Najla Mhiri

Application of a continuously stirred tank bioreactor (CSTR) for bioremediation of hydrocarbon-rich industrial wastewater effluents

A continuously stirred tank bioreactor (CSTR) was used to optimize feasible and reliable bioprocess system in order to treat hydrocarbon-rich industrial wastewaters. A successful bioremediation was developed by an efficient acclimatized microbial consortium. After an experimental period of 225 days, the process was shown to be highly efficient in decontaminating the wastewater. The performance of the bioaugmented reactor was demonstrated by the reduction of COD rates up to 95%. The residual total petroleum hydrocarbon (TPH) decreased from 320 mg TPH l(-1) to 8 mg TPH l(-1). Analysis using gas chromatography-mass spectrometry (GC-MS) identified 26 hydrocarbons. The use of the mixed cultures demonstrated high degradation performance for hydrocarbons range n-alkanes (C10-C35). Six microbial isolates from the CSTR were characterized and species identification was confirmed by sequencing the 16S rRNA genes. The partial 16S rRNA gene sequences demonstrated that 5 strains were closely related to Aeromonas punctata (Aeromonas caviae), Bacillus cereus, Ochrobactrum intermedium, Stenotrophomonas maltophilia and Rhodococcus sp. The 6th isolate was affiliated to genera Achromobacter. Besides, the treated wastewater could be considered as non toxic according to the phytotoxicity test since the germination index of Lepidium sativum ranged between 57 and 95%. The treatment provided satisfactory results and presents a feasible technology for the treatment of hydrocarbon-rich wastewater from petrochemical industries and petroleum refineries.

Characterization of a novel biosurfactant produced by Staphylococcus sp. strain 1E with potential application on hydrocarbon bioremediation.

A biosurfactant‐producing bacterium (Staphylococcus sp. strain 1E) was isolated from an Algerian crude oil contaminated soil. Biosurfactant production was tested with different carbon sources using the surface tension measurement and the oil displacement test. Olive oil produced the highest reduction in surface tension (25.9 dynes cm–1). Crude oil presented the best substrate for 1E biosurfactant emulsification activity. The biosurfactant produced by strain 1E reduced the growth medium surface tension below 30 dynes cm–1. This reduction was also obtained in cell‐free filtrates. Biosurfactant produced by strain 1E showed stability in a wide range of pH (from 2 to 12), temperature (from 4 to 55 °C) and salinity (from 0 to 300 g l–1) variations. The biosurfactant produced by strain 1E belonged to lipopeptide group and also constituted an antibacterial activity againt the pathogenic bacteria such as Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and Bacillus subtilis. Phenanthrene solubility in water was enhanced by biosurfactant addition. Our results suggest that the 1E biosurfactant has interesting properties for its application in bioremediation of hydrocarbons contaminated sites. (© 2012 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

Isolation and Characterization of Hydrocarbon-Degrading Yeast Strains from Petroleum Contaminated Industrial Wastewater.

Two yeast strains are enriched and isolated from industrial refinery wastewater. These strains were observed for their ability to utilize several classes of petroleum hydrocarbons substrates, such as n-alkanes and aromatic hydrocarbons as a sole carbon source. Phylogenetic analysis based on the D1/D2 variable domain and the ITS-region sequences indicated that strains HC1 and HC4 were members of the genera Candida and Trichosporon, respectively. The mechanism of hydrocarbon uptaking by yeast, Candida, and Trichosporon has been studied by means of the kinetic analysis of hydrocarbons-degrading yeasts growth and substrate assimilation. Biodegradation capacity and biomass quantity were daily measured during twelve days by gravimetric analysis and gas chromatography coupled with mass spectrometry techniques. Removal of n-alkanes indicated a strong ability of hydrocarbon biodegradation by the isolated yeast strains. These two strains grew on long-chain n-alkane, diesel oil, and crude oil but failed to grow on short-chain n-alkane and aromatic hydrocarbons. Growth measurement attributes of the isolates, using n-hexadecane, diesel oil, and crude oil as substrates, showed that strain HC1 had better degradation for hydrocarbon substrates than strain HC4. In conclusion, these yeast strains can be useful for the bioremediation process and decreasing petroleum pollution in wastewater contaminated with petroleum hydrocarbons.

Anaerobic co-digestion of Tunisian green macroalgae Ulva rigida with sugar industry wastewater for biogas and methane production enhancement

Ulva rigida is a green macroalgae, abundantly available in the Mediterranean which offers a promising source for the production of valuable biomaterials, including methane. In this study, anaerobic digestion assays in a batch mode was performed to investigate the effects of various inocula as a mixture of fresh algae, bacteria, fungi and sediment collected from the coast of Sfax, on biogas production from Ulva rigida. The results revealed that the best inoculum to produce biogas and feed an anaerobic reactor is obtained through mixing decomposed macroalgae with anaerobic sludge and water, yielding into 408mL of biogas. The process was then investigated in a sequencing batch reactor (SBR) which led to an overall biogas production of 375mL with 40% of methane. Further co-digestion studies were performed in an anaerobic up-flow bioreactor using sugar wastewater as a co-substrate. A high biogas production yield of 114mL g-1 VSadded was obtained with 75% of methane. The co-digestion proposed in this work allowed the recovery of natural methane, providing a promising alternative to conventional anaerobic microbial fermentation using Tunisian green macroalgae. Finally, in order to identify the microbial diversity present in the reactor during anaerobic digestion of Ulva rigida, the prokaryotic diversity was investigated in this bioreactor by the denaturing gradient gel electrophoresis (DGGE) method targeting the 16S rRNA gene.

Biological treatment of fish processing wastewater: A case study from Sfax City (Southeastern Tunisia).

The present work presents a study of the biological treatment of fish processing wastewater at salt concentration of 55 g/L. Wastewater was treated by both continuous stirred-tank reactor (CSTR) and membrane bioreactor (MBR) during 50 and 100 days, respectively. These biological processes involved salt-tolerant bacteria from natural hypersaline environments at different organic loading rates (OLRs). The phylogenetic analysis of the corresponding excised DGGE bands has demonstrated that the taxonomic affiliation of the most dominant species includes Halomonadaceae and Flavobacteriaceae families of the Proteobacteria (Gamma-proteobacteria class) and the Bacteroidetes phyla, respectively. The results of MBR were better than those of CSTR in the removal of total organic carbon with efficiencies from 97.9% to 98.6%. Nevertheless, salinity with increasing OLR aggravates fouling that requires more cleaning for a membrane in MBR while leads to deterioration of sludge settleability and effluent quality in CSTR.

Characterization of Sporohalobacter salinus sp. nov., an anaerobic, halophilic, fermentative bacterium isolated from a hypersaline lake.

Halophilic, obligately anaerobic, Gram-stain-negative bacterial strains were isolated from a sediment sample taken from under the salt crust of El-Jerid hypersaline lake in southern Tunisia by using tryptone or glucose as the substrate. One strain, CEJFT1B(T), was characterized phenotypically and phylogenetically. Cells were non-motile, non-spore-forming, short rods. Strain CEJFT1B(T) was able to grow in the presence of 5-30 % (w/v) NaCl (optimum 20 %) and at 30-60 °C (optimum 45 °C). It grew at pH 5.5-7.8 and the optimum pH for growth was 6.8. The isolate required yeast extract for growth. Substrates utilized by strain CEJFT1B(T) as the sole carbon source included glucose, fructose, sucrose, pyruvate, Casamino acids and starch. Individual amino acids such as glutamate, lysine, methionine, serine, tyrosine, and amino acid mixtures formed by the Stickland reaction such as alanine-glycine, valine-proline, leucine-proline, isoleucine-proline were also utilized. Products of glucose fermentation were acetate (major product), butyrate, H2 and CO2. The genomic DNA G+C content of strain CEJFT1B(T) was 32.3 mol%. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain CEJFT1B(T) should be assigned to the genus Sporohalobacter. The sequence similarity between strain CEJFT1B(T) and Sporohalobacter lortetii was 98.5 %, but DNA-DNA hybridization between the two strains revealed a relatedness value of 56.4 %, indicating that they are not related at the species level. The combination of phylogenetic analysis, DNA-DNA hybridization data, and differences in substrate utilization support the view that strain CEJFT1B(T) represents a novel species of the genus Sporohalobacter, for which the name Sporohalobacter salinus sp. nov. is proposed. The type strain is CEJFT1B(T) ( = DSM 26781(T) = JCM 19279(T)).

Biodegradation of malodorous thiols by a Brevibacillus sp. strain isolated from a Tunisian phosphate factory

Hydrogen sulfide (H2S) and thiols (RSH) generated by the phosphate industry cause harmful effects on human health and quality of life. The present study aims to investigate and evaluate a bacterial strain CAT37 isolated from gas-washing wastewaters in terms of its properties and ability to degrade malodorous thiols. Gas-washing wastewater samples were submitted to physicochemical analyses and used for the isolation of thiol-degrading bacteria. The results from gas chromatography-mass spectrometry (GC-MS) analysis revealed that the isolated strain CAT37 was able to oxidize ∼99% of each thiol, decanethiol and dodecanethiol used as sole carbon and energy sources after 30 days of incubation at 37°C. The strain CAT37 displayed a biodegradative potential on several thiols known by their toxicity and odors. The results from phylogenetic and phenotypic analysis revealed that the CAT37 isolate belonged to the genus Brevibacillus, showing the highest sequence similarity to Brevibacillus agri. Overall, the results indicated that the strain CAT37 exhibited a number of attractive biodegradation abilities against thiols and could be considered a promising candidate for industrial application in future thiol biodeodorization strategies.

Bioremediation of Petroleum Contaminated Water and Soils in Tunisia

The petrochemical industry generates series of liquid and solid wastes containing large amounts of priority pollutants during the petroleum-refining process. These residues must be treated through depuration processes. The bioremediation process, presenting countless advantages in relation to other processes employed, is an evolving method for the removal and the transformation of many environmental pollutants including those produced by the petroleum industry. In a first step, a continuously stirred tank bioreactor (CSTR) was used to optimize feasible and reliable bioprocess system for successful bioremediation of industrial effluent and to develop an efficient microbial consortium for the degradation of petroleum hydrocarbons. After an experimental period of 175 days, the process was shown to be highly efficient in decontaminating the wastewater. The performance of the bio augmented reactor was demonstrated by the reduction of COD rates up to 95%. Six microbial isolates from the CSTR were characterized and species identification was confirmed by sequencing the 16 S rRNA genes. Besides, the treated wastewater could be considered as non toxic according to the micro-toxicity test. In a second step, bioremediation of a refinery soil containing hydrocarbons climate was investigated. The objective of this study was to assess the ability of bioremediation technique in the presence of the acclimatized consortium to reduce the total petroleum hydrocarbon (TPH) content in the contaminated soil. Results clearly demonstrated that an enhanced bioremediation was carried when the acclimatized bacterial consortium was added to the hydrocarbons contaminated soil. The proposed bioremediation technology has proved significantly higher hydrocarbons removal efficiencies. TPH analysis showed that 50% of the hydrocarbons were eliminated during the first 15 days of bio remediation. TPH removal reached 96% at the end of the treatment. Further, GC/MS profile has proved that the acclimatized bacterial consortium could effectively remove the medium- and long-chain alkanes in the contaminated soil such as the alkanes were undetectable after a 30-day of incubation period. In a third step, a Halomonas sp. strain C2SS100 had been isolated and characterized from Sercina petroleum reservoir. The strain had shown potential hydrocarbon degradation under halophilic condition (100 g 1−1 NaCl). During growth on n-Hexadecane (C16), C2SS100 produced biosurfactant that could solubilise phenanthrene, a three-ring aromatic hydrocarbon. The halophilic character of this bacterium could add further advantages for its use in marine and saline environments-oil bioremediation.

Abundance and diversity of prokaryotes in ephemeral hypersaline lake Chott El Jerid using Illumina Miseq sequencing, DGGE and qPCR assays

Chott El Jerid is the largest hypersaline ephemeral lake in southern Tunisian Sahara desert and is one of the biggest depressions at the North of Africa. This study aimed to investigate the diversity and abundance of microbial communities inhabiting Chott El Jerid during wet season (when it was flooded), using molecular methods [Illumina Miseq sequencing, DGGE and qPCR (qPCR)]. 16S rRNA gene analyses revealed that bacterial community was dominated by Proteobacteria (especially Ralstonia species), followed by Firmicutes, Bacteroidetes, Cyanobacteria, Actinobacteria and Verrucomicrobia. The results obtained using prokaryotic universal primers showed low relative abundance of Archaea dominated by few OTUs related to Methanosarcinaceae and Methanomassiliicoccaceae families and the presence of sulfate-reducing Archaea affiliated with Archaeoglobus. However, the results obtained using Archaea-specific primers showed that archaeal community was mainly composed of aerobic Halobacteria (especially Halorubrum species) and anaerobic members of Methanomicrobia. These results also provided evidence for the presence of members of the genus Halohasta in this environment. qPCR results revealed that Archaea were more abundant in studied samples than Bacteria. The sulfate-reducing Bacteria were also found abundant (~ one-third of the bacterial community) and outnumbered methanogens, suggesting their potential important role in this sulfate-rich and hypersaline ecosystem.