Role of Caox on soil pH and composition of oxalotrophic communities
Concentrations of Caox observed in different soils in which OCP has been characterized range from 0.015 to up to 0.175 mg g−1 of soil (Martin et al., 2012). More recently, we have observed that values in litter can be up to 1.3 mg g−1 of soil (unpublished results). The maximum concentration of Caox used in the present and previous microcosm studies (Martin et al., 2012) is largely above these values (4 mg g−1 of soil). This Caox concentration is the same used for the isolation and culturing of oxalotrophic bacteria (Tamer & Aragno, 1980; Braissant et al., 2002) and therefore is expected not to be toxic for bacterial growth. The present study shows that the addition of even concentrations as low as 0.5% of Caox stimulates oxalotrophic activity, leading to a shift in the local soil pH (7.2). The increase in pH from 6.4 to 7.2 or 7.7 (for 1% and 4 % of Caox, respectively) demonstrates experimentally for the first time that the input of Caox is one of the limiting factors for bacterial oxalotrophic activity in soil.
Surprisingly, the comparison of different concentrations of Caox showed that a change in the concentration of the amended carbon source is not a driving force that modifies drastically the composition of the oxalotrophic community. Previous studies have demonstrated the selection of similar microbial communities by structurally similar carbon sources, which are metabolized by related biochemical pathways (Wawrik et al., 2005). Nonetheless, the same study has shown that if a soil community is enriched on more than one carbon source, changes in the composition of the enriched community are observed. Indeed, several carbon sources have been shown to modify drastically the composition of active metabolically soil bacteria (Monard et al., 2008). Although other natural carbon sources can be expected in the microcosm experiment, in the case of oxalotrophic bacteria, only Caox amendment appears to be significant.
Identification of active oxalotrophic bacteria
Although the incorporation of BrdU is reported not to be equally effective in all bacteria (Borneman, 1999) and, thus, some groups can be underestimated, in this study, we highlight the use of the BrdU assay to identify a diverse assemblage of active oxalotrophs in microcosm experiments. The 20 mobility species identified were affiliated to bacteria related to Actinobacteria and the divisions Alphaproteobacteria and Betaproteobacteria of the class Proteobacteria. Certain genera such as Methylobacterium, Xanthobacter, Bradyrhizobium, Burkholderia, Azospirillum, and Janthinobacterium have been previously identified as oxalotrophic using culture methods (Sahin et al., 2008). Some specific species such as Methylobacterium extorquens are model bacteria with high metabolic rate when grown on potassium oxalate (consumption rate of 0.32 μM h−1; (Bravo et al., 2011). Nonetheless, this is the first time that their active metabolic contribution to oxalate catabolism in soil has been demonstrated. Moreover, this is the first time that groups such as Kribbella and Starkeya are shown as active oxalotrophic bacteria. These bacteria are probably unable to grow in vitro, because they have never been reported in studies dealing with the diversity of cultured oxalotrophic bacteria (Sahin, 2003).
Nonetheless, the limitations of the BrdU method need to be considered in the analysis of the results. For example, groups such as K, B, and N that appear at all the concentrations in unlabeled DNA only appear in one concentration in the labeled DNA. Likewise, the absence of mobility species A, K, B, N, D, and J in BrdU-labeled DNA, and the presence of the same mobility species in unlabeled DNA, is a clear evidence of a bias in the BrdU-frc detection at 0% of Caox. The opposite was observed with the presence of mobility species C, T, G, and S in labeled DNA, and their absence, in the unlabeled DNA in the absence of Caox. Further experiments using a quantitative approach for the detection of specific frc mobility species could help to improve the resolution of these results, as well as to elucidate the detection limit of oxalotrophic mobility species. Thus, the idea that certain populations remain at low intensity or are poorly labeled when no stimulation of Caox is carried out in the system (0%) should be taken into account.
Another issue that needs to be considered is the repeatability of the results. We conducted a rigorous sampling program and considered biological replicates in our analysis. Those replicates were consistent in terms of pH evolution, as well as with our previous results for African soils (Martin et al., 2012). Nevertheless, the microcosms approach is still a method to try to model activity in situ, but does not necessarily reproduce it entirely (Bowling et al., 1980; Fraser & Keddy, 1997; Fraser, 1999). Therefore, it would be important to validate the results obtained, first in other microcosms with other OCP soils and, more importantly, in the field. This is still technically challenging, but should be targeted as a priority for future experiments.
The role and metabolic capability of Actinobacteria such as Streptomyces and Kribbella as oxalotrophs are worth discussing in more detail. It has been demonstrated that a Streptomyces sp. (strain BV1M3), isolated from a tropical soil, has also a large activity when grown on Kox as sole carbon source (consumption rate of 0.26 μM h−1; (Bravo et al., 2011). Many studies on the role of Actinobacteria as oxalotrophic bacteria have been primarily concerned with the enumeration and taxonomy of Streptomyces (Lechevalier & Lechevalier, 1970; Sahin, 2003, 2004). This group is known to be saprophyte (Goodfellow & Williams, 1983). The filamentous morphology and spore dispersion by rain (Gobat et al., 2004) or attached to arthropods (Ensign, 1978) make Streptomyces (and Actinobacteria in general) ideal microorganisms to exploit habitat heterogeneity influenced by the availability of any given substrate (Kassen, 2002), and this could be particularly true for Caox due to its low solubility (Cromack et al., 1977), probably explaining the importance of Streptomyces as active oxalotrophs in the soil microcosms.
Although DGGE (Muyzer & Smalla, 1998) is a technique at the basis of the development of microbial ecology, it is increasingly being displaced by high-throughput sequencing approaches. However, for exploratory experiments in which the amount of sampling and the correct timing to observe a meaningful effect (i.e. maximum Caox oxidation) are unknown variables, DGGE is a pertinent compromise of analytical investment. It is clear that the amount of time devoted to obtain a limited number of sequences after band excision is not comparable to the massive amount of data that can be obtained by novel sequencing approaches. Nonetheless, the data generated here constitute a suitable basis for future more targeted experiments, in which obtaining a comprehensive view of the oxalotrophic community would be justifiable. Finally, attention needs to be paid to the fact that the fragment used in this study is very short and that, ideally for a phylogenetic reconstruction of the community, a more complete amplicon of the frc gene would be a better approach. We expect that this will be possible by improvement in our knowledge of bacterial oxalotrophy in a near future, contributing to take full advantage of the potential of new screening and characterizing techniques.