Appl Biochem Biotechnol DOI 10.1007/s12010-015-1657-4
Interactions Between Autotrophic and Heterotrophic Strains Improve CO2 Fixing Efficiency of Non-photosynthetic Microbial Communities Jiajun Hu 1,2 & Lei Wang 1 & Shiping Zhang 3 & Xuefei Xi 1 & Yiquan Le 1 & Xiaohua Fu 1 & Yiufai Tsang 4 & Mintian Gao 2
Received: 21 January 2015 / Accepted: 28 April 2015 # Springer Science+Business Media New York 2015
Abstract Five autotrophic strains isolated from non-photosynthetic microbial communities (NPMCs), which were screened from oceans with high CO2 fixing capability, were identified as Ochrobactrum sp. WH-2, Stenotrophomonas sp. WH-11, Ochrobactrum sp. WH-13, Castellaniella sp. WH-14, and Sinomicrobium oceani WH-15. The CO2 fixation pathways of all these strains were Calvin-Benson-Bassham pathway. These strains could metabolize multifarious organic compounds, which allowed switching them to autotrophic culture after enrichment in heterotrophic culture. The central composite response surface method indicated that these strains possessed many interactive effects, which increased the CO2 fixing efficiency of a combined community composed of these strains by 56 %, when compared with that of the single strain. Furthermore, another combined community composed of these autotrophic strains and NPMC had richer interactive relationships, with CO2 fixing efficiency being 894 % higher than that of the single strain and 148 % higher than the theoretical sum of the CO2 fixing efficiency of each of its microbial components. The interaction between strictly heterotrophic bacteria in NPMC and isolated autotrophic strains played a crucial role in Electronic supplementary material The online version of this article (doi:10.1007/s12010-015-1657-4) contains supplementary material, which is available to authorized users.
* Lei Wang [email protected] 1
State Key Laboratory of Pollution Control and Resource Reuse, School of Environmental Science and Engineering, Tongji University, Shanghai 200092, China
2
Shanghai Key Laboratory of Bio-Energy Crops, School of Life Sciences, Shanghai University, Shanghai 200444, China
3
Low-Carbon Conversion Science and Engineering Center, Shanghai Advanced Research Institute, Chinese Academy of Sciences, Shanghai 201210, China
4
Department of Science and Environmental Studies, The Hong Kong Institute of Education, Hong Kong 999077, China
Appl Biochem Biotechnol
improving the CO2 fixing efficiency, which not only eliminated self-restraint of organic compounds generated during the growth of autotrophic bacteria but also promoted its autotrophic pathway. Keywords Autotrophic strain . CO2 fixing . Combined community . Interaction . Heterotrophic bacteria
Introduction Microbial CO2 fixation is considered as an important method of environmental protection and resource exploitation based on the entire carbon cycle [1–3]. Photosynthetic microorganisms, especially algae, have attracted increasing attention [4, 5] not only for its CO2 fixation capacity but also for its added value, such as its application in microbial fuel cell [6]. Nevertheless, CO 2 fixation by nonphotosynthetic microorganisms is also considered to be an important process, especially in soil environment without light [7]. When compared with photosynthetic microorganisms, non-photosynthetic microorganisms exhibit a wider range of physiological and ecological traits [8]. As non-photosynthetic microorganisms do not need light during the CO2 fixing process, they have a wider range of applications and farreaching effects. It has been reported that CO2 fixation by chemoautotrophic microorganisms (such as sulfur bacteria and nitrifying bacteria) is inefficient because their generation time is from several hours to even close to 1 day [9, 10]. Alternatively, hydrogen-oxidizing bacteria are considered to be potential candidates for CO2 fixation. However, high concentration of H2 is required for the cultivation of hydrogen-oxidizing bacteria, which limits their large-scale application [11]. When compared with a single strain, NPMC has been found to have a higher CO2 fixing efficiency [12]. This could be mainly owing to the fact that NPMC possesses richer metabolic pathway than a single strain because the interactions between different strains, such as syntrophy, resulting in higher CO2 fixing efficiency. Syntrophy is the combination of the metabolic capabilities of two or more organisms to catabolize a substrate that cannot be catabolized by either one of them [13]. This enables the microorganisms to utilize resources more efficiently for supporting their growth, which might also occur in NPMC for CO2 fixation. In a previous study, it was found that there are numerous strictly heterotrophic bacteria in a NPMC with high CO2 fixing efficiency. This suggests that the metabolism of NPMC may be beyond the scope of chemoautotrophic bacteria with more interactive relationships not only between autotrophic bacteria but also between autotrophic and heterotrophic bacteria. In the present study, multiple chemoautotrophic strains were isolated from NPMC and their carbon metabolism and 16S rDNA were examined. Subsequently, the interaction of autotrophic strains and their effects were studied by constructing a mathematical model. Furthermore, the influence of strictly heterotrophic bacteria on the CO2 fixing efficiency of the combined community was also investigated. An attempt was made to explore the mechanism of interactive relationships in NPMC to improve the CO2 fixing efficiency and also find a combined community with high CO2 fixing efficiency. The experimental results may provide technical support for the
Appl Biochem Biotechnol
development of an alternative biopathway to efficiently fix and reuse CO2, thus mitigating global warming.
Materials and Methods Non-photosynthetic CO2 Fixing Microorganisms NPMC capable of fixing CO2 were isolated from the surface seawater of four oceans around the world, including Australia, China, Japan, France, Thailand, Equator, Antarctic Pole, and Arctic Pole [14].
Autotrophic Culture The autotrophic culture medium (ACM) contained (g/L): Na2CO3, 1.0; NaHCO3, 1.0; (NH4)2SO4, 5.0; KH2PO4, 1.0; K2HPO4, 2.0; MgSO4·7H2O, 0.2; NaCl, 20; CaCl2, 0.01; and FeSO4·7H2O, 0.01. To this ACM, 2 mL of trace element solution were added, which contained (mg/L): Na2MoO4·2H2O, 1.68; H3BO3, 0.4; ZnSO4·7H2O, 1.0; MnSO4·5H2O, 1.0; CuSO4·5H2O, 7.0; CoCl2·6H2O, 1.0; and NiSO4·7H2O, 1.0. Serum bottles (150 mL) containing 40 mL of the medium were prepared and autoclaved for 20 min at 121 °C. For normal experiments, the bacterial inoculum concentration was 2.5 % (v/v). The serum bottle was sealed with a silicon stopper and filled with 20 % (v/v) CO2 by using a syringe (air:CO2 ratio of approximately 80:20). All the cultures were incubated at 30 °C and 120 rpm without light for 96 h. The atmosphere within the serum bottles was readjusted to the initial ratios after culturing for 48 h.
Screening and Isolation of Single Autotrophic Strain For screening and isolation of single autotrophic strains, media with different electron donors were prepared by adding 0.1 % (w/v) NaNO2, Na2S2O3, Na2S, FeSO4, or none to the ACM. Subsequently, 2 % (w/v) agar was added to the media and the culture plates were autoclaved for 20 min at 121 °C. The following steps were conducted under sterile conditions to avoid contamination. A total of 100 μL of NPMC was inoculated onto the culture plates and incubated at 30 °C. After culturing for about 10–15 days, a single colony was picked and streaked onto new culture plates and incubated at 30 °C for 10–15 days, and the same process was repeated until isolation of a single strain. The selected single strain was inoculated into sterile EP tube (4 mL) with 2 mL of the medium and cultured for about 15 days. The isolated strains were cultured according to the autotrophic culture method described earlier and their CO2 fixing efficiency was examined. Strains with weak CO2 fixing capacity were eliminated. Strains have been deposited in China Center for Industrial Culture Collection (CICC).
Heterotrophic Enrichment Culture A total of 2.5 % (v/v) microbial culture (single strain or NPMC) was inoculated into LuriaBertani medium and incubated at 30 °C and 120 rpm for 48–96 h. Two blank samples were prepared for each heterotrophic enrichment process to examine whether the bacteria were contaminated.
Appl Biochem Biotechnol
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., Japan).
16S rDNA Sequence Determination and Phylogenetic Analysis The bacterial DNA was extracted by using a PowerSoil DNA Isolation Kit (MO BIO Laboratories, Inc., Canada) according to the manufacturer’s instructions. Primers 8f and 1492r [14] were used to amplify the bacterial 16S rDNA. The PCR products were purified by using a B type Mini-DNA Rapid Purification Kit (BioDev, China), after which they were cloned by using the PMD18-T plasmid vector system (TaKaRa, Japan). The DNA sequences were then determined by a commercial service (BGI, China) with bidirectional sequencing. The vector sequence was cut off and the remaining nucleotides were compared with those available in GenBank by using the BLAST program to identify the most similar 16S rDNA fragments. The phylogenetic analysis was conducted by using MEGA 6 [15].
Determination of the CO2 Fixing Pathway of the Isolated Autotrophic Strain and Its Capacity to Metabolize Different Organic Matters The carbon fixation pathway was examined through quantitative PCR to detect the presence of key enzyme genes in CO2 fixation pathway in the microbial DNA extracts, including form I and form II ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) genes, cbbL [16] and cbbM [17], for Calvin-Benson-Bassham (CBB) pathway; ATP-dependent citrate lyase gene, aclB [18], for reductive tricarboxylic acid cycle; and methyl coenzyme M reductase α-subunit gene, mcrA [19], for reductive acetyl-CoA pathway. The organic carbon source utilization was examined by using the API ID 32GN Test Kit (bioMérieux, France), and the results were obtained by using ATB Expression (bioMérieux, France).
Response Surface Method Design Preliminary experiments were conducted to screen the appropriate strains. The statistical software package, Design Expert 8.0.6 (Stat-Ease, Inc., USA), was used to design the experiments and analyze the results. The RSM design involving a central composite design (CCD) was adopted to match the ratio of five strains to improve the CO2 fixing efficiency of the combined community. The minimum and maximum range of the variables investigated and the complete experimental plan with respect to the actual and coded forms were as follows: each strain inoculum concentration levels of −1 and +1 were 0 and 2 % (v/v), respectively. For statistical calculations, the actual values of the variable Xi were coded as xi according to the following relationship: . xi ¼ ðX i – X 0 Þ ΔX 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 ΔX indicates the step change.
Appl Biochem Biotechnol
The experimental data were analyzed by RSM using the following second-order polynomial equation: y ¼ β0 þ
n X i¼1
βi X i þ
n X i¼1
β ii X i 2 þ
n XX
βi X i X j
i< j¼1
where y is the response (TOC, mg/L), Xi and Xj are the coded independent variables, and β0, βi, βii, and βij are the intercept, linear, quadratic, and interaction constant coefficients, respectively. Design Expert was used for regression analysis and analysis of variance (ANOVA). A total of 0.46 % NaNO2, 0.50 % Na2S2O3, and 1.25 % Na2S were added to the medium as electron donors to provide adequate energy for the microorganisms in the RSM experiments.
Statistical Analysis Cluster analysis is a mathematical method that can be used to determine objects that are similar in a group [20]. The purpose of cluster analysis is to identify subsets of a data set, which contain similar points. Replacement of these subsets by their aggregate properties, such as means and standard deviations, for example, creates a compact representation of the data set as a set of clusters. The cluster properties can then be used for comparative data analysis [21]. This approach has been considered suitable to examine the differences in carbon metabolism of the strains. In the present study, cluster analysis was carried out by using SPSS 21 (IBM, USA).
Results and Discussion Identification and Carbon Metabolism Characterization of the Dominant Autotrophic Strains Isolated from NPMC Autotrophs grow by using CO2 as their sole carbon source and generate biomass on which all other organisms thrive [22]. The present study was focused on chemoautotrophic microorganisms, which might play a major role in CO2 fixation by NPMC. First, NPMC with high CO2 fixing efficiency without H2 as the electron donor was screened from the marine environment. Then, about 300 autotrophic bacterial colonies were isolated from the NPMC. Based on the inorganic carbon assimilation efficiency, bacterial morphology, and culture conditions, a total of 30 strains were selected. After molecular biology identification, five strains with high CO2 fixing efficiency were selected for further examination. The five strains were Ochrobactrum sp. WH-2 (CICC 23802, GenBank accession number: KM502331), Stenotrophomonas sp. WH-11 (CICC 23803, GenBank accession number: KM975671), Ochrobactrum sp. WH-13 (CICC 23804, GenBank accession number: KM975672), Castellaniella sp. WH-14 (CICC 23805, GenBank accession number: KM975673), and Sinomicrobium oceani WH-15 (CICC 23806, GenBank accession number: KM975674). Except S. oceani WH-15 belonged to Bacteroidetes, other strains all belonged to Proteobacteria, the largest group of bacteria with rich metabolic diversity. Besides the aerobic pathway, anaerobic pathway was also detected because the interactions between bacteria could overcome oxidative stress under aerobic condition [2]. Quantitative
Appl Biochem Biotechnol
PCR results showed that the carbon fixation pathways of these five strains were mainly by CBB pathway without other carbon fixation pathways (such as reductive tricarboxylic acid cycle and reductive acetyl-CoA pathway). Two types of RubisCO, namely, form I and form II, could be observed in these strains. It can be seen from Fig. 1a that four strains exhibited form I RubisCO, whereas Castellaniella sp. WH-14 presented form II RubisCO. Despite the difference in their primary structure, it has been indicated that form II RubisCO could catalyze the same reaction as form I RubisCO. In addition, some other differences between form I and form II RubisCO have been reported. Most importantly, form II RubisCO has a distinct physiological role, because it is used primarily to enable the CBB pathway to balance the redox potential of the cell under select growth conditions [23]. Figure 1b, c shows that the five strains could metabolize a variety of organics, including short- and long-chain organics (with respect to carbon number), sugars, organic acids, vitamins, and amino acids (with respect to carbon structure). The ability of Castellaniella sp. WH-14 to metabolize organics with six or more carbon atoms was found to be far lower than that of the other strains. The results obtained showed that all the five strains were facultative autotrophic microorganisms and possessed rich heterotrophic metabolic pathways. The CO2 fixing efficiency of these isolated bacteria was found to be low. Under conventional culture conditions, the general CO2 fixing efficiency of these strains was 1–2 mg C/L and up to about 5 mg C/L after the culture period. This efficiency was much lower than the maximum efficiency of the NPMC [12]. These findings implied that there might be multiple interactive relationships between the microorganisms in NPMC. The results of cluster analysis showed a difference in inorganic and organic carbon metabolism of the strains on squared Euclidean distance metric (see Fig. 1d). Furthermore, the phylogenetic tree showed the different evolutionary distances between the isolated strains (see Fig. 1e). As indicated by Morris et al. [24], rich interactions could improve autotrophic metabolism and combined community with high CO2 fixing efficiency might be obtained by mixing multiple strains together. Rich interactions signify rich and efficient metabolic pathways, which may be obtained by the strains with different evolutionary distance (different species) or different ability of carbon metabolism (metabolizable organic carbon types and CO2 fixation pathways). Hence, the five strains isolated in the present study were selected to construct a combined community with interactive relationships. If these strains could constitute a collaborative carbon fixation system, then their feature as facultative autotrophic strains could provide another advantage; i.e., they could be cultured heterotrophically to achieve high cell density and can be transferred to an inorganic environment for autotrophic culture. This characteristic was conducive for the enrichment and development of a combined community quickly and easily.
Interactive Effects of the Isolated Autotrophic Strains on CO2 Fixation Interactive effects between microorganisms, such as microbial syntrophy, could enable the chemical reaction with positive free energy change to support microbial growth [24]. Syntrophy between different strains, such as ethanol fermentation bacteria and methanogens [25], signifies several reactions occurring simultaneously in different bacteria, resulting in the overall reaction to be exergonic and more effective. In the present study, to examine the presence of interaction among the five strains isolated and their effect on CO2 fixation, central composite response surface method (RSM) experimentation was used to evaluate the
Appl Biochem Biotechnol
Fig. 1 a Carbon fixing pathway of the strains. b, c Utilization of organic carbon sources by the strains. , , negative. d Dendrogram obtained by using average linkage between groups (hierarchical positive; cluster analysis algorithms, squared Euclidean distance metric) based on inorganic and organic carbon metabolism by the strains. e Branch graph of phylogenetic tree for 16S rDNA sequence of the samples. cbbL form I RubisCO gene, cbbM form II RubisCO, aclB ATP-dependent citrate lyase gene, mcrA methyl coenzyme M reductase α-subunit gene, ACE acetate, PROP propionate, LAT DL-lactate, SER L-serine, ALA L-alanine, MNT malonate, 3OBU 3-hydroxy butyrate PRO L-proline, ARA L-arabinose, RIB D-ribose, VALT valerate, ITA itaconic acid, CIT citrate, GLU D-glucose, RHA rhamnose, FUC L-fucose, INO inositol, HIS histidine, 2KG 2-ketogluconate, GLYG glycogen, MAN mannitol, SOR D-sorbitol, 5KG 5-keto-gluconate, pOBE 4-hydroxy benzoate, SAL salicylic acid, mOBE 3-hydroxy benzoate, NAG N-acetylglucosamine, SUB suberate CAP caprate, SAC sucrose, MAL maltose, MEL D-melibiose
Appl Biochem Biotechnol
interaction between or among the different strains and identify the combined effect of these interactions. The coefficients of the regression equation were calculated according to the results presented in Fig. 2 by using Design Expert and were utilized to evaluate the experimental results. After evaluating the reliability of the established model Eq. (S1), it was confirmed that Eq. (S1) fitted the experimental data well (see Supplementary material). A p value
Interactions Between Autotrophic and Heterotrophic Strains Improve CO2 Fixing Efficiency of Non-photosynthetic Microbial Communities Jiajun Hu 1,2 & Lei Wang 1 & Shiping Zhang 3 & Xuefei Xi 1 & Yiquan Le 1 & Xiaohua Fu 1 & Yiufai Tsang 4 & Mintian Gao 2
Received: 21 January 2015 / Accepted: 28 April 2015 # Springer Science+Business Media New York 2015
Abstract Five autotrophic strains isolated from non-photosynthetic microbial communities (NPMCs), which were screened from oceans with high CO2 fixing capability, were identified as Ochrobactrum sp. WH-2, Stenotrophomonas sp. WH-11, Ochrobactrum sp. WH-13, Castellaniella sp. WH-14, and Sinomicrobium oceani WH-15. The CO2 fixation pathways of all these strains were Calvin-Benson-Bassham pathway. These strains could metabolize multifarious organic compounds, which allowed switching them to autotrophic culture after enrichment in heterotrophic culture. The central composite response surface method indicated that these strains possessed many interactive effects, which increased the CO2 fixing efficiency of a combined community composed of these strains by 56 %, when compared with that of the single strain. Furthermore, another combined community composed of these autotrophic strains and NPMC had richer interactive relationships, with CO2 fixing efficiency being 894 % higher than that of the single strain and 148 % higher than the theoretical sum of the CO2 fixing efficiency of each of its microbial components. The interaction between strictly heterotrophic bacteria in NPMC and isolated autotrophic strains played a crucial role in Electronic supplementary material The online version of this article (doi:10.1007/s12010-015-1657-4) contains supplementary material, which is available to authorized users.
* Lei Wang [email protected] 1
State Key Laboratory of Pollution Control and Resource Reuse, School of Environmental Science and Engineering, Tongji University, Shanghai 200092, China
2
Shanghai Key Laboratory of Bio-Energy Crops, School of Life Sciences, Shanghai University, Shanghai 200444, China
3
Low-Carbon Conversion Science and Engineering Center, Shanghai Advanced Research Institute, Chinese Academy of Sciences, Shanghai 201210, China
4
Department of Science and Environmental Studies, The Hong Kong Institute of Education, Hong Kong 999077, China
Appl Biochem Biotechnol
improving the CO2 fixing efficiency, which not only eliminated self-restraint of organic compounds generated during the growth of autotrophic bacteria but also promoted its autotrophic pathway. Keywords Autotrophic strain . CO2 fixing . Combined community . Interaction . Heterotrophic bacteria
Introduction Microbial CO2 fixation is considered as an important method of environmental protection and resource exploitation based on the entire carbon cycle [1–3]. Photosynthetic microorganisms, especially algae, have attracted increasing attention [4, 5] not only for its CO2 fixation capacity but also for its added value, such as its application in microbial fuel cell [6]. Nevertheless, CO 2 fixation by nonphotosynthetic microorganisms is also considered to be an important process, especially in soil environment without light [7]. When compared with photosynthetic microorganisms, non-photosynthetic microorganisms exhibit a wider range of physiological and ecological traits [8]. As non-photosynthetic microorganisms do not need light during the CO2 fixing process, they have a wider range of applications and farreaching effects. It has been reported that CO2 fixation by chemoautotrophic microorganisms (such as sulfur bacteria and nitrifying bacteria) is inefficient because their generation time is from several hours to even close to 1 day [9, 10]. Alternatively, hydrogen-oxidizing bacteria are considered to be potential candidates for CO2 fixation. However, high concentration of H2 is required for the cultivation of hydrogen-oxidizing bacteria, which limits their large-scale application [11]. When compared with a single strain, NPMC has been found to have a higher CO2 fixing efficiency [12]. This could be mainly owing to the fact that NPMC possesses richer metabolic pathway than a single strain because the interactions between different strains, such as syntrophy, resulting in higher CO2 fixing efficiency. Syntrophy is the combination of the metabolic capabilities of two or more organisms to catabolize a substrate that cannot be catabolized by either one of them [13]. This enables the microorganisms to utilize resources more efficiently for supporting their growth, which might also occur in NPMC for CO2 fixation. In a previous study, it was found that there are numerous strictly heterotrophic bacteria in a NPMC with high CO2 fixing efficiency. This suggests that the metabolism of NPMC may be beyond the scope of chemoautotrophic bacteria with more interactive relationships not only between autotrophic bacteria but also between autotrophic and heterotrophic bacteria. In the present study, multiple chemoautotrophic strains were isolated from NPMC and their carbon metabolism and 16S rDNA were examined. Subsequently, the interaction of autotrophic strains and their effects were studied by constructing a mathematical model. Furthermore, the influence of strictly heterotrophic bacteria on the CO2 fixing efficiency of the combined community was also investigated. An attempt was made to explore the mechanism of interactive relationships in NPMC to improve the CO2 fixing efficiency and also find a combined community with high CO2 fixing efficiency. The experimental results may provide technical support for the
Appl Biochem Biotechnol
development of an alternative biopathway to efficiently fix and reuse CO2, thus mitigating global warming.
Materials and Methods Non-photosynthetic CO2 Fixing Microorganisms NPMC capable of fixing CO2 were isolated from the surface seawater of four oceans around the world, including Australia, China, Japan, France, Thailand, Equator, Antarctic Pole, and Arctic Pole [14].
Autotrophic Culture The autotrophic culture medium (ACM) contained (g/L): Na2CO3, 1.0; NaHCO3, 1.0; (NH4)2SO4, 5.0; KH2PO4, 1.0; K2HPO4, 2.0; MgSO4·7H2O, 0.2; NaCl, 20; CaCl2, 0.01; and FeSO4·7H2O, 0.01. To this ACM, 2 mL of trace element solution were added, which contained (mg/L): Na2MoO4·2H2O, 1.68; H3BO3, 0.4; ZnSO4·7H2O, 1.0; MnSO4·5H2O, 1.0; CuSO4·5H2O, 7.0; CoCl2·6H2O, 1.0; and NiSO4·7H2O, 1.0. Serum bottles (150 mL) containing 40 mL of the medium were prepared and autoclaved for 20 min at 121 °C. For normal experiments, the bacterial inoculum concentration was 2.5 % (v/v). The serum bottle was sealed with a silicon stopper and filled with 20 % (v/v) CO2 by using a syringe (air:CO2 ratio of approximately 80:20). All the cultures were incubated at 30 °C and 120 rpm without light for 96 h. The atmosphere within the serum bottles was readjusted to the initial ratios after culturing for 48 h.
Screening and Isolation of Single Autotrophic Strain For screening and isolation of single autotrophic strains, media with different electron donors were prepared by adding 0.1 % (w/v) NaNO2, Na2S2O3, Na2S, FeSO4, or none to the ACM. Subsequently, 2 % (w/v) agar was added to the media and the culture plates were autoclaved for 20 min at 121 °C. The following steps were conducted under sterile conditions to avoid contamination. A total of 100 μL of NPMC was inoculated onto the culture plates and incubated at 30 °C. After culturing for about 10–15 days, a single colony was picked and streaked onto new culture plates and incubated at 30 °C for 10–15 days, and the same process was repeated until isolation of a single strain. The selected single strain was inoculated into sterile EP tube (4 mL) with 2 mL of the medium and cultured for about 15 days. The isolated strains were cultured according to the autotrophic culture method described earlier and their CO2 fixing efficiency was examined. Strains with weak CO2 fixing capacity were eliminated. Strains have been deposited in China Center for Industrial Culture Collection (CICC).
Heterotrophic Enrichment Culture A total of 2.5 % (v/v) microbial culture (single strain or NPMC) was inoculated into LuriaBertani medium and incubated at 30 °C and 120 rpm for 48–96 h. Two blank samples were prepared for each heterotrophic enrichment process to examine whether the bacteria were contaminated.
Appl Biochem Biotechnol
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., Japan).
16S rDNA Sequence Determination and Phylogenetic Analysis The bacterial DNA was extracted by using a PowerSoil DNA Isolation Kit (MO BIO Laboratories, Inc., Canada) according to the manufacturer’s instructions. Primers 8f and 1492r [14] were used to amplify the bacterial 16S rDNA. The PCR products were purified by using a B type Mini-DNA Rapid Purification Kit (BioDev, China), after which they were cloned by using the PMD18-T plasmid vector system (TaKaRa, Japan). The DNA sequences were then determined by a commercial service (BGI, China) with bidirectional sequencing. The vector sequence was cut off and the remaining nucleotides were compared with those available in GenBank by using the BLAST program to identify the most similar 16S rDNA fragments. The phylogenetic analysis was conducted by using MEGA 6 [15].
Determination of the CO2 Fixing Pathway of the Isolated Autotrophic Strain and Its Capacity to Metabolize Different Organic Matters The carbon fixation pathway was examined through quantitative PCR to detect the presence of key enzyme genes in CO2 fixation pathway in the microbial DNA extracts, including form I and form II ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) genes, cbbL [16] and cbbM [17], for Calvin-Benson-Bassham (CBB) pathway; ATP-dependent citrate lyase gene, aclB [18], for reductive tricarboxylic acid cycle; and methyl coenzyme M reductase α-subunit gene, mcrA [19], for reductive acetyl-CoA pathway. The organic carbon source utilization was examined by using the API ID 32GN Test Kit (bioMérieux, France), and the results were obtained by using ATB Expression (bioMérieux, France).
Response Surface Method Design Preliminary experiments were conducted to screen the appropriate strains. The statistical software package, Design Expert 8.0.6 (Stat-Ease, Inc., USA), was used to design the experiments and analyze the results. The RSM design involving a central composite design (CCD) was adopted to match the ratio of five strains to improve the CO2 fixing efficiency of the combined community. The minimum and maximum range of the variables investigated and the complete experimental plan with respect to the actual and coded forms were as follows: each strain inoculum concentration levels of −1 and +1 were 0 and 2 % (v/v), respectively. For statistical calculations, the actual values of the variable Xi were coded as xi according to the following relationship: . xi ¼ ðX i – X 0 Þ ΔX 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 ΔX indicates the step change.
Appl Biochem Biotechnol
The experimental data were analyzed by RSM using the following second-order polynomial equation: y ¼ β0 þ
n X i¼1
βi X i þ
n X i¼1
β ii X i 2 þ
n XX
βi X i X j
i< j¼1
where y is the response (TOC, mg/L), Xi and Xj are the coded independent variables, and β0, βi, βii, and βij are the intercept, linear, quadratic, and interaction constant coefficients, respectively. Design Expert was used for regression analysis and analysis of variance (ANOVA). A total of 0.46 % NaNO2, 0.50 % Na2S2O3, and 1.25 % Na2S were added to the medium as electron donors to provide adequate energy for the microorganisms in the RSM experiments.
Statistical Analysis Cluster analysis is a mathematical method that can be used to determine objects that are similar in a group [20]. The purpose of cluster analysis is to identify subsets of a data set, which contain similar points. Replacement of these subsets by their aggregate properties, such as means and standard deviations, for example, creates a compact representation of the data set as a set of clusters. The cluster properties can then be used for comparative data analysis [21]. This approach has been considered suitable to examine the differences in carbon metabolism of the strains. In the present study, cluster analysis was carried out by using SPSS 21 (IBM, USA).
Results and Discussion Identification and Carbon Metabolism Characterization of the Dominant Autotrophic Strains Isolated from NPMC Autotrophs grow by using CO2 as their sole carbon source and generate biomass on which all other organisms thrive [22]. The present study was focused on chemoautotrophic microorganisms, which might play a major role in CO2 fixation by NPMC. First, NPMC with high CO2 fixing efficiency without H2 as the electron donor was screened from the marine environment. Then, about 300 autotrophic bacterial colonies were isolated from the NPMC. Based on the inorganic carbon assimilation efficiency, bacterial morphology, and culture conditions, a total of 30 strains were selected. After molecular biology identification, five strains with high CO2 fixing efficiency were selected for further examination. The five strains were Ochrobactrum sp. WH-2 (CICC 23802, GenBank accession number: KM502331), Stenotrophomonas sp. WH-11 (CICC 23803, GenBank accession number: KM975671), Ochrobactrum sp. WH-13 (CICC 23804, GenBank accession number: KM975672), Castellaniella sp. WH-14 (CICC 23805, GenBank accession number: KM975673), and Sinomicrobium oceani WH-15 (CICC 23806, GenBank accession number: KM975674). Except S. oceani WH-15 belonged to Bacteroidetes, other strains all belonged to Proteobacteria, the largest group of bacteria with rich metabolic diversity. Besides the aerobic pathway, anaerobic pathway was also detected because the interactions between bacteria could overcome oxidative stress under aerobic condition [2]. Quantitative
Appl Biochem Biotechnol
PCR results showed that the carbon fixation pathways of these five strains were mainly by CBB pathway without other carbon fixation pathways (such as reductive tricarboxylic acid cycle and reductive acetyl-CoA pathway). Two types of RubisCO, namely, form I and form II, could be observed in these strains. It can be seen from Fig. 1a that four strains exhibited form I RubisCO, whereas Castellaniella sp. WH-14 presented form II RubisCO. Despite the difference in their primary structure, it has been indicated that form II RubisCO could catalyze the same reaction as form I RubisCO. In addition, some other differences between form I and form II RubisCO have been reported. Most importantly, form II RubisCO has a distinct physiological role, because it is used primarily to enable the CBB pathway to balance the redox potential of the cell under select growth conditions [23]. Figure 1b, c shows that the five strains could metabolize a variety of organics, including short- and long-chain organics (with respect to carbon number), sugars, organic acids, vitamins, and amino acids (with respect to carbon structure). The ability of Castellaniella sp. WH-14 to metabolize organics with six or more carbon atoms was found to be far lower than that of the other strains. The results obtained showed that all the five strains were facultative autotrophic microorganisms and possessed rich heterotrophic metabolic pathways. The CO2 fixing efficiency of these isolated bacteria was found to be low. Under conventional culture conditions, the general CO2 fixing efficiency of these strains was 1–2 mg C/L and up to about 5 mg C/L after the culture period. This efficiency was much lower than the maximum efficiency of the NPMC [12]. These findings implied that there might be multiple interactive relationships between the microorganisms in NPMC. The results of cluster analysis showed a difference in inorganic and organic carbon metabolism of the strains on squared Euclidean distance metric (see Fig. 1d). Furthermore, the phylogenetic tree showed the different evolutionary distances between the isolated strains (see Fig. 1e). As indicated by Morris et al. [24], rich interactions could improve autotrophic metabolism and combined community with high CO2 fixing efficiency might be obtained by mixing multiple strains together. Rich interactions signify rich and efficient metabolic pathways, which may be obtained by the strains with different evolutionary distance (different species) or different ability of carbon metabolism (metabolizable organic carbon types and CO2 fixation pathways). Hence, the five strains isolated in the present study were selected to construct a combined community with interactive relationships. If these strains could constitute a collaborative carbon fixation system, then their feature as facultative autotrophic strains could provide another advantage; i.e., they could be cultured heterotrophically to achieve high cell density and can be transferred to an inorganic environment for autotrophic culture. This characteristic was conducive for the enrichment and development of a combined community quickly and easily.
Interactive Effects of the Isolated Autotrophic Strains on CO2 Fixation Interactive effects between microorganisms, such as microbial syntrophy, could enable the chemical reaction with positive free energy change to support microbial growth [24]. Syntrophy between different strains, such as ethanol fermentation bacteria and methanogens [25], signifies several reactions occurring simultaneously in different bacteria, resulting in the overall reaction to be exergonic and more effective. In the present study, to examine the presence of interaction among the five strains isolated and their effect on CO2 fixation, central composite response surface method (RSM) experimentation was used to evaluate the
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Fig. 1 a Carbon fixing pathway of the strains. b, c Utilization of organic carbon sources by the strains. , , negative. d Dendrogram obtained by using average linkage between groups (hierarchical positive; cluster analysis algorithms, squared Euclidean distance metric) based on inorganic and organic carbon metabolism by the strains. e Branch graph of phylogenetic tree for 16S rDNA sequence of the samples. cbbL form I RubisCO gene, cbbM form II RubisCO, aclB ATP-dependent citrate lyase gene, mcrA methyl coenzyme M reductase α-subunit gene, ACE acetate, PROP propionate, LAT DL-lactate, SER L-serine, ALA L-alanine, MNT malonate, 3OBU 3-hydroxy butyrate PRO L-proline, ARA L-arabinose, RIB D-ribose, VALT valerate, ITA itaconic acid, CIT citrate, GLU D-glucose, RHA rhamnose, FUC L-fucose, INO inositol, HIS histidine, 2KG 2-ketogluconate, GLYG glycogen, MAN mannitol, SOR D-sorbitol, 5KG 5-keto-gluconate, pOBE 4-hydroxy benzoate, SAL salicylic acid, mOBE 3-hydroxy benzoate, NAG N-acetylglucosamine, SUB suberate CAP caprate, SAC sucrose, MAL maltose, MEL D-melibiose
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interaction between or among the different strains and identify the combined effect of these interactions. The coefficients of the regression equation were calculated according to the results presented in Fig. 2 by using Design Expert and were utilized to evaluate the experimental results. After evaluating the reliability of the established model Eq. (S1), it was confirmed that Eq. (S1) fitted the experimental data well (see Supplementary material). A p value
