7
17.7 ± 5.0
0.5 ± 0.3
0.06 ± 0.05
68.0 ± 10.1
1.5 ± 3.0
0.06 ± 0.03
0.2 ± 0.3
Effect of B12 concentration and pH on CF biodegradation by DHM-1
Effect of DHM-1 inoculum level on CF biodegradation
Biodegradation of mixtures of halomethanes
The feasibility of using bioremediation to clean up halogenated solvents in the vicinity of nonaqueous phase liquids depends in part on the ability of microbes to grow in the presence of contaminant concentrations that approach their aqueous solubility limit. The results of this study indicate that the DHM-1 enrichment culture grows in the presence of at least 4000 mg/L CF, approximately one half its aqueous solubility, and the culture retains its ability to biodegrade CF at 2000 mg/L. When transforming 500 mg/L of CF, the two Pantoea spp. isolated from DHM-1 adapted their growth by alteration of their membrane fluidity or homeoviscous and homeophasic adaptation (Shan et al. [2010a]). A similar response at the higher CF concentrations evaluated in this study seems likely. Tolerance of high concentrations of halogenated compounds appears to be a characteristic of Enterobacter species (Sharma and McCarty ), which are closely related to Pantoea (Shan et al. [2010a]). Several bioaugmentation cultures that are enriched in Dehalococcoides and used to treat chlorinated ethenes also possess the ability to grow at the high solvent concentrations found near nonaqueous phase liquids (ESTCP 2005).
DHM-1 was effective in transforming CT (~11 mg/L) and CFC-11 (~24 mg/L) as well as CF. Based on experiments with 14C-labeled CT and CF, the primary transformation products are environmentally benign (CO2, CO and organic acids). Similar results were obtained with DHM-1 when it was in an earlier stage of development (Shan et al. [2010b]). Although [14C]CFC-11 was not used in this study, the majority of the CFC-11 consumed was accounted for as fluoride and no volatile products were detected, suggesting the primary transformation products were nonhazardous. DHM-1 did not produce significant levels of DCM and CM from CT and CF. DCM and CM are not necessarily problematic, since both are readily fermentable (Mägli et al. ). In contrast, little is known about the anaerobic biodegradability of HCFC-21 and HCFC-31; if significant levels are formed from CFC-11, it is unclear how quickly they can be degraded. Preliminary experiments indicate an anaerobic culture that grows on DCM as its sole substrate was unable to utilize HCFC-31 (Shan ).
Halomethanes are often found in mixtures at hazardous waste sites. DHM-1 was effective in transforming mixtures of CT, CFC-11 and CF. Consistent with general expectations for halomethanes (Wackett et al. ), CT was transformed first, followed by CFC-11 and then CF. The inhibitory effect of CFC-11 on CF is a potential concern, since the average rate of CFC-11 transformation by DHM-1 was approximately 40-fold slower than CF.
An important consideration for cultures used in bioaugmentation is the required inoculum level. For chlorinated ethenes, a commonly used target is 107 cells/L of Dehalococcoides (ESTCP ). Assuming a unit mass of 1.6 × 10−14 g/cell (Cupples et al. ) and one half of the cell mass is protein, this equates to an inoculum of 8 × 10−5 mg/L protein. DHM-1 was able to biodegrade 500 mg/L of CF at a high rate with a volumetric addition as low as 10−8 percent; this equates to a concentration of 10−8 mg/L protein, several orders of magnitude lower than the target for Dehalococcoides. This suggests that achieving an adequate inoculum in situ will not be problematic.
The catalytic degradation of halogenated compounds by B12 and other transition metal coenzymes under low redox conditions has long been recognized (e.g., Gantzer and Wackett ; Wackett et al. ). One of the concerns with using B12 along with DHM-1 for bioaugmentation is the culture’s relatively high requirement for B12. In a previous study we used a B12 molar dose of 3%, i.e., 0.03 mol B12 per mol CF (Shan et al. [2010a]). Varying the B12 dose in this study indicated a half saturation value of 0.005 mol B12/mol CF. A ratio 0.005 mol B12/mol CT also significantly improved the rate of CT degradation in a methanogenic sludge consortium, at a CT concentration of 15.4 mg/L (Guerrero-Barajas and Field ). Nevertheless, for halomethane concentrations in the hundreds of mg/L, the B12 required by DHM-1 is several orders of magnitude higher than what is needed to grow Dehalococcoides (He et al. ), for which B12 functions as a cofactor for reductive dehalogenases (Schipp et al. ).
Aquifer pH is a significant concern for bioaugmentation, since many cultures lose effectiveness at pH levels below 6 or above 8. The highest pH evaluated in this study was 7.6, which yielded a higher rate than in the circumneutral pH region (6.3-7.3; Figure 4). CF biodegradation rates decreased significantly below 6.0 and activity essentially ceased at pH 5.0. This is similar to the behavior of many Dehalococcoides enrichment cultures (ESTCP 2005; Vainberg et al. ). The difficulties associated with adjusting aquifer pH include non-homogenous distribution of the buffering agent and the potential for clogging due to precipitation when pH is increased.
For contaminant plumes with high concentrations of halomethanes that are not undergoing natural attenuation, the options for bioremediation are limited. The benefits of using a culture such as DHM-1 include its high rate of CF transformation; its ability to transform mixtures of CT, CF, and CFC-11; the conversion of these halomethanes to environmentally benign products; its growth on an inexpensive primary substrate (corn syrup); and its ability to grow in the presence of CF at levels at least as high as 4000 mg/L. The culture also retains its ability to anaerobically transform CF after exposure to air for as long as one day (Additional file 1: Figure S3). Further studies are needed to validate the use of DHM-1 under field conditions, and to determine if lower cost formulations of B12 can be developed, e.g., using the fermentation product from cultures that synthesize B12 with a lesser degree of purification.
HS, HW, RY and PJ were supported by teaching assistantships from Clemson University while this research was conducted.
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