Atef Jaouani

Hydrocarbonoclastic bacteria isolated from petroleum contaminated sites in Tunisia: isolation, identification and characterization of the biotechnological potential

Petroleum hydrocarbons are important energy resources used by industry and in our daily life, whose production contributes highly to environmental pollution. To control such risk, bioremediation constitutes an environmentally friendly alternative technology that has been established and applied. It constitutes the primary mechanism for the elimination of hydrocarbons from contaminated sites by natural existing populations of microorganisms. In this work, a collection of 125 strains, adapted to grow on minimal medium supplemented with crude oil, was obtained from contaminated sediments and seawater from a refinery harbor of the Bizerte coast in the North of Tunisia. The diversity of the bacterial collection was analyzed by amplification of the internal transcribed spacers between the 16S and the 23S rRNA genes (ITS-PCR) and by 16S rRNA sequencing. A total of 36 distinct ITS haplotypes were detected on agarose matrix. Partial 16S rRNA gene sequencing performed on 50 isolates showed high level of identity with known sequences. Strains were affiliated to Ochrabactrum, Sphingobium, Acinetobacter, Gordonia, Microbacterium, Brevundimonas, Novosphingobium, Stenotrophomonas, Luteibacter, Rhodococcus, Agrobacterium, Achromobacter, Bacilllus, Kocuria and Pseudomonas genera. Acinetobacter and Stenotrophomons were found to be the most abundant species characterized by a marked microdiversity as shown through ITS typing. Culture-independent approach (DGGE) showed high diversity in the microbial community in all the studied samples with a clear correlation with the hydrocarbon pollution rate. Sequencing of the DGGE bands revealed a high proportion of Proteobacteria represented by the Alpha and Gamma subclasses. The predominant bacterial detected by both dependent and independent approaches were the Proteobacteria. The biotechnological potential of the isolates revealed a significant production of biosurfactants with important emulsification activities useful in bioremediation. The highest emulsification activity was detected in Pseudomonas geniculata with 52.77% of emulsification. Our overall results suggest that the obtained bacterial isolates may constitute potential candidates for bioremediation and can be useful for biotechnological applications.

Isolation and characterization of non-Frankia actinobacteria from root nodules of Alnus glutinosa, Casuarina glauca and Elaeagnus angustifolia

Actinobacterial isolates randomly obtained on nitrogen-free BAP medium from surface sterilized root nodules of Alnus glutinosa, Casuarina glauca and Elaeagnus angustifolia sampled from fields were reported. They were assigned on the basis of partial 16S rRNA sequences to Micromonospora, Nocardia and Streptomyces genera. The isolates have been screened for hydrolytic activities, indole acetic acid (IAA) and siderophores production, phosphate solubilization and antagonistic activities. Results suggest putative traits as plant growth promoting bacteria proprieties of the isolates that occur in unique association in root nodules of the three analysed actinorhizal host species.

Olive Oil Mill Wastewater Purification by Combination of Coagulation- Flocculation and Biological Treatments

In order to define an efficient pre-treatment of Olive Oil Mill Wastewater (OOMW) to overcome major obstacles to biological treatment, various organic and mineral coagulants have been tested. In particular, the application of quicklime until a pH around 12 – 12.4 was reached, allowed the reduction of almost 37 % of the initial COD, and approximately 88 % and 71 % of the colour and phenolic content of the waste. Hence, further biological treatments with an adapted aerobic consortium (AC) and a white rot fungus (WRF) strain were improved. The WRF Coriolopsis polyzona was more efficient than AC to reduce colour and polyphenols when the waste was prior diluted or pre-treated; however, it was less effective in COD removal. The combined treatment: lime – AC of OOMW having initial COD of 102 g l−1 led to the elimination of about 77, 91 and 63 %, of the COD, phenols and colour, respectively. Interestingly, the opposite combination AC – lime permitted better COD, phenols and colour reduction to respectively, 21, 11 and 11% of the initial values. This latter condition is technically recommended since only one step separation was needed and no pH correction was necessary before undergoing aerobic treatment. Moreover, the process would produce a sludge potentially rich in organic matter, and consequently, useful as an agricultural amendment or/and as an additive in animal nutrition.

Esterase as an enzymatic signature of Geodermatophilaceae adaptability to Sahara desert stones and monuments.

Aim:  To assess esterase profiling of members of Geodermatophilaceae isolated from desert stones and monuments in Tunisia and Egypt.

Effect of culturing processes and copper addition on laccase production by the white-rot fungus Fomes fomentarius MUCL 35117.

Aim:  To produce high laccase activities from the white‐rot fungus Fomes fomentarius.

Allochthonous bioaugmentation in ex situ treatment of crude oil-polluted sediments in the presence of an effective degrading indigenous microbiome

Oil-polluted sediment bioremediation depends on both physicochemical and biological parameters, but the effect of the latter cannot be evaluated without the optimization of the former. We aimed in optimizing the physicochemical parameters related to biodegradation by applying an ex-situ landfarming set-up combined with biostimulation to oil-polluted sediment, in order to determine the added effect of bioaugmentation by four allochthonous oil-degrading bacterial consortia in relation to the degradation efficiency of the indigenous community. We monitored hydrocarbon degradation, sediment ecotoxicity and hydrolytic activity, bacterial population sizes and bacterial community dynamics, characterizing the dominant taxa through time and at each treatment. We observed no significant differences in total degradation, but increased ecotoxicity between the different treatments receiving both biostimulation and bioaugmentation and the biostimulated-only control. Moreover, the added allochthonous bacteria quickly perished and were rarely detected, their addition inducing minimal shifts in community structure although it altered the distribution of the residual hydrocarbons in two treatments. Therefore, we concluded that biodegradation was mostly performed by the autochthonous populations while bioaugmentation, in contrast to biostimulation, did not enhance the remediation process. Our results indicate that when environmental conditions are optimized, the indigenous microbiome at a polluted site will likely outperform any allochthonous consortium.

Application of Asymetrical and Hoke Designs for Optimization of Laccase Production by the White-Rot Fungus Fomes fomentarius in Solid-State Fermentation

Statistical approaches were employed for the optimization of different cultural parameters for the production of laccase by the white rot fungus Fomes fomentarius MUCL 35117 in wheat bran-based solid medium. first, screening of production parameters was performed using an asymmetrical design 2533//16, and the variables with statistically significant effects on laccase production were identified. Second, inoculum size, CaCl2 concentration, CuSO4 concentration, and incubation time were selected for further optimization studies using a Hoke design. The application of the response surface methodology allows us to determine a set of optimal conditions (CaCl2, 5.5 mg/gs, CuSO4, 2.5 mg/gs, inoculum size, 3 fungal discs (6 mm Ø), and 13 days of static cultivation). Experiments carried out under these conditions led to a laccase production yield of 150 U/g dry substrate.

Purification and characterization of a highly thermostable esterase from the actinobacterium Geodermatophilus obscurus strain G20

Intracellular thermostable esterase produced by the extremophilic Geodermatophilus obscurus G20 was purified to homogeneity by a heat treatment, followed by an anion‐exchange chromatography, and then characterized. The molecular weight of the purified enzyme determined by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) was shown to be approximatively 55 kDa. The enzyme showed an optimal activity between pH 8.0 and 9.0 and was stable in the pH range 7.0–10.0. Moreover, it is highly thermostable, with a residual activity greater than 90% after incubation at 80 °C for more than 10 h. The enzyme showed preference for esters of p ‐nitrophenol with short chain fatty acid. When the p ‐nitrophenyl acetate (C2) was used as substrate, the Michaelis–Menten constant (Km) and maximum velocity for the reaction (Vmax) of esterase were 400 μM and 2500 U/mg protein, respectively. The effect of phenylmethanesulphonyl fluoride (PMSF), a serine‐specific inhibitor, on the enzyme activity suggested that the thermostable esterase belong to the serine hydrolase group. Because of its high thermostability, activity at alkaline pH, tolerance to methanol and various metal ions and specificity for short chain fatty acids, this enzyme showed high potential for use in biocatalysis. (© 2012 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

Decolorization of Solophenyl Red 3BL Polyazo Dye by Laccase-Mediator System: Optimization through Response Surface Methodology

The decolorization of direct Solophenyl red 3BL (SR), a polyazo dye extensively used in textile industry was studied. The Fomes fomentarius laccase alone did not decolorize SR. The natural redox mediator, acetosyringone (AS), was necessary for decolorization to occur. Box-Behnken design was used to evaluate the effects of three parameters, namely, enzyme concentration (0.5–2.5 U mL−1), redox mediator concentration (3–30 μM), and incubation time (1–24 h), on the SR decolorization yield. The fitted mathematical model allowed us to plot response surfaces as well as isoresponse curves and to determine optimal decolorization conditions. The results clearly indicated that the AS concentration was the main factor influencing the SR decolorization yield. The selected optimal conditions were enzyme concentration 0.8 U mL−1, mediator concentration 33 μM, and time 14 h 30 min. These conditions allowed 79.66% of SR decolorization versus 80.70% for the predicted value. These results showed a promising future of applying laccase-AS system for industrial wastewater bioremediation.

Newly isolated yeasts from Tunisian microhabitats: Lipid accumulation and fatty acid composition

Newly isolated yeasts from different Tunisian microhabitats, such as soil, milk, olive brine, vinegar, and from olive mill wastewater‐contaminated biotopes were extensively studied for their biochemical arsenal and morphological features, i.e. cell, ascospore, and lipid body morphology. All strains were classified into the Ascomycota phylum. However, they showed great functional diversity, including different morphological and biochemical features, lipid production ability, and fatty acid profiles. Accordingly, the strains were placed in three different groups: Group I, which includes Candida species; Group II (Pichia and related); and Group III (Kluyveromyces marxianus strain CC1). Group I and II were characterized by a high percentage of oleic acid (41.6–65.3% of total lipids) while in Group III, linoleic acid was the major fatty acid (37.2%). Members of Group I and II were further grouped into subgroups according to their fatty acid composition. Among the newly isolated strains, Pichia etchellsii BM1 was able to accumulate around 25% wt/wt lipid per dry cell mass and thus characterized as oleaginous. Some other strains, such as Candida metapsilosis strain EL2, C. parapsilosis strain LV2, C. pararugosa strain BM24, and K. marxianus strain CC1, which are able to produce extracellular lipases, may be of interest for specific environmental applications and/or for the production of novel lipases.