Matching Different Inorganic Compounds as Mixture of Electron

Jul 20, 2010 - YIQUAN LE. School of Environmental Science and Engineering, State Key. Laboratory of Pollution Control and Resource Reuse, Tongji...
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Environ. Sci. Technol. 2010, 44, 6364–6370

Matching Different Inorganic Compounds as Mixture of Electron Donors to Improve CO2 Fixation by Nonphotosynthetic Microbial Community without Hydrogen JIAJUN HU, LEI WANG,* SHIPING ZHANG, XIAOHUA FU, AND YIQUAN LE School of Environmental Science and Engineering, State Key Laboratory of Pollution Control and Resource Reuse, Tongji University, Shanghai 200092, China

Received January 29, 2010. Revised manuscript received July 1, 2010. Accepted July 2, 2010.

The dominant bacteria in nonphotosynthetic microbial community (NPMC) isolated from the ocean were identified by PCR-DGGE. The results revealed that the dominant microorganisms in cultures of NPMC differed when Na2S, Na2S2O3, and NaNO2 were used as the electron donor to reduce CO2. These findings implied that different microorganisms in the NPMC respond to different inorganic compound as suitable electron donor, indicating that matching of Na2S, Na2S2O3, and NaNO2 may provide mixed electron donors that increase the ability of NPMC to fix CO2. Accordingly, the central composite response surface method (RSM) was used to predict the optimal concentration and match of Na2S, Na2S2O3, and NaNO2 as mixed electron donors to improve CO2 fixation efficiency under aerobic and anaerobic conditions without hydrogen. The results indicated that 0.46% NaNO2, 0.50% Na2S2O3, and 1.25% Na2S were the optimal match under aerobic conditions, while 1.04% NaNO2, 1.07% Na2S2O3, and 0.98% Na2S were the optimal match under anaerobic conditions. Under these conditions, the fixed CO2 by NPMC was determined to be 387.51 and 512.57 mg/L, respectively, which obviously exceeded those values obtained prior to optimization (5.94 and 7.14 mg/ L, respectively), as well as that obtained when hydrogen was used as the electron donor (91.60 mg/L).

1. Introduction The absorption and resource reuse of CO2 is one of the most important measures currently used to reduce the rate and magnitude of global warming (1, 2). Microbial CO2 fixation is a significant method of environmental protection and resource exploitation based on the material flow and energy flow of the entire biosphere (3, 4). To date, most studies that have been conducted to evaluate CO2 fixing by microorganisms have primarily focused on algae and hydrogen-oxidizing bacteria (5, 6). However, algae require light during culture and cannot resist high concentrations of CO2 (7, 8), while hydrogen-oxidizing bacteria require large amounts of hydrogen gas (9, 10), which has limited the application of these two methods. Therefore, it is necessary to isolate highly efficient CO2 fixing microorganisms that do not require light * Corresponding author e-mail: [email protected]. 6364

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or hydrogen to enable a wider range of applications and far-reaching effects. The energy of nonphotosynthetic microbial CO2 fixation only occurs via oxidation of inorganic compounds used as electron donors that can release energy and electrons when CO2 is the sole carbon source (11). The energy material is a crucial factor involved in microbial CO2 fixing (12). In our previous study (13), two NPMCs with CO2 fixing capability without hydrogen were obtained by screening seawater and sediment samples collected from the Yellow Sea, East China Sea, South China Sea, and Antarctic waters. The use of NaNO2, Na2S2O3, and Na2S as electron donors was found to be capable of improving the efficiency of CO2 fixing, but each of their fixation efficiencies was still lower than that of hydrogen gas, which was considered a highly effective electron donor (14). The CO2 fixing pathway of a microbial community is much more complicated than that of a single strain. This may result in the inorganic compounds as electron donors becoming more varied due to different microorganisms in the microbial community producing different responses to various electron donors. Therefore, mixed electron donors may be more suitable for reduction of CO2 by NPMC. To determine if matching different inorganic compounds to develop a mixture of electron donors is more suitable to NPMC to enable efficient fixation of CO2 without hydrogen, the variability of the dominant organisms in NPMC in response to each electron donor was evaluated by PCR-DGGE (denaturing gradient gel electrophoresis). These results were then used as a basis to optimize the concentrations and ratios of the inorganic compounds tested based on a statistical experimental design to develop a mixture of electron donors suitable for NPMC. The statistical experimental design could not only clarify the interactive effects of the electron donors, but also aid in understanding the true potentials of CO2 fixing microbes. The results may provide technical support for the development of an alternative biopathway to efficiently fix and reuse CO2, thus mitigating global warming.

2. Materials and Methods 2.1. Nonphotosynthetic CO2 Fixing Microorganism. See Supporting Information. 2.2. Cultivation Conditions. NPMCs were cultured in 150-mL serum bottle with 40 mL of medium under aerobic or anaerobic conditions for 96 h. The details were described in the Supporting Information. 2.3. DNA Extraction and PCR-DGGE. See Supporting Information. 2.4. Sequencing of DGGE Bands. See Supporting Information. 2.5. Estimation of CO2 Fixing Efficiency. The total organic carbon (TOC) value reflecting the microbial CO2 fixing efficiency was analyzed using a Shimadzu TOC-VCPH total organic carbon analyzer (Shimadzu Seisakusho Co. Ltd., Kyoto, Japan). To reduce the impact of inorganic carbon on the analyses, the pH of the sample was adjusted to about 4.0 prior to TOC analysis. 2.6. Experimental Design. Preliminary experiments were conducted to screen the appropriate parameters and determine the experimental domain of each electron donor concentration under aerobic and anaerobic conditions. The statistical software package, Design-Expert 7.1.3 (Stat-Ease, Inc., Minneapolis, MN), was used to design the experiments and analyze the results. The response surface method design involving a central composite design (CCD) was adopted to match the ratio of NaNO2, Na2S2O3, and Na2S to improve the CO2 fixing efficiency by NPMC. A set of 20 experiments 10.1021/es1002499

 2010 American Chemical Society

Published on Web 07/20/2010

FIGURE 1. DGGE fingerprint: (a) The bacteria were screened from seawater, where I was the initial bacteria, II was the bacteria cultured under aerobic condition with hydrogen gas and Na2S, III was the bacteria cultured under aerobic condition with Na2S, IV was the bacteria cultured under aerobic condition with Na2S2O3, V was the bacteria cultured under anaerobic condition with Na2S, VI was the bacteria cultured under anaerobic condition with Na2S2O3. (b) The bacteria were screened from sediments, where I was the initial bacteria, II was the bacteria cultured under aerobic condition with hydrogen gas and Na2S, III was the bacteria cultured under aerobic condition with Na2S, IV was the bacteria cultured under anaerobic condition with Na2S2O3, V was the bacteria cultured under anaerobic condition with NaNO2. The numbered bands were excised, reamplified, and sequenced. (c) Dice coefficient of the lanes, where a and b correspond to the results in parts a and b, respectively, and II, III, IV, V, and VI refer to the lane number in parts a and b. including six center points were conducted under aerobic and anaerobic conditions. All variables were taken at a central coded value of zero. The minimum and maximum range of variables investigated and the full experimental plan with respect to the actual and coded forms were as follows: NaNO2 levels of -1 and +1 were 0.25% and 0.75% w/v, respectively, while Na2S2O3 levels of -1 and +1 were 0.50% and 1.00% w/v, respectively, and Na2S levels of -1 and +1 were 0.75% and 1.25% w/v, respectively, under aerobic conditions. The NaNO2 levels of -1 and +1 were 0.55% and 1.05% w/v, respectively, while the Na2S2O3 levels of -1 and +1 were 0.60% and 1.10% w/v, respectively, and the Na2S levels of -1 and +1 were 0.75% and 1.25% w/v, respectively, under anaerobic conditions. For statistical calculations, the actual values of variables Xi were coded as xi according to the following relationship xi ) (Xi - X0)/∆X where xi is the dimensionless value of the independent variable, Xi represents the real value of the independent variable, X0 is the value of Xi at the center point, and ∆X presents the step change. Experimental data were analyzed by the RSM using the following second-order polynomial equation n

y ) β0 +

n

∑βX + ∑β X

2 ii i

i i

i)1

i)1

n

+

∑ ∑ βXX

i i j

i0.05 indicates not significant), respectively. It should be noted that the lack of fit value was not significant relative to the pure error. ANOVA analysis confirmed that the models were good and fit the experimental data well. The p-value of the model terms obtained from all parameters except B, AC, and C2 under aerobic modeling conditions was lower than 0.0001. The values for B, AC, and C2 were 0.0008, 0.4364, and 0.0351, respectively. The p-values of the model terms under aerobic modeling conditions were all lower than 0.0001. The term was significant when the p-value was less than 0.05. The P-values of AB and BC in the aerobic model were 0.10, which meant that the interaction between A and C could be ignored in the reaction system. In the anaerobic model, the P-values of AB, AC, BC, and ABC were all far less than 0.05, indicating that the interaction between any two of the three electron donors or between all three were significant in the reaction system. The two interactions (AB, BC) were both significant under aerobic/anaerobic conditions. An interaction between NaNO2 and Na2S2O3 was also observed by Baalsrud. Specifically, it was reported that in the presence of nitrite, thiosulphate was oxidized very rapidly while the nitrite was oxidized to nitric oxide, which indicated that the oxidation of these two electron donors was probably coupled in an obligatory way (18). In addition, S0 was produced during oxidization of S2-, and the thiosulfate disproportionation reaction occurred, which may have led to the interaction between S2- and S2O32-. Moreover, S2O32- was produced by the thiosulfate disproportionation reaction (11). S0 can be produced by a reaction between SO32- and S2- under acidic conditions (11). However, the two interactions (AC, ABC) were only significant under anaerobic conditions, and were negligible under aerobic conditions. Many electron acceptors substitute O2, such as NO3-, which may be the reason for the interactions between AC and ABC. Another reason for the interactions may be that a symbiotic effect occurred between different bacterium of the NPMC using different electron donors. The 3-D response surface curves were plotted according to the regression equation (eqs 1 and 2) to further investigate

the interaction between the factors and to determine the optimum levels of each factor required for maximum TOC. The results shown in Figure 2a demonstrate that, in the aerobic model, the interaction between Na2S2O3 and Na2S was significant when there was a lower concentration of Na2S2O3 and higher concentration of Na2S, with the strongest interaction being observed in response to 0.50% Na2S2O3 and 1.25% Na2S. As shown in Figure 2b, the interaction between Na2S2O3 and NaNO2 was obvious when the concentrations of Na2S2O3 and NaNO2 were high, with the strongest effect being observed in response to 1.00% Na2S2O3 and 0.75% NaNO2. In the anaerobic model, the interaction between Na2S2O3 and Na2S was similar to that of the aerobic model (see Figure 2c). Specifically, the strongest effects were observed in response to 0.60% Na2S2O3 and 1.25% Na2S. As shown in Figure 2d, the interaction between Na2S2O3 and NaNO2 was strongest in response to 1.10% Na2S2O3 and 1.05% NaNO2, which was similar to the results observed when the aerobic model was used. The interaction between NaNO2 and Na2S in the reaction system could be ignored in the aerobic model, while it was significant under anaerobic conditions (see Figure 2e). The strongest interaction was observed in response to 0.90% NaNO2 and 1.25% Na2S. Comparison of the results shown in Figures 2 reveals that the interaction of the electron donor under anaerobic conditions was generally stronger than under aerobic conditions. Additionally, the trend in the interaction with changes in the concentration of electron donors under aerobic and anaerobic conditions differed significantly. On the basis of the results of the experiments above, the CO2 fixing efficiency of the NPMC was obviously affected by the oxygen concentration. The effects of mixed electron donors were stronger under anaerobic conditions. There may be two possible reasons for this. First, it is well-known there are many key enzymes involved in the CO2 fixation pathway which were sensitive to oxygen. For example, 4-hydroxybutyryl-CoA dehydratase is the key enzyme involved in the 3-hydroxypropionate/4-hydroxybutyrate pathway. This enzyme is sensitive to oxygen exposure due to inactivation of the clusters that reside in its active center (19). Although the microorganisms also implement additional measures to prevent inactivation under oxygen-rich conditions, the entire pathway is still affected by the oxygen concentration to a certain extent. This occurs via the fixation pathway. With respect to the electron donors, there may be different utilization efficiencies and pathways for microorganisms with and without oxygen gas. The mechanism through which interactions with different electron donors influence CO2 fixation by NPMC is currently being studied through enzymological analysis. Another reason may be that the reduction potential of the dipole pair 1/2O2/H2O is very high (+0.82 V). O2 has a strong tendency to accept electrons and can release more energy than other electron acceptors. For some facultative anaerobes, such as denitrifying bacteria, aerobic respiration is dominant in the presence of O2, but substituted electron acceptors are reduced in the absence of O2. The common electron acceptors, NO3-, SO42-, and CO2, which were present in the culture system employed here, may be utilized as electron acceptors simultaneously. Accordingly, SO42- was the final product for the multistep oxidation of S2O32- and S2-, and NO3- was the final product for the oxidation of NO2- (11). These findings may indicate that, in addition to releasing energy via oxidation, the energy materials could regenerate to some extent and be utilized again. This recycling may be achieved by different bacteria using different electron donors. For example, Thiobacillus denitrificans can oxidize S2- and reduce NO3- simultaneously (20). The simultaneous oxidation of NO2- and the reduction of SO42- may be completed by the cooperation of specific VOL. 44, NO. 16, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 2. Response surface plots for TOC showing (a) the interacting effect of Na2S2O3 and Na2S concentration under aerobic conditions, (b) the interacting effect of Na2S2O3 and NaNO2 concentration under aerobic conditions, (c) the interacting effect of Na2S2O3 and Na2S concentration under anaerobic conditions, (d) the interacting effect of Na2S2O3 and NaNO2 concentration under anaerobic conditions, and (e) the interacting effect of NaNO2 and Na2S concentration under anaerobic conditions. (a-e) Another factor of the three was set at the center point of the concentration. and nonspecific strains as discussed in section 3.1. Thus, the overall energy released from the substitute electron acceptors was higher than that released from O2. To study the different manifestations of the electron donors under aerobic and anaerobic conditions directly, the data generated under the aerobic and anaerobic conditions were merged together and the gas phase condition was used as a categoric factor for subsequent analysis using Design Expert (detailed information and discussions are provided in the description of the mergence of aerobic and anaerobic model, Supporting Information). The results indicated that the oxygen concentration influenced the effect of the electron donors significantly. 3.3. Optimization of Microbial CO2 Fixing Efficiency by Matching Different Inorganic Compounds as a Mixture of Electron Donors. To match NaNO2, Na2S2O3, and Na2S as mixed electron donors to improve the CO2 fixation efficiency of NPMC under aerobic and anaerobic conditions, the optimal values of each factor for maximum TOC under aerobic and anaerobic conditions were predicted by Design Expert based on numerical optimization (see Figure 3a,b). 6368

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Numerical optimization is a tool used to identify the optimal settings of factors used in the design space by the established models. As shown in Figure 3a, there were two higher areas (TOC > 85) and a highest area (TOC > 95 mg/L) when a lower concentration of Na2S2O3 and a moderate concentration of NaNO2 were used under aerobic conditions. The optimal level given by numerical optimization was 0.46% NaNO2, 0.50% Na2S2O3, and 1.25% Na2S, which gave a predicted TOC value of 102.80 mg/L. The result obtained during the validation experiment was 105.76 mg/L and the TOC value of control sample was 1.62 mg/L. Figure 3b showed that the highest area (TOC > 125 mg/L) appeared when the concentration of NaNO2 and Na2S2O3 was higher under anaerobic conditions. The optimal level given by numerical optimization was 1.04% NaNO2, 1.07% Na2S2O3, and 0.98% Na2S, and the predicted TOC value was 141.94 mg/L. The result of the validation experiment was 139.89 mg/L, and the TOC value of the control sample was 1.95 mg/L. Using this mixture of electron donors, the maximum CO2 fixing efficiency of NPMC was 387.51 and 512.57 mg/L under aerobic or anaerobic conditions, respectively, which was

FIGURE 3. Response contour plot for the predicted maximum TOC under (a) aerobic conditions (Na2S concentration was set at 1.25%) and (b) anaerobic conditions (Na2S concentration was set at 0.98%). much higher than that before optimization (5.94 and 7.14 mg/L) and that when hydrogen was used as the electron donor (91.60 mg/L). Scott et al. found that the maximum efficiency of CO2 fixing by endosymbiotic chemoautotrophs was approximately 50.8 µmol min-1 g-1 protein (21). Assuming that the value of the carbon concentration of the dry biomass in our experiment was equal to that of the protein concentration (the initial value under the aerobic and anaerobic condition was 0.576 mg protein L-1 and 0.643 mg protein L-1, respectively), the maximum CO2 fixing efficiency of the NPMC under aerobic and anaerobic conditions was approximately 2.65 and 3.14 mmol min-1 g-1 protein, respectively, values which were far higher than the values of endosymbiotic chemoautotrophs observed by Scott et al. Lo´pez et al. reported that the efficiency of CO2 fixing by the photosynthetic microbe, cyanobacteria Anabaena sp. ATCC 33047, was about 1.45 g CO2 L-1 day-1 (22). When the photobacterium was cultured for 96 h, this value corresponded to 5.8 g CO2 L-1, which was 10 times greater than the value observed in our experiment. But carbon fixing by the cyanobacteria Anabaena sp. ATCC 33047 was conducted in discontinuous mode using a constant biomass concentration of 1 g L-1 (22). This difference may have occurred because the initial biomass used in their experiment was much greater than in our experiment. All of the above results indicated that there are different responses of the NPMC structure to different electron donors, which was the basis of improving the microbial CO2 fixing efficiency with a mixture of electron donors. The stronger interactions between electron donors in the system could improve the microbial CO2 fixing efficiency remarkably. The CO2 fixing efficiencies of the NPMC under aerobic conditions or anaerobic conditions were both improved significantly to the optimal level using the mixture of electron donors. Therefore, the application of a statistical experimental design to match different inorganic compounds as mixed electron donors is a useful method for enhancing the CO2 fixation efficiency of NPMC under aerobic and anaerobic conditions without hydrogen. At present, pilot-scale cultivation of the NPMC and recycling of the mixture of electron donors are being evaluated.

Acknowledgments The work was financially supported by National Key Scientific and Technological Project, China (2006BAC01A14).

Supporting Information Available Bacteria source, cultivation conditions, identification of microorganisms, mergence of aerobic and anaerobic models (Figure S1), and results of microorganisms sequence (Tables

S1). This material is available free of charge via the Internet at http://pubs.acs.org.

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