Siqin Han

Abundance and diversity of Sphingomonas in Shenfu petroleum-wastewater irrigation zone, China

IntroductionMembers of the genus Sphingomonas have raised increasing attention due to their ability for polycyclic aromatic hydrocarbon (PAH) degradation and their ubiquity in the environment. However, few studies have revealed the ecological information on the abundance and diversity of Sphingomonas in the environment.Materials and methodsA primer set targeting the Sphingomonas 16S rRNA gene was designed. The specificity was tested with four petroleum-contaminated soils by construction of clone libraries and further restriction fragment length polymorphism analysis. Subsequently, real time PCR and denaturing gradient gel electrophoresis (DGGE) assays were used to evaluate the abundance and diversity of Sphingomonas in the Shenfu irrigation zone, China.ResultsA genus-specific primer set SA/429f-933r was developed, and 90% of the sequences retrieved from soil clone libraries were related to Sphingomonas. Members of the genus Sphingomonas were detected in all soils, and significant correlation (p < 0.05) was observed between the Sphingomonas abundance and the ratios of PAHs to total petroleum hydrocarbon (TPH). DGGE profiles revealed Sphingomonas population structures differed greatly in different sites. The Sphingomonas diversity was not statistically (p > 0.05) correlated with the contamination level. Some of the soil-derived sequences were not grouped phylogenetically with sequences of known Sphingomonas, indicating new members of the Sphingomonas genus might be present in the Shenfu irrigation zone.ConclusionThe newly designed Sphingomonas-selective primers were specific and practicable for analyzing Sphingomonas abundance and diversity in petroleum-contaminated soils. The significant correlation between the abundance and the ratios of PAHs to TPH suggested an important role of Sphingomonas in PAH bioremediation.

Sphingomonas from petroleum-contaminated soils in Shenfu, China and their PAHs degradation abilities

Members of the Sphingomonas genus are often isolated from petroleum-contaminated soils due to their unique abilities to degrade polycyclic aromatic hydrocarbons (PAHs), which are important for in situ bioremediation. In this study, a combined phenotypic and genotypic approach using streptomycin-containing medium and Sphingomonas-specific PCR was developed to isolate and identify culturable Sphingomonas strains present in petroleum-contaminated soils in the Shenfu wastewater irrigation zone. Of the 15 soil samples examined, 12 soils yielded yellow streptomycin-resistant colonies. The largest number of yellow colony-forming units (CFUs) could reach 105 CFUs g−1 soil. The number of yellow CFUs had a significant positive correlation (p < 0.05) with the ratio of PAHs to total petroleum hydrocarbons (TPH), indicating that Sphingomonas may play a key role in degrading the PAH fraction of the petroleum contaminants at this site. Sixty yellow colonies were selected randomly and analyzed by colony PCR using Sphingomonas-specific primers, out of which 48 isolates had PCR-positive signals. The 48 positive amplicons generated 8 distinct restriction fragment length polymorphism (RFLP) patterns, and 7 out of 8 phylotypes were identified as Sphingomonas by 16S rRNA gene sequencing of the representative strains. Within these 7 Sphingomonas strains, 6 strains were capable of using fluorene as the sole carbon source, while 2 strains were phenanthrene-degrading Sphingomonas. To the best of our knowledge, this is the first report to evaluate the relationship between PAHs contamination levels and culturable Sphingomonas in environmental samples.

Development of genus-specific primers for better understanding the diversity and population structure of Sphingomonas in soils

Genus Sphingomonas has received increasing attentions due to its somewhat unique metabolic versatilities in the contaminated environment. However, due to the lack of genus‐specific primers, the ecological significance of Sphingomonas in polluted soils has been rarely documented by 16S rDNA finger‐printing methods. In this study, three genus‐specific primer sets targeted at the 16S rRNA gene of Sphingomonas were developed and their specificities were tested with four contaminated soils from Shenfu petroleum‐wastewater irrigation zone by constructing clone libraries, amplified ribosomal DNA restriction analysis (ARDRA) and sequencing the represented ARDRA patterns. Meanwhile, the newly designed primer sets and a previously reported primer set were compared, and the results showed that the newly developed primer set SA/429f‐933r could detect a larger spectrum (90%) of Sphingomonas strains with higher specificity. Despite the superiority of primer set SA/429f‐933r in specifically detecting Sphingomonas from contaminated soils, we cannot blink the fact that different primer sets preferentially amplified different dominant species. Therefore, two or more primer sets are recommended for evaluating the diversity and population structure of genus Sphingomonas. Additionally, a proportion (9.7%) of the cloned sequences discovered in this study were different from known Sphingomonas sequences, suggesting that new Sphingomonas sequences might present in soils from Shenfu irrigation zone.