Microbial Diversity in Sulfate-Reducing Marine Sediment Enrichment Cultures Associated with Anaerobic Biotransformation of Coastal Stockpiled Phosphogypsum (Sfax, Tunisia)

Abstract

Anaerobic biotechnology using sulfate-reducing bacteria (SRB) is a promising alternative for reducing long-term stockpiling of phosphogypsum (PG), an acidic (pH ~3) by-product of the phosphate fertilizer industries containing high amounts of sulfate. The main objective of this study was to evaluate, for the first time, the diversity and ability of anaerobic marine microorganisms to convert sulfate from PG into sulfide, in order to look for marine SRB of biotechnological interest. A series of sulfate-reducing enrichment cultures were performed using different electron donors (i.e., acetate, formate, or lactate) and sulfate sources (i.e., sodium sulfate or PG) as electron acceptors. Significant sulfide production was observed from enrichment cultures inoculated with marine sediments, collected near the effluent discharge point of a Tunisian fertilizer industry (Sfax, Tunisia). Sulfate sources impacted sulfide production rates from marine sediments as well as the diversity of SRB species belonging to Deltaproteobacteria. When PG was used as sulfate source, Desulfovibrio species dominated microbial communities of marine sediments, while Desulfobacter species were mainly detected using sodium sulfate. Sulfide production was also affected depending on the electron donor used, with the highest production obtained using formate. In contrast, low sulfide production (acetate-containing cultures) was associated with an increase in the population of Firmicutes. These results suggested that marine Desulfovibrio species, to be further isolated, are potential candidates for bioremediation of PG by immobilizing metals and metalloids thanks to sulfide production by these SRB.

Keywords: Desulfovibrio; anaerobes; anaerobic biotechnology; bioremediation; marine sediment; next-generation sequencing; phosphogypsum; sulfate-reducing bacteria.

Figure 1

Compositions of microbial communities in the coastal marine sediment and phosphogypsum samples of Sfax (Tunisia). Relative phylogenetic abundance was based on frequencies of 16S rRNA gene sequences affiliated with Archaea and major bacterial phyla or proteobacterial classes in the microbial communities of marine sediment (MS) and phosphogypsum (PG).

Figure 2

Profiles of hydrogen sulfide production during 14 days of enrichment cultures from coastal marine sediment (as inoculum) using different electron donors (AC, acetate; FOR, formate; LAC, lactate; C, control without electron donor) and sulfate sources: sodium sulfate (A) or phosphogypsum (B). Values are means of two biological replicates ± confidence intervals (error bars).

Figure 3

Compositions of microbial communities in sulfate-reducing enrichment cultures from marine sediment using different electron donors and sulfate sources after 14 days. Relative phylogenetic abundance was based on frequencies of 16S rRNA gene sequences affiliated with Archaea and major bacterial phyla or proteobacterial classes in the microbial communities. The names of enrichment cultures (duplicates 1 and 2) have been abbreviated as follows: SF, SL, SA, and SC for Na₂SO₄ enrichment cultures with formate, lactate, acetate and without electron donor, respectively; SPF, SPL, SPA, and SPC for PG enrichment cultures with formate, lactate, acetate, and without electron donor, respectively.

Figure 4

Principal Component Analysis (PCA) biplot showing the variation among the enrichment cultures based on hydrogen sulfide production performances and the relative abundance of microbial taxa. Black circles represent PG enrichment cultures and black triangles represent Na₂SO₄ enrichment cultures. The names of enrichment cultures (duplicates 1 and 2) have been abbreviated as follows: SF, SL, and SA, for Na₂SO₄ enrichment cultures with formate, lactate, and acetate as electron donors, respectively; SPF, SPL, and SPA, for PG enrichment cultures with formate, lactate, and acetate as electron donors, respectively. Arrows indicate the direction of maximum increase and strength (through the length) of each variable to the overall distribution. The blue arrows are indicators of hydrogen sulfide production (pH, P_max, V_max) and the red arrows represent the microbial taxa. Among these latter, α-Prot, β-Prot, δ-Prot, ε-Prot stand for Alphaproteobacteria, Betaproteobacteria, Deltaproteobacteria, and Epsilonproteobacteria. The first two principal axes explained 61.3% of the variance.

Figure 5

Maximum-likehood (ML) tree based on 16S rRNA gene sequences showing the phylogenetic position of Deltaproteobacteria enriched from marine sediment (as inoculum) using different electron donors and sulfate sources (sodium sulfate or phosphogypsum). Representative sequences in the tree were obtained from GenBank (accession number in the brackets). Bootstrap values >75% are indicated at nodes. The bars represent the relative abundance of each OTU affiliated with Deltaproteobacteria in the enrichment cultures. The blue bars indicate the relative abundance of OTUs in sodium sulfate cultures, whereas the red bars represent the relative abundance of OTUs in PG cultures.

Figure 6

Maximum-likehood (ML) tree based on 16S rRNA gene sequences showing the phylogenetic position of Firmicutes from marine sediment (as inoculum) using different electron donors and sulfate sources (sodium sulfate or phosphogypsum). Representative sequences in the tree were obtained from GenBank (accession number in the brackets). Bootstrap values >75% are indicated at nodes. The bars represent the relative abundance (in %) of each OTU affiliated with Firmicutes in the enrichment cultures. The blue bars indicate the relative abundance of OTUs in sodium sulfate cultures, whereas the red bars represent the relative abundance of OTUs in PG cultures.