Treatment of olive mill wastewater through employing sequencing batch reactor: performance and microbial diversity assessment

Abstract

This work describes the performance of a sequencing batch reactor (SBR) and the involvement of a novel reconstituted bacterial consortium in olive mill wastewater (OMW) treatment. The organic loading rate applied to the SBR was serially increased in terms of initial COD from 10 to 75 g L^-1 to allow gradual acclimatization of activated sludge to high concentrations of toxic compounds in OMW. After the acclimatization period, up to 60% of the total COD content were effectively biodegraded from OMW at 75 g L^-1 COD within 30 day hydraulic retention time. The diversity and community composition of cultivable bacteria participating in the aerobic process of treating OMW were further assessed. A total of 91 bacterial strains were isolated from the reactor and analyzed by amplification of the 16S-23S rRNA internal transcribed spacer (ITS) region and by 16S rRNA gene sequencing. The most abundant phylum was Firmicutes (57.1%) followed by Proteobacteria (35.2%) and Actinobacteria (7.7%). The use of the Biolog® Phenotype Microarray system to evaluate the ability of isolated strains to utilize OMW phenolic compounds is reported in this work for the first time. Interestingly, results showed that all species tested were able to utilize phenolics as sole carbon and energy sources. The removals of COD and phenolics from undiluted OMW by the reconstituted bacterial consortium were almost similar to those obtained by the acclimatized activated sludge, which suggest that cultivable bacteria play the major role in OMW biodegradation. Phytotoxicity assays using tomato seeds showed a significant improvement of seed germination values for treated OMW. Our overall results suggest that the novel developed bacterial consortium could be considered as a good prospect for phenolics-rich wastewaters bioremediation applications.

Keywords: Acclimatized aerobic consortium; Biolog ® phenotyping; Olive mill wastewater; Phenolic compounds; Phytotoxicity; Sequencing batch reactor.

Fig. 1

COD removal during repetitive batch experiments carried out at 75 g L⁻¹ COD. Each batch run was for 30 days

Fig. 2

Phylogenetic tree based on the 16S rRNA gene of representative isolates of each haplotype group (A–V) and reference sequences from GenBank (http://www.ncbi.nlm.nih. gov/genbank). Phylogenetic relationships among taxa were evaluated by performing bootstrap analysis of 1000 data sets using MEGA version 6.0. Numbers in parentheses represent the sequences accession numbers. Bar, 0.02 changes per nucleotide position

Fig. 3

Comparison of Area Under the Respiration Curve (AUC) data from the Biolog bacterial phenotypic microarray. Group I: (OM56) Bacillus nealsonii strain OM56; (OM24) Ochrobactrum tritici strain OM24; (OM26) Ochrobactrum tritici strain OM26; (OM14) Ochrobactrum haematophilum strain OM14; (OM9) Bacillus thioparans strain OM9; (OM18) Roseomonas mucosa strain OM18; (OM8) Paenibacillus xylanilyticus strain OM8; Group II: (OM61) Kocuria rosea strain OM61; (OM50) Brevibacillus laterosporus strain OM50; Group III: (OM48) Bacillus amyloliquefaciens strain OM48; (OM84) Klebsiella oxytoca strain OM84; (OM88) Pseudomonas aeruginosa strain OM88; (OM79) Cellulosimicrobium cellulans strain OM79; (OM73) Lysinibacillus macroids strain OM73; (OM43) Bacillus cereus strain OM43; (OM33) Rhodococcus zopfii strain OM33; (OM60) Bacillus thuringencis strain OM60; (OM22) Rhodococcus pyridinivorans strain OM22

Fig. 4

Average of phenolics and COD removal efficiencies of OMW at 75 g L⁻¹ COD by the reconstituted bacterial consortium (RC) and acclimatized activated sludge (AS). Data are presented as mean values from duplicate experiments. a, b Means without a common letter differ (p < 0.05)

Fig. 5

Effect of OMW dilution and treatment with the reconstituted bacterial consortium on germination index of S. lycopersicum seeds. Untreated OMW (UOMW), treated OMW (TOMW), number under the slash indicates the dilution ratio. Data are presented as mean values from duplicate experiments. a–e Means without a common letter differ (p < 0.05)