High-Rate, High-Yield Production of Methanol by Ammonia

Toward Nucleating the Concept of the Water Resource Recovery Facility (WRRF): Perspective from the Principal Actors. Erik R. Coats and Patrick I. Wils...
0 downloads 0 Views 268KB Size
Download PDF
Article pubs.acs.org/est

High-Rate, High-Yield Production of Methanol by AmmoniaOxidizing Bacteria Edris Taher and Kartik Chandran* Department of Earth and Environmental Engineering, Columbia University, 500 West 120th Street, New York, New York 10027, United States S Supporting Information *

ABSTRACT: The overall goal of this study was to develop an appropriate biological process for achieving autotrophic conversion of methane (CH4) to methanol (CH3OH). In this study, we employed ammonia-oxidizing bacteria (AOB) to selectively and partially oxidize CH4 to CH3OH. In fedbatch reactors using mixed nitrifying enrichment cultures from a continuous bioreactor, up to 59.89 ± 1.12 mg COD/L of CH3OH was produced within an incubation time of 7 h, which is approximately ten times the yield obtained previously using pure cultures of Nitrosomonas europaea. The maximum specific rate of CH4 to CH3OH conversion obtained during this study was 0.82 mg CH3OH COD/mg AOB biomass COD-d, which is 1.5 times the highest value reported with pure cultures. Notwithstanding these positive results, CH4 oxidation to CH3OH by AOB was inhibited by NH3 (the primary substrate for the oxidative enzyme, ammonia monooxygenase, AMO) as well as the product, CH3OH, itself. Further, oxidation of CH4 to CH3OH by AOB was also limited by reducing equivalents supply, which could be overcome by externally supplying hydroxylamine (NH2OH) as an electron donor. Therefore, a potential optimum design for promoting CH4 to CH3OH oxidation by AOB could involve supplying NH3 (needed to maintain AMO activity) uncoupled from the supply of NH2OH and CH4. Partial oxidation of CH4-containing gases to CH3OH by AOB represents an attractive platform for the conversion of a gaseous mixture to an aqueous compound, which could be used as a commodity chemical. Alternately, the nitrate and CH3 OH thus produced could be channeled to a downstream anoxic zone in a biological nitrogen removal process to effect nitrate reduction to N2, using an internally produced organic electron donor.

■

INTRODUCTION

On the other hand, ammonia-oxidizing bacteria (AOB) can oxidize CH4 to CH3OH via the nonspecific action of the enzyme ammonia monooxygenase (AMO).1 The other benefit of using bacterial conversion of CH4 to CH3OH is that the contaminants such as moisture and CO2, which need to be removed from anaerobic digestion gas or biogas for chemical conversion to CH3OH, do not pose a limitation for biological conversion. In fact autotrophic AOB can also utilize the CO2 contained in gas mixtures for cell synthesis. Metabolism of AOB. The oxidation of ammonia-nitrogen (NH3-N) to nitrite-nitrogen (NO2−-N) serves as the primary source of energy for AOB. NH3-N is oxidized to hydroxylamine (NH2OH) by membrane-bound ammonia monooxygenase (AMO). NH2OH is oxidized to NO2−-N by hydroxylamine oxidoreductase (HAO) (Figure 1). The oxidation of NH3-N and NH2OH are intimately linked; the reducing power generated in HAO-mediated NH2OH oxidation is essential for AMO-driven NH3-N oxidation.2 All the energy obtained by

There are intense efforts globally to develop biobased fuels, chemicals, and energy. While ethanol has been of primary focus in the past few years, it should be noted that other chemicals and biofuels such as methanol can be also attractive. In addition to being used in gasoline blends, methanol can be used in fuel cells, combined with long-chain fatty acids and lipids to form biodiesel, or chemically dimerized to dimethyl ether (DME, also a fuel). Methanol is also one of the most widely used chemicals for enhancing denitrification in wastewater treatment. Methanol is commonly produced from natural gas, by chemical catalysis. The chemical pathway first involves the oxidation of CH4 to CO2 and H2 and subsequent reduction of CO2 to CH3OH, and is quite economically and energy intensive and redundant. Given that natural gas reserves are finite, it might be more sustainable to look toward alternate sources of CH4 to produce CH3OH, such as anaerobic digester gas, biogas, or landfill gas, which in addition contain moisture and CO2. However, the primary limitation to the more widespread use of such gas mixtures is the cost and energy required to purify the CH4 present and the challenges of handling a gaseous stream. © XXXX American Chemical Society

Received: October 20, 2012 Revised: March 6, 2013 Accepted: March 8, 2013

A

dx.doi.org/10.1021/es3042912 | Environ. Sci. Technol. XXXX, XXX, XXX−XXX

Environmental Science & Technology

Article

Figure 1. Metabolic pathways of N. europaea. Unshaded enzymes (AMO and HAO) represent nitrogen oxidation pathways, and shaded enzymes (NirK and Nor) represent nitrogen reduction pathways.

AOB is derived from the oxidation of NH2OH to NO2−-N3 and subsequent electron transfer phosphorylation. The dominant mode of energy generation by AOB is via aerobic metabolic pathways1 (unshaded enzymes in Figure 1). However, under oxygen-limiting and anoxic conditions, AOB can utilize alternate electron acceptors such as NO2−, dimeric nitrogen dioxide (N2O4) and produce N2O and NO, but not nitrogen gas (N2) (enzymes shaded gray in Figure 1, as summarized elsewhere4). Fortuitous Oxidation of Organic Compounds by AOB. The capability of ammonia-oxidizing bacteria to oxidize other energy-providing compounds including organic chemicals is by virtue of the broad substrate base of ammonia monooxygenase (AMO). AOB such as Nitrosomonas europaea and Nitrosococcus oceani are also capable of oxidizing various hydrocarbon compounds,5 including methane,6,7 methanol,8 ethylene,9 and methyl bromide.10 The ability of AMO to oxidize methane is because it is related to methane monooxygenase (MMO) from an evolutionary perspective.11 Why Use AOB to Oxidize Methane to Methanol? The consideration for using AOB over methane-oxidizing bacteria (MOB) is rather straightforward. MOB oxidize methane completely to CO2, which cannot be used readily as a fuel. Therefore, if MOB are to be used for CH3OH production, then the metabolic pathways that further process CH3OH will need to be selectively inhibited, which may not be trivial. On the other hand, AOB only oxidize CH4 partially to CH3OH as documented by different studies5−7,12 and possibly to trace amounts of formaldehyde (HCHO).8 However, HCHO itself is highly toxic to AOB and feedback inhibits any further oxidation of CH3OH to HCHO.8 Additionally, the genes coding for conversion of formate (HCOOH) to CO2 are completely missing in AOB such as N. europaea and N. eutropha.13,14 Notwithstanding the potential of using AOB for CH4 oxidation to CH3OH, it must be recognized that AOB do not derive any energy or reducing equivalents from this process. Additionally, since AMO requires reducing power to function, continued CH4 oxidation can likely be limited by reducing power unless supplied externally. NH3 is not an ideal or direct source of reducing power, since it can competitively inhibit

CH4 oxidation. Therefore, it was hypothesized that the use of an alternate reducing power source such as NH2OH could promote AOB-mediated CH4 oxidation to CH3OH. It was additionally hypothesized that uncoupling NH3 and CH4 feeding strategies could promote CH4 oxidation to CH3OH by avoiding competition between these two substrates for AMO. The primary objectives of this study were to 1. identify factors (such as substrate feeding strategies) that could be optimized from a process engineering perspective to maximize CH4 to CH3OH conversion by AOB 2. determine the rates of AOB-mediated CH4 oxidation to CH3OH and quantify the impact of substrates (NH3), intermediates (NH2OH), and products (CH3OH) on CH4 oxidation to CH3OH

■

MATERIALS AND METHODS A mixed nitrifying enrichment culture consisting of AOB and nitrite-oxidizing bacteria (NOB) was developed in a continuous flow reactor (V = 11.18 L) as described previously.15 Briefly, the reactor was fed with inorganic medium containing 500 mg NH4+-N/L as the sole energy source and devoid of any organic carbon. The reactor was housed in a constant temperature chamber maintained at 22 °C and was operated at a hydraulic retention time (HRT) and a solids retention time (SRT) of 1 d and 5 d, respectively. The reactor pH was automatically controlled at 7.5 ± 0.1 using a 40 g/L solution of sodium bicarbonate. The reactor biomass was mixed using mechanical stirring and aeration was provided using lab-air at 3L/min. Biomass samples were withdrawn from the reactor and used for CH4 oxidation tests in 400-mL fed-batch vessels. Reactor performance and the progress of CH4 oxidation in batch vessels were monitored by measuring NH3 (gas-sensing electrode, Corning, Corning, NY), NO2− (diazotization-colorimetry), NO3− (ion selective electrode, Fisher, Waltham, MA), and NH2OH (spectrophotometry), CH3OH (gas-chromatography with flame ionization detection, GC-FID), and dissolved oxygen (YSI Clark type electrodes) concentrations. Biomass B

dx.doi.org/10.1021/es3042912 | Environ. Sci. Technol. XXXX, XXX, XXX−XXX

Environmental Science & Technology

Article

Figure 2. Impact of differing biomass−substrate combination regimes on the yields (A) and rates (B) of CH3OH production from CH4 by AOB in batch bioreactors.

in AOB (SI). The objective of supplying only the minimum amount of NH3 to sustain maintenance energy requirements was to minimize competition between NH3 and CH4 oxidation, while concurrently retaining AOB (sensu-stricto, AMO enzyme) activity. The second feeding strategy (FS2) involved continuous NH2OH feed at the same rate of 9 mg-N/h to simulate excess reducing equivalents supply. The third feeding strategy (FS3) involved cofeeding an equi-molar mixture of NH3 and NH2OH at a total N-loading rate of 9 mg-N/h. In the fourth feeding strategy (FS4), NH3 was fed at 9 mg-N/h only during the O2 feeding phase (30 min every hour, primarily to sustain AMO activity), following which it was replaced with NH2OH for 30 min again at 9 mg-N/h (summarized in SI Figure S1). Due to the much higher flow rate of O2 (500 mL/min) compared to that of CH4 (30 mL/min), CH4 gas−liquid mass transfer likely limited biological consumption during the O2 feeding phase. For the fourth feeding strategy alone, two objectives were thus concurrently achieved: minimizing competition between NH3 and CH4 oxidation for AMO and providing non-NH3 derived reducing equivalents for CH4 to CH3OH oxidation in the form of NH2OH. High-Rate Methanol Production. During the high-rate experiments, both NH2OH and CH4 were continuously fed to a 400-mL batch vessel fed at 9 mg-N/h and 30 mL/min, respectively. The O2 supply was controlled in the range of approximately 100 mL/min to maintain DO concentrations in the range 4−5 mg O2/L to render it nonlimiting (based on an

concentrations were approximated using total chemical oxygen demand (tCOD, Hach Chemical Co.). First-Generation Experiments. For all CH3OH production experiments, nitrifying biomass was withdrawn from the parent reactor and washed three times by settling the biomass quiescently, decanting the supernatant, and replacing with ammonia-free reactor feed medium buffered at a pH of 7.5. CH3OH production experiments were conducted in the same constant temperature chamber (T = 22 °C) where the parent nitrifying reactor was housed. The first-generation experiments consisted of batch incubations (V = 400 mL) of nitrifying biomass with 100 mg NH4+-N/L, with and without 1.4 mg NH2OH-N/L and saturated with three different initial concentrations of dissolved O2 and dissolved CH4 using pure O2 and CH4 (Supporting Information (SI) Table S-I). Subsequently, a continuous gas-feeding strategy for both CH4 and O2 was adopted, but still with an initial pulse of NH4+-N and NH2OH at 98.6 and 1.4 mg-N/L, respectively. Second-Generation Experiments. The second-generation experiments were also conducted in 400-mL batch vessels at T = 22 °C and a pH of 7.5 (as summarized in SI Table S-II). During the second-generation experiments, four different feeding strategies were adopted. All four strategies involved continuous CH4 feeding at 30 mL/min for operational ease and pulsing of pure O2 at 500 mL/min for 30 min every hour. The first feeding strategy (FS1) involved continuous NH3 feed at approximately 9 mg-N/h, to satisfy maintenance requirements C

dx.doi.org/10.1021/es3042912 | Environ. Sci. Technol. XXXX, XXX, XXX−XXX

Environmental Science & Technology

Article

O2 half-saturation coefficient of 0.5 mg O2/L16). In addition, after approximately every 2 h, biomass was withdrawn from the test vessel and the spent reaction medium was separated from the biomass by filtration through 0.2-μm nominal pore size membrane. Fresh biomass of identical volume and concentration from the parent continuously operated nitrifying bioreactor was added to the test vessel after washing, in an effort to overcome inhibition by CH3OH. Inhibition of NH3 and NH2OH Oxidation by CH3OH. For inhibition assays, washed nitrifying biomass was exposed to 50 mg COD/L and 100 mg COD/L of methanol for 7 h. These concentrations were chosen since they span the CH3OH concentrations observed during the fed-batch CH 3OH production experiments, which lasted 7 h. After the 7-h exposure period, the impact of CH3OH on the test biomass was determined via extant respirometry.17 Briefly, the respirometric assays were initiated with a 5 mg-N/L substrate spike (NH3 or NH2OH) into duplicate respirometric vessels. The oxygen uptake resulting from substrate oxidation was measured online using two dissolved oxygen (DO) electrodes (YSI model 5331, Yellow Springs, OH), which were connected to a DO monitor (YSI 5300, Yellow Springs, OH) and interfaced to a personal computer. The maximum specific growth rates (μmax ) associated with NH3 and NH2OH oxidation were computed by normalizing the initial slope of the oxygen uptake rate (OUR) profile to the biomass concentration and multiplying by a stoichiometric factor, (Y/1 − Y), where Y is the growth yield of AOB (0.1 mg biomass COD/mg N-oxidized, as described earlier15,17). The impact of CH3OH on NH3 oxidation was described using a noncompetitive inhibition model. The noncompetitive inhibition coefficient KI,CH3OH was determined via nonlinear least-squares estimation as described elsewhere.18

feeding just NH3 (FS1) resulted in the lowest stable CH3OH concentrations after a 7-h incubation period of 23.47 ± 0.50 mg CH3OH COD/L (calculated from the final four time points in Figure 2a, also summarized in SI Table S-II). The finite CH3OH production capacity was likely mainly due to limitation of reducing equivalents and some competition between NH3 and CH4 oxidation. Continuously feeding just NH2OH (FS2) resulted in statistically higher stable CH3OH concentrations (27.50 ± 0.78 mg CH3OH COD/L, α = 0.05) after a 7-h incubation period (Figure 2a). The higher CH3OH concentration with continuous NH2OH feed can be explained by the lack of competition between CH4 and NH3 oxidation, as well as the supply of reducing equivalents from NH2OH oxidation. However, the finite CH3OH production capacity still pointed to a possible reduction in AMO activity owing to NH3 starvation. As described previously, it might be possible that the kinetics of ammonia oxidation and the expression of AMO are negatively impacted by ammonia limitation or starvation.18,19 However, it is not likely that NH3 starvation for 7 h is sufficient to result in the decline in CH4 oxidation rates observed herein. On the other hand, it is more likely that the accumulated CH3OH directly inhibited AMO (as discussed below). Further, from a practical perspective, the operating costs for FS2 are rather exorbitant. During the 7-h experiment t h e r a t i o o f t h e co st o f h y d r o x y l a m in e - s u l f a t e ((NH2OH)2H2SO4) of $2.50/kg to the cost recovery from the produced CH3OH at $1.55 per gallon was calculated to equal 97:1 (Table S-II). The cofeeding of NH3 and NH2OH (FS3) resulted in CH3OH concentrations (31.52 ± 1.19 mg CH3OH COD/L, Figure 2a), which were statistically higher than those obtained with feeding either NH3 or NH2OH individually. A finite CH3OH production capacity was still observed (Figure 2a) possibly owing to competition between CH4 and NH3 oxidation. The initial rate of CH3OH production with the cofeeding of NH3 and NH2OH was similar to that with just NH3 or NH2OH feeding (Figure 2b). Based on similar calculations as for FS2, the ratio of the cost of (NH2OH)2H2SO4 to the cost recovery from the produced CH3OH was calculated to equal 42:1 (SI Table S-II). The fourth strategy of uncoupling NH3 oxidation and NH2OH oxidation (FS4) resulted in the highest CH3OH concentrations (40.71 ± 0.16 mg CH3OH COD/L, Figure 2a) after an incubation time of 7.5 h. Notably, the finite CH3OH production capacity was not as pronounced as with strategies FS1−FS3 (Figure 2a). For the fourth strategy, based on the stabilization in the DO profiles, it was inferred that NH3 oxidation was complete well within the period of O2 supply (shaded sections, SI Figure S1). Therefore, CH4 and NH2OH were oxidized in the absence of NH3 oxidation (unshaded sections, SI Figure S1). The independent and alternating oxidation of NH3 and (CH4 and NH2OH) together thus minimized competition for AMO between NH3 and CH4 due to the vigorous oxygenation applied (500 mL/min O2 as opposed to 30 mL/min CH4), which likely resulted in lower gas−liquid CH4 transfer and therefore conversion to CH3OH during oxygenation. From a cost perspective, the ratio of the cost of (NH2OH)2H2SO4 to the cost recovery from the produced CH3OH was 33:1 (SI Table S-II). Based on the second-generation experiments, maximum CH4 to CH3OH oxidation rates in the range 0.20−0.30 mg CH3OH COD/mg biomass COD-d were obtained (derived from the

■

RESULTS AND DISCUSSION First-Generation Experiments. Co-incubation of AOB cultures with NH3 saturated with O2 and CH4 in the absence of NH2OH did not produce detectable CH3OH concentrations after an incubation period of 5 h (SI Table S-I). The addition of 1.4 mg-N/L of NH2OH under similar incubation conditions led to low and statistically similar production of CH3OH (α = 0.05, SI Table S-I). The extent of NH3 oxidation in the presence of NH2OH was also slightly lower, presumably due to CH4 conversion at the expense of NH3 (SI Table S-I). The absence of substantial CH3OH production in these experiments points to limitation for a common cosubstrate pool (likely reducing equivalents) during competitive co-oxidation of NH3 and CH4. Additionally, O2 limitation likely contributed to a finite transformation capacity of NH3 and CH4, which necessitated continuous O2 feed during the subsequent first-generation experiments. Indeed, the continuous feeding strategy for both CH4 and O2 with an initial pulse of 98.6 mg NH4+-N/L and 1.4 mg NH2OH-N/L resulted in significantly higher CH3OH concentrations (19 ± 0.45 mg COD/L) at t = 5 h (SI Figure S2). However, despite the presence of excess NH3, CH4, and O2 concentrations, a finite transformation yield of CH3OH was still observed, pointing to either inhibition of CH3OH production by CH3OH itself or a limitation in reducing equivalents. The second-generation experiments described next attempted to overcome the limitations observed during the first-generation experiments. Second-Generation Experiments. Of the four strategies tested during the second-generation experiments, continuously D

dx.doi.org/10.1021/es3042912 | Environ. Sci. Technol. XXXX, XXX, XXX−XXX

Environmental Science & Technology

Article

Table 1. Comparison of Microbial CH3OH Production by Different AOB Cultures maximum CH3OH production rate (mg CH3OH COD/mg biomass COD-d)

peak CH3OH concentration attained (mg COD/L)

0.21

23.47 ± 0.50

0.30

27.50 ± 0.78

0.22

31.52 ± 1.19

0.20

40.71 ± 0.16

0.82

59.89 ± 1.12

0.37 0.31−0.54 0.02−0.1

28.8 NA 6.2 ± 4.9

microbial system used

reference

mixed nitrifying cultures NH3 only feed (FS1)

this study mixed nitrifying cultures NH2OH only feed (FS2) this study mixed nitrifying cultures NH3 and NH2OH cofeed (FS3) this study mixed nitrifying cultures NH3 and NH2OH alternating feed this (FS4) study mixed nitrifying cultures NH2OH only feed with biomass this replenishment (high rate) study pure suspended cultures of N. europaea 7 pure suspended cultures of N. europaea 22 pure immobilized cultures of N. europaea 21

biomass replacement, likely due to inhibition by the accumulated CH3OH, by the point of biomass addition (t = 2h). However, the decrease in the CH3OH production rate after the first biomass replacement was not as pronounced as the decrease without replacement (for NH2OH feed, FS2, Figure 2b). The higher rates of CH3OH production during the high-rate feeding strategy were therefore due to the lower aeration rate imposed, which promoted higher CH4 gas−liquid mass transfer and therefore increased conversion of CH4 to CH3OH. Therefore, these results underline the significance of maximizing the rate of CH4 gas−liquid transfer to maximize the kinetics and yields of CH3OH production. A finite CH3OH production capacity was still observed and after two replacements, the CH3OH production rates were similar to the second-generation experiments without biomass replacement (Figure 2b). The reduction in CH3OH production rates suggests that CH3OH inhibition of AMO was likely sustained and could be a limiting factor for this concept as a whole, unless addressed by adequate process engineering approaches and reactor configurations. From a cost perspective, the ratio of the cost of (NH2OH)2H2SO4 to the cost recovery from the produced CH3OH was 45:1 (SI Table S-II). Reactor Configurations. At this point, given the low concentrations of CH3OH produced, separation and purification of CH3OH to produce a commodity chemical do not appear to be viable options. If, on the other hand, the objective were to use CH3OH as a stimulant for denitrification, then the produced methanol could be channeled to a downstream anoxic reactor to enhance denitrification, where it would be consumed. In addition, cycling between oxic and anoxic conditions also leads to internal production of NH2OH by AOB,4 possibly reducing the need for supplying it externally. However, the cost and energy benefits of each of these strategies need to be individually evaluated in the overall framework of any given wastewater treatment or resource recovery facility. Another option to enhance overall process efficiency is by channeling the off-gas from the CH4 to CH3OH conversion bioreactor to a cogeneration unit, given that a fraction of the supplied CH4 will eventually be released to the off-gas, without conversion to CH3OH. It must also be mentioned that none of the feeding strategies described above are fully optimized. Indeed, one of the primary objectives of this study was to just identify via experimentation the factors to be optimized for maximizing the conversion of CH4 to CH3OH. As described above, the two crucial factors identified include the feeding strategy itself and improved gas−

initial slopes of the methanol accumulation profiles in Figure 2a). The initial rate of CH3OH production with just NH2OH feeding was the highest among all the second-generation experiments likely owing to the supply of excess reducing equivalents, prior to reduction in AMO activity (Figure 2b). These rates are expectedly lower than the specific oxidation rates of NH3 (the primary energy substrate for AOB) by this same reactor by a factor of approximately five15 and are likely limited by mass transfer of gaseous CH4 as well as inhibition of NH3 and CH4 oxidation by CH4 20 and CH3OH (as described below). High-Rate Methanol Production. During the biomass replacement tests, the initial CH3OH production rate (0.80 mg CH3OH COD/mg biomass COD-d, Figure 2b) and CH3OH concentrations after 7 h (59.89 ± 1.12 mg CH3OH COD/L, Figure 2a) when fed with just NH2OH were significantly higher than the corresponding second-generation experiments (Figure 2a and b). When compared to FS2, which was similar in terms of NH2OH feed, but with a higher O2 flow rate, the high-rate strategy resulted in statistically higher CH3OH concentrations. This difference is likely due to the higher O2 sparging rate and the resulting lower proportion of CH4 introduced into the reactor during FS2. These maximum CH4 to CH3OH oxidation rates achieved are about 1.5 times the highest reported by AOB in previous studies (Table 1). The yield was ten times that reported by immobilized pure cultures of N. europaea21 and approximately 2.5 times that reported for suspended pure cultures of N. europaea.7 The higher rates and yields observed herein compared to previous studies could be likely due to a combination of the feeding strategies employed as well as differences in factors such as gas−liquid transfer and the physiological state of the organisms themselves. The objective of providing fresh biomass periodically was to explore an alternate strategy to achieve sustained AMO activity. In this case, AMO activity would result from active NH3 oxidation in the continuous parent reactor, rather than in the CH4 to CH3OH “side” fed-batch reactor. Yet another advantage of replenishing AOB biomass would be the possible reduction in CH3OH-mediated toxicity to AOB resulting from continued exposure. Biomass replenishment could be also possible and automated in a continuous plant by periodically pumping fresh biomass from an autotrophic nitrifying reactor (without CH4 supply) into the biomethanol reactor. Contrary to expectation, replacing the biomass did not result in sustained high rates of CH3OH production and there was actually a decrease in the CH3OH production rate even after the first E

dx.doi.org/10.1021/es3042912 | Environ. Sci. Technol. XXXX, XXX, XXX−XXX

Environmental Science & Technology

Article

of this process could potentially allow wastewater treatment plants to offset some of their CH3OH costs. Consequently, the overall greenhouse footprint of wastewater treatment plants (by lowering CH4 release as well as recovering CH3OH) could be reduced. At the same time, through this microbially mediated approach, redundancies in currently followed chemical conversion of CH4 to CH3OH can be avoided. Further mechanistic and modeling studies are needed to understand the substrate and product fluxes during AOB mediated oxidation of CH4 to CH3OH oxidation and to maximize the kinetics and yield of CH3OH.

liquid mass-transfer for CH4, which will be addressed during follow-up studies. Inhibition of Ammonia and Hydroxylamine Oxidation by Methanol. Exposure to CH3OH for 7 h resulted in a decrease in the μmax estimates of NH3 oxidation but not NH2OH oxidation by AOB (Figure 3). For NH3 oxidation, the

■

ASSOCIATED CONTENT

■

AUTHOR INFORMATION

S Supporting Information *

Calculations, tables, and figures as mentioned in the text. This material is available free of charge via the Internet at http:// pubs.acs.org. Figure 3. Impact of 7 h methanol exposure on the maximum specific growth rate of AOB associated with NH3 oxidation and NH2OH oxidation.

Corresponding Author

*Phone: (212) 854 9027; fax: (212) 854 7081; e-mail: [email protected]; URL: www.columbia.edu/∼kc2288. Notes

The authors declare no competing financial interest.

estimated noncompetitive inhibition coefficient, KI,CH3OH was 62.8 mg COD/L (Figure 3), which is considerably lower than a previously reported value of 240 mg COD/L, using pure cultures of N. europaea.20 Therefore, the reduction in CH3OH production rates with increasing incubation times (Figure 2a and b) was possibly due to CH3OH inhibition of AMO, perhaps also compounded by factors such as reducing equivalent limitation or even down-regulation of AMO in the absence of NH3. The mechanism of CH3OH-mediated inhibition of AOB cannot be conclusively determined from the results of this study. Nevertheless, from a process engineering perspective, it might thus be beneficial to develop strategies for CH3OH consumption or removal to alleviate AOB inhibition, as discussed above in the section on Reactor Configurations. An additional consideration is that the results from this study were obtained from a nitrifying enrichment culture cultivated using NH3 as the sole electron donor and only exposed to CH4 and CH3OH during the batch tests, which lasted for a maximum of 7 h. It is conceivable that long-term cultivation of AOB on CH4 might result in adaptation of the AOB to CH4 and CH3OH, possibly resulting in higher yields and kinetics of CH3OH. Clearly, longer-term continuous operation of the CH4 to CH3OH process needs further investigation. Finally, pure O2 and pure CH4 were only used to maximize CH3OH production for this study. Subsequent studies will indeed employ representative gas mixtures to simulate biogas and air. In sum, a biological process for utilizing the ability of autotrophic AOB to convert CH4 to CH3OH was developed, admittedly with the addition of external reducing equivalents. The primary factors to maximize CH4 conversion to CH3OH include the selection of appropriate feeding (or supply) strategies for CH4, NH3, and reducing equivalents, maximizing CH4 gas−liquid mass transfer and minimizing the impacts of cosubstrate (CH4) and product (CH3OH) inhibition. The results obtained highlight the metabolic versatility of AOB to convert CH4 to CH3OH and point to the possibility of developing engineered processes to promote the production and utilization of CH4 as a chemical needed for enhanced denitrification. Once optimized, the successful implementation

■ ■

ACKNOWLEDGMENTS This work was supported by the Paul Busch award to K.C. REFERENCES

(1) Chain, P.; Lamerdin, J.; Larimer, F.; Regala, W.; Lao, V.; Land, M.; Hauser, L.; Hooper, A.; Klotz, M.; Norton, J.; Sayavedra-Soto, L.; Arciero, D.; Hommes, N.; Whittaker, M.; Arp, D. Complete genome sequence of the ammonia-oxidizing bacterium and obligate chemolithoautotroph Nitrosomonas europaea. J. Bacteriol. 2003, 185 (9), 2759−2773. (2) Hooper, A. B.; Vannelli, T.; Bergmann, D. J.; Arciero, D. M. Enzymology of the oxidation of ammonia to nitrite by bacteria. Antonie van Leeuwenhoek 1997, 71, 59−67. (3) Hooper, A. B. Biochemistry of the nitrifying lithoautotrophic bacteria. In Autotrophic Bacteria; Schlegel, H. G., Bowien, B., Eds.; Springer Verlag: Berlin, 1989; pp 239−265. (4) Chandran, K.; Stein, L. Y.; Klotz, M. G.; van Loosdrecht, M. C. M. Nitrous oxide production by lithotrophic ammonia-oxidizing bacteria and implications for engineered nitrogen-removal systems. Biochem. Soc. Trans. 2011, 39 (6), 1832−1837. (5) Hyman, M. R.; Murton, I. B.; Arp, D. J. Interaction of Ammonia Monooxygenase from Nitrosomonas europaea with Alkanes, Alkenes, and Alkynes. Appl. Environ. Microbiol. 1988, 54 (12), 3187−3190. (6) Jones, R. D.; Morita, R. Y. Methane Oxidation by Nitrosococcus oceanus and Nitrosomonas europaea. Appl. Environ. Microbiol. 1983, 45 (2), 401−410. (7) Hyman, M. R.; Wood, P. M. Methane oxidation by Nitrosomonas europaea. Biochem. J. 1983, 212 (1), 31−37. (8) Voysey, P. A.; Wood, P. M. Methanol and Formaldehyde Oxidation by an Autotrophic Nitrifying Bacterium. J. Gen. Microbiol. 1987, 133 (2), 283−290. (9) Hyman, M. R.; Wood, P. M. Ethylene oxidation by Nitrosomonas europaea. Arch. Microbiol. 1984, 137 (2), 155−158. (10) Duddleston, K. N.; Bottomley, P. J.; Porter, A. J.; Arp, D. J. New Insights into Methyl Bromide Cooxidation by Nitrosomonas europaea Obtained by Experimenting with Moderately Low Density Cell Suspensions. Appl. Environ. Microbiol. 2000, 66 (7), 2726−2731. (11) Holmes, A. J.; Costello, A.; Lidstrom, M. E.; Murrell, J. C. Evidence that particulate methane monooxygenase and ammonia monooxygenase may be evolutionarily related. FEMS Microbiol. Lett. 1995, 132 (3), 203−208. F

dx.doi.org/10.1021/es3042912 | Environ. Sci. Technol. XXXX, XXX, XXX−XXX

Environmental Science & Technology

Article

(12) Ward, B. B. Kinetic studies on ammonia and methane oxidation by Nitrosococcus oceanus. Arch. Microbiol. 1987, 147 (2), 126−133. (13) Stein, L. Y.; Arp, D. J.; Berube, P. M.; Chain, P. S. G.; Hauser, L.; Jetten, M. S. M.; Klotz, M. G.; Larimer, F. W.; Norton, J. M.; Camp, H. J. M. O. d.; Shin, M.; Wei, X. Whole-genome analysis of the ammonia-oxidizing bacterium, Nitrosomonas eutropha: Implications for niche adaptation. Environ. Microbiol. 2007, 9 (12), 2993−3007. (14) Chain, P.; Lamerdin, J.; Larimer, F.; Regala, W.; Lao, V.; Land, M.; Hauser, L.; Hooper, A.; Klotz, M.; Norton, J. Complete genome sequence of the ammonia-oxidizing bacterium and obligate chemolithoautotroph Nitrosomonas europaea. J. Bacteriol. 2003, 185, 2759− 2773. (15) Ahn, J. H.; Yu, R.; Chandran, K. Distinctive microbial ecology and biokinetics of autotrophic ammonia and nitrite oxidation in a partial nitrification bioreactor. Biotechnol. Bioeng. 2008, 100 (6), 1078− 1087. (16) Rittmann, B. E.; McCarty, P. L. Environmental BiotechnologyPrinciples and Applications; McGraw Hill: New York, 2001. (17) Chandran, K.; Smets, B. F. Optimizing experimental design to estimate ammonia and nitrite oxidation biokinetic parameters from batch respirograms. Water Res. 2005, 39 (20), 4969−4978. (18) Chandran, K.; Love, N. G. Physiological state, growth mode, and oxidative stress play a role in Cd(II)-mediated inhibition of Nitrosomonas europaea 19718. Appl. Environ. Microbiol. 2008, 74 (8), 2447−2453. (19) Yu, R.; Chandran, K. Strategies of Nitrosomonas europaea 19718 to counter low dissolved oxygen and high nitrite concentrations. BMC Microbiol. 2010, 10 (1), 70. (20) Hooper, A. B.; Terry, K. R. Specific inhibitors of ammonia oxidation in Nitrosomonas. J. Bacteriol. 1973, 115 (2), 480−485. (21) Thorn, G. J. S. Development of an immobilized Nitrosomonas europaea bioreactor for the production of methanol from methane. Master’s Thesis, University of Canterbury, Christchurch, New Zealand, 2007. (22) Hyman, M. R.; Murton, I. B.; Arp, D. J. Interaction of ammonia monooxygenase from Nitrosomonas europaea with alkanes, alkenes and alkynes. Appl. Environ. Microbiol. 1988, 54 (12), 3187−3190.

G

dx.doi.org/10.1021/es3042912 | Environ. Sci. Technol. XXXX, XXX, XXX−XXX