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Environ Monit Assess (2017) 189:49 Pagc 3 of 13 49

Fig. 1 Study sitcs and locadon of sampling statioas

Conkey (Marchal et al. 1982). The identification was based on morphological and biochemical analyses (Api 20E and 20NE strips. Biomerieux) and on molecular PCR amplification and sequencing of 16S rDNA gene with primers 27f and 1401 r as described in Batisson et al. (2009). Phylogenetic analysis of the strains was also performed (Wang et al. 2007). The evolutionary relationship between ditferent strains was deduced using the neighbor-joining method (Saitsou and Nei 1987).

In order to see their growth and development. tlie isolated indigenous bacterial strains were then grown on synthetic seawater agar consisting of Tris (2 g L ^!), NaCl (23 g L^_l), NH₄C1 (1 g L~‘), MgS0₄.7H₂0

la hic 1 Mcasuremcnt mcthods of hydrocarboas, total nitrogcn. and total phosphates

Paramctcrs	Mcthods	Standard
Total	Chemical oxygen	S pcctrophotomctry
hydro car-	dcmand(COD)	N931241
bons	dctcmiination
(mg L ‘)	aftCT cxtraction with pcntanc
Total nitrogcn (mg L ‘)	Digcstion	Digcstion DIN EN ISO
	pcroxodisu!fatc	11905-1H36
	2,6-	Dctcmiination ISO 38405
	dimcthylphcnol	7890 l.DIN
Total	Molybdcnum bluc	EPA 365.2 + 3, APHA
phosphate		4500-PE; DIN EN ISO
(mg L^1)		6878-D11

(6 g L"¹), MgCl₂.6H₂0 (5 g L~‘), CaCl₂ 21I₂() (1 g L^-1), and agar (15 g L~‘) (Soltani 2004) supple-mented with 1 ml of hydrocarbon (crude oil supplied by the Skikda Oil Refinery). The selection of strains was obtained atter incubating on the Petri dishes at 30 °C for a period ranging from 24 h to 7 days (Leahy and Colwell 1990).

Tests of bacteria growth in the presence of alkanes and refined hydrocarboas

Among the isolated and identified strains, four were subjected to growth tests in the presence of hydrocar-bons and biostimulation with nitrogen and phosphates. respecth ely. equal to 1.5 g KNO5 and 45 mg NalI₂ P0₄ in a finał volume of 300 ml, respectively. The cultures w ere carried out in stenie synthetic seawater with added 1 ml alkanes, hexane C₆H_l4, heptanes    decane

C_ioH₂₂, cyclohexane C₆H_I2, or refined hydrocarbon (gasoline) as tlie sole carbon and eneigy source. The media w^rere incubated in a bacteriological incubator at 30 °C with stirring at 150 rpm in aerobic condition. Control cultures were performed without biostimulation. The growth kinetics was estimated through the turbidity media by measuring optical densities at 600 nm (Peny et al. 2004). Finally, the hydrocarbon concentrations were detennined (Table 1) before aiKi after bacterial growth. A multivariate analysis of

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