ISOLATION AND USE OF EXTREMOPHILES IN THE DETERMINATION OF BIOCHEMICAL OXYGEN DEMAND IN SALINE WASTEWATER

MELINA GROSSELLI ; LILIANA VILLEGAS; PATRICIA GONZÁLEZ; CÉSAR ALMEIDA

CORDOBA

Congreso; XI Congreso Argentino de Microbiología General | SAMIGE 2015; 2015

Biochemical oxygen demand (BOD) is a useful parameterfor assessing the biodegradability of dissolved organic matter in water. At thesame time, this parameter is used to evaluate the efficiency with which certainprocesses remove biodegradable natural organic matter. However, the values ofBOD in saline water are very low because NaCl destroy the seed. Therefore theincorporation of a suitable selection of bacteria (which is not always easy toobtain) is required.  In consequence,this method used to poorly develop. In this sense the extremophile bacteria,particularly halophilic bacteria or halophytes can contribute to thedevelopment of this technique. The objective of this work was to develop a BODanalysis protocol for the analysis of saline effluent. Within this context ahalophilic microorganisms was isolated from Salinas del Bebedero, San Luis,Argentina. Salt sample was cultured in agar Dussault and Lachance.Subsequently, the isolated colonies were cultured in medium Dussault andLachance broth for 7 days at 37ºC with constant stirring (180 rpm).Subsequently the nucleic acid extraction method was performed by Brosius etal., based on the lysis of the cells with glass beads, and 10% SDS. To amplifythe variable region V3-V5, 16S rDNA primers described as F344-R915 (Stahl etal, 1991) were used: 344F: 5´- ACG GGG CGC YGCAGCAGG GA-3 ´and 915R: 5´- GTGCTC CCC CGCCAATTC CT-3 ´; Halorubrumsalsolis DNA was used as control. The size of the reaction products wascharacterized on agarose gel 1% and evaluated on an image analyzer. Themolecular weight was determined by comparison with molecular weight marker 100bp DNA Ladder (Promega). The PCR fragments were sequenced by Macrogen (Korea),with Reaction Kit READY PRIMS a model ABI prisma373A sequencer (PE AppliedBiosystems).The sequencing results were analyzed by comparison with 16S rRNA genesdatabases (GenBank). The organism under study under study showed a 100%identity to gender Haloarcula sp. Onemilliliter of the strain (106 cell per mL) was used as seed in theBOD analysis protocol. Standard solution of glucose-glutamic acid (BOD = 20 mgL-1) with different concentrations of NaCl (5%, 10%, 20%, 30% and40%) were used. BOD bottles were incubated at 20 °C for 5 days. No significantdifferences were found among differents samples, since their organic mattercontent was similar (CV <2.1%). In conclusion, Haloarcula sp can be used in the determination of BOD in salineeffluent.