ISOLATION OF LIPOLYTIC BACTERIA FROM COLOMBIAN ANDEAN SOILS: A TARGET FOR BIOPROSPECTING

oil (1%). Two isolates identified as Pseudomonas psychrophila and Stenotrophomonas rhizophila produced the largest hydrolysis halos, with an optimal activity at pH 8 and 50°C. Only the S. rhizophila extracellular fraction hydrolyzed short and long chain pNP-esters, including pNP-palmitate. The broader substrate specificity of this isolate is probably due to the simultaneous presence of lipase and esterase activity in the crude extract. This is the first report of lipolytic activity in S. rhizophila , and the first preliminary characterization of the novel lipolytic activity in S. rhizophila and P. psychrophila. Further work is needed to purify and completely characterize the esterases and lipases produced by both species.


INTRODUCTION
Lipases are important biocatalysts with innumerable applications in food, dairy, detergent and pharmaceutical industries.The biological function of these enzymes is to catalyze the hydrolysis of triacylglycerols to release free fatty acids, diacylglycerols, mono-acylglycerols and glycerol (Patil et al., 2011).Lipolytic enzymes include esterases commonly defined as carboxyesterases (EC.3.1.1.1)and lipases, originally called true lipases (Sangeetha et al., 2011).Over the last few decades, lipases have gained special attention for their ability to act in micro-aqueous environments and catalyze esterification, trans-esterification, aminolysis and acidolysis reactions (Joseph et al., 2008).Bacterial lipases have valuable properties such as a wide variety of substrates, selectivity, stability in organic solvents, and activity in harsh conditions.Other advantages that promote research on these enzymes are their high yield, low production costs and the possibility of genetic manipulation (Glogauer et al., 2011;Meilleur et al., 2009).Most of the lipases in industry are lipoproteins found in genera such as: Achromobacter, Acinetobacter, Bacillus, Burkholderia, Pseudomonas, Staphylococcus, Chromobacterium, among others (Castilla et al., 2017;Soliman et al., 2007).These lipases can be used in different applications including production of fats and oils, detergents, fine chemicals, pharmaceutical drugs, pulp and paper, as well as use in bioremediation, waste treatment, oil biodegradation and medical applications (Stergiou et al., 2013).The low stability of well-characterized mesophilic enzymes under extreme conditions has prompted the search for new enzymes with better properties (Tirawongsaroj et al., 2008;Jiménez et al., 2012;López et al., 2014).Thus, the isolation of novel microorganisms capable of producing stable lipases for industrial applications is required (Messiasa et al., 2009;Abol et al., 2016).Extreme environments with low temperatures are the most common places to find psychrophilic bacteria with lipolytic activity (De Pascale et al., 2012; De Santi et al., 2016).Cold-adapted microorganisms produce lipases which function effectively at cold temperatures with high rates of catalysis, in comparison to those lipases synthesized by mesophiles or thermophiles that show low, or no activity at low temperatures (Joseph et al., 2008).Some of the adaptations found in enzymes from extreme environments include structural flexibility, higher content of alpha helix compared to beta-sheets and temperature independent reaction rates (Dalmaso et al., 2015).Previous studies have reported direct functional screening to isolate lipolytic microorganisms through enrichment cultures with lipid substrates including oils (olive oil, palm oil, oil mill), fatty acids and glycerol (Yao et al., 2013;Gombert et al., 1999).Hence the enrichment cultivation in olive oil used in this study, was carried out based on the assumption that microorganisms able to survive on this substrate can synthesize lipases (Gaoa et al., 2000).One of the advantages of this technique is the accumulation of microorganisms with useful enzymes in growthcontrolled conditions (Ekkers et al., 2012).The major aim of this study was to isolate lipolytic bacteria from low temperature environs found in the paramo and glacier soils from "Los Nevados" National Natural Park.The screening of lipolytic bacteria through the enrichment culture strategy, followed by the bacterial identification by 16s rDNA sequencing, and the preliminary enzyme characterization at different conditions (temperatures, pH and substrates) were the main steps that allowed us to bioprospect for putative novel lipases.
Microbial enrichments with a substrate of interest could enhance the possibility of finding certain desired metabolic activities.As lipases are one of the most important enzymes in industrial applications, the Colombian Andean soils were explored as a source of lipolytic microorganisms.Two Andean soils under low temperatures were sampled: paramo and glacier soils from "Los Nevados" National Natural Park.Both soils were enriched through a fed-batch fermentation using olive oil as the inductor substrate.Forty-three lipolytic isolates were obtained and their taxonomic assignments were performed on the basis of 16S rDNA gene sequencing.In both cases, the phylum Proteobacteria represented the majority of the isolates.Qualitative assays to measure the lipolytic activity were performed by using tributyrin, triolein or olive oil (1%).Two isolates identified as Pseudomonas psychrophila and Stenotrophomonas rhizophila produced the largest hydrolysis halos, with an optimal activity at pH 8 and 50°C.Only the S. rhizophila extracellular fraction hydrolyzed short and long chain pNP-esters, including pNP-palmitate.The broader substrate specificity of this isolate is probably due to the simultaneous presence of lipase and esterase activity in the crude extract.This is the first report of lipolytic activity in S. rhizophila, and the first preliminary characterization of the novel lipolytic activity in S. rhizophila and P. psychrophila.Further work is needed to purify and completely characterize the esterases and lipases produced by both species.

Enrichment cultures
The inoculum preparation started with the samples being washed in mineral salt medium (MSM), supplemented with tween 20 (0.5%) and olive oil 1%.This procedure was performed with eight grams of soil for each environment, followed by a 24 hours incubation at 140 rpm and at 22°C (Wang et al., 2005).After sedimentation, the supernatant was used as inoculum (10%) for the enrichment culture.This was performed with the same supplemented medium, in a fermenter batch adjusted to 1L bioreactor.The effective volume was 70%, and the cultures conditions were 22ºC, 140 rpm, pH 6.9 and 1.6 L min -1 of aeration during 3 weeks (Lanser et al., 2002;Henne et al., 2000).Every 5 days the bioreactor was fed with MSM ensuring the final effective volume.CFU mL -1 was estimated in nutrient agar (MoBio), (Köhler, 2007;Wang et al., 2005).

Isolation and morphological description
Functional screening was carried out at the end of the enrichment in MSM agar supplemented with olive oil or tributyrin at 22°C after three days (Bunterngsook et al., 2010;Ertugrul et al., 2007).Colonies with hydrolysis halos were isolated and preserved in 15% glycerol solution at -80°C.The morphological description of colonies was performed and Gram staining was also included to verify cell shape and cell wall properties.

Lipase assay
Lipase production and secretion was evaluated in triplicate by agar diffusion assay.Briefly, MSM agar plates were supplemented with 1% of tributyrin, olive oil or triolein (Chakravarthy & Narasu, 2012).As preinoculum, one full loop of the isolated colonies was transferred in 10 ml LB broth at 20°C for 24 hours.After incubation, 5 μl of the inoculum was then transferred on filter paper disks and incubated on agar plates at 20°C for 8 days.The strains were examined daily for their ability to produce clarified zones (Golani et al., 2016).

DNA extraction, amplification and sequencing of 16S rDNA gene
Total DNA was extracted from the isolates as previously described (Spanevello et al., 2002).Universal primers 27F (5'-AGAGTTTGATCMTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3') (Sarkar et al., 2014) were used to obtain a PCR product of 1.5 Kb.A 100 μL reaction contained buffer 1X, MgCl 1.5 mM, dNTPs 0.2 mM, 0.3 μM of each primer, 0.5% of BSA and Taq Polymerase Promega 1U.PCR was carried out by an initial denaturation at 94ºC for 5 min; then 30 cycles of denaturation at 94ºC for 30 sec, annealing at 57ºC for 45 sec, extension at 72ºC for 1 min y 30 sec and finally an extension cycle at 72ºC for 7 min.PCR products were analyzed by electrophoresis 1.5% (wt/vol) agarose gels with SyBR-safe (InvitroGen TM ).The PCR products were sequenced via the Sanger method by Macrogen Inc. Korea.

Sequence alignments and phylogenetic analysis
The 16S rDNA sequences were analyzed using BLAST (http://www.ncbi.nlm.nih.gov/BLAST) and the best hit was assigned to the query.Distance trees were constructed using the UPGMA algorithm, (Bisht & Panda, 2011) with the MEGA 7.0 ® package.The alignments were performed through CLUSTAL omega http://www.ebi.ac.uk/Tools/msa/clustalo0w2/).Bootstrap resampling analysis for 1000 replicates was performed to estimate the confidence of tree topologies (Felsenstein, 1985).The sequences were submitted to the European Molecular Biology Laboratory (EMBL-ENA) Nucleotide Sequence Database (http://www.ebi.ac.uk/embl/Submission/webin.html).The accession numbers of the nucleotide sequence data are listed in Table 1.

Growth curve experiments
The three strains that produced the largest hydrolysis halos, Stenotrophomonas rhizophila (strain USBA 843E), Pseudomonas psychrophila (strains USBA 844A, USBA 846E) were inoculated in Bioscreen® plates in triplicate with 200 μl as final volume and an OD540 adjusted to 0.02.Optical density was determined at 20°C each 6 h for 80 hours in order to obtain the their growth curves.

Lipase activity
USBA 843E, USBA 844A and USBA 846E strains were cultured in LB broth supplemented with olive oil 1% as inductor substrate at 20°C for 72 hours and 140 rpm.Then, they were centrifuged at 10.000 rpm for 7 minutes to recover the supernatant.The protein content was determined using the Bradford method (Bradford, 1976) and the enzymatic reaction was measured in triplicate in the Bioscreen®.For this reaction, 20 μl of supernatant were added to 180 μl of buffer Tris-HCl 0.1 M and 0.1 mM of lipid substrate emulsified in isopropanol (Rashid et al., 2001).The pNP-esters evaluated were: acetate, butyrate, decanoate and palmytate at 5ºC, 10ºC, 20ºC, 30ºC, 40ºC, 50ºC and 60ºC.The optimum pH of lipolytic activity of the strains was determined between 3 and 9, using 0.1M Glycine-HCl pH 3; 0.1M Acetate pH 4 and 5; 0.1 M phosphate pH 6; 0.1M Tris-HCl pH 7.8 and 9.This assay was carried out for 1 h at 30°C including biological triplicates.The activity was evaluated measuring the liberated p-nitrophenol at 405 nm.One unit of activity was defined as the amount of enzyme needed to release 1 μmol of p-nitrophenol per min.

Statistical analyses
The results obtained from the lipase activity assay were analyzed through the SPSS software (version 12.0).The analyses were performed using the Shapiro-Wilk Normality Test, and the pairwise comparison Kruskal-Wallis (Nonparametric) test to establish differences between the three substrates.The homogeneous subgroups test was used to select the strains with the best lipolytic activity.

Enrichment culture and isolation of lipolytic bacteria
The initial pH was adjusted to 7 + 0.2, and it decreased around 6.0 after 3 days.Regarding cell density, the paramo enrichment culture increased its initial cell count from10 5 CFU mL -1 to 10 9 CFU mL -1 , while the glacier enrichment increased from 10 3 CFU mL -1 to 10 8 CFU mL -1 .After the screening a total of 43 colonies were isolated, 30 from the paramo enrichment and 13 from the glacier.

Morphological description
The most common colony morphologies for the bacterial isolates were: wrinkled, mucoid, viscous, and translucent, with irregular and regular shapes, and with white, pale yellow or yellow tones.Exopolysaccharide production was observed in some isolates.Some colonies could change the morphotype after transferring to a new agar plate.Nevertheless, the microscopic observation showed the same Gram-negative, rod-shaped bacteria.

Lipase assay
After 8 days of incubation at 20ºC, six of the 43 isolates presented significant hydrolysis halos (statistical analysis).Isolates USBA 851A (Pseudomonas fragi), USBA 852C (Ralstonia pickettii), USBA 843E (Stenotrophomonas rhizophila), and USBA 844A (Pseudomonas psychrophila) produced the major hydrolysis halos in the tributyrin agar plates with diameters between 7 mm to 11 mm (Excluding the colony diameter).In triolein, the isolates USBA 844A and USBA 846E (Pseudomonas psychrophila) showed halos between 8 mm and 10 mm.In olive oil, the isolates USBA 851A (Pseudomonas fragi), USBA 851D (Pseudomonas fragi) and USBA 846E (Pseudomonas psychrophila) produced halos between 5 and 7 mm of diameter (Fig 1).As previously mentioned, Stenotrophomonas rhizophila and Pseudomonas psychrophila, among other isolates, produced the largest hydrolysis halos on the evaluated substrates.We note this fact because the lipolytic activity from Stenotrophomonas rhizophila has not been reported until now.This species of Stenotrophomonas is characterized by the absence of lipase and β-glucosidase production (Wolf et al., 2002).Additionally, the lipolytic activity of Pseudomonas psychrophila has been reported but it has not been evaluated.This finding led us to choose these isolates (USBA 843E, USBA 844A/USBA 846E) to perform the enzymatic activity assays on, in order to find novel lipases from psychrophilic bacteria.

Sequence alignments and phylogenetic analysis
The 16S rDNA gene sequences were compared with sequences reported in the GeneBank database using BLAST and the Ribosomal Database Project (RDPII), in order to determine the taxonomic assignment of the 43 isolates.The percent identities obtained were 99% and 100%, while the e-value was equal to zero (Tab 1).).The trees were generated using the UPGMA method and were rooted with Methanogenium frigidum (NR_104790.1) and Bacillus subtillis (JQ433875.1)as out groups.Scale bar indicates number of substitutions per site and the bootstrap analysis was performed on the basis of 1000 replicates

Growth curve experiments
The late stationary phase from the isolates USBA 843E (Stenotrophomonas rhizophila), USBA 844A and USBA 846E (Pseudomonas psychrophila) was reached after 72 hours of incubation.In this phase, the extracellular fraction was obtained to perform the p-nitrophenyl ester assays.
The lipase activity essay was carried out for 1 h at 30°C with four lipid substrates: pNP-acetate, pNP-butyrate, pNP-decanoate and pNP-palmitate.Shortchain pNP-esters (C2 and C4) were hydrolyzed for the three isolates.Only the USBA 843E (S. rhizophila) showed activity with the four substrates including pNP-palmitate.The broader substrate specificity of this isolate is probably due to the simultaneous presence of lipase and esterase activity in the crude extract (Fig 3).

Effect of temperature on enzyme activity
The USBA 843E (S. rhizophila) enzyme fraction showed maximal activity at 50°C in the four lipid substrates.Its highest activity was in short chain (C2 and C4) fatty acids.The extracellular fraction from USBA 844A (P.psychrophila) had high activity at 50°C in pNP-butyrate and at 40°C in pNP-acetate.Finally, the USBA 846E (P.psychrophila) showed its optimum activity at 50°C in pNPacetate, pNP-butyrate and pNP-decanoate, and its highest activity was in short chain (C2 and C4) fatty acids (Fig 3).The pH used during the temperature assays was 7.

Effect of pH on lipase activity
The three isolates showed their optimal lipolytic activity at pH 8 in all the lipid substrates except for pNP-butyrate that was highly hydrolyzed at pH 9.For the isolate USBA 843E (S. rhizophila), the hydrolytic activity was observed at pH 7 in C2 and C4, pH 8 in all the lipid substrates, and pH 9 in C4 and C10 (Fig 3).The lipase from USBA 844A (P.psychrophila) showed activity at pH 7-8 in C2, and at pH 8-9 in C4 (Fig 3).For the isolate USBA 846E (P.psychrophila), the C2 hydrolysis was carried out at pH 6 to 9; of C4 at pH 8-9 and C10 at pH 8 (Fig 3).The temperature used during the pH assays was 20°C.Pseudomonas and Stenotrophomonas were the most abundant genera recovered from the enrichments, and they also exhibited the largest hydrolysis halos.This shows how their ability to metabolize the given substrate allowed them to increase their population compared to other lipolytic bacteria.Bardgett et al. (2005) reported that the diversity tends to fall when the better-adapted microorganism dominates the community.In our case, the better-adapted microorganisms with the ability and required enzymes to degrade the nonconventional substrate, dominated the enrichment culture communities.Furthermore, when we compared the lipolytic isolates from both soils, we found greater species diversity in the glacier enrichment culture than the one obtained from paramo.This is counter intuitive to expectation if we consider that the optimal environmental conditions, in terms of temperature, humidity and nutrient availability were present in the paramo soil (Buytaert et al., 2006).In this case it is necessary to take into account that the enrichment method involved changes in the dynamics of the community.This could be caused predominantly by the microbial interactions within the bioreactor and the selection pressure in olive oil In their study they reported that the three strains evaluated showed a high antifungal activity and no lipase or βglucosidase production, which differs from the characteristics observed from the 13 strains of S. maltophila.In addition, the growth rate of S. rhizophila at 4ºC was 6.25 times higher than the observed with S. maltophila, which lead us to predict an even higher potential for the purified enzyme of S. Our possible "novel" alkaline enzymes should be purified and tested for a complete characterization in order to explore all the potential in terms of stability, substrate specificity and possible applications.

CONCLUSION
Exposing microorganisms to specific conditions in order to screen for capabilities that are not usually explored in non-conventional environments is a useful tool to discover biotechnological applications.Our study reports the first preliminary characterization of the lipolytic activity of Stenotrophomonas rhizophila and Pseudomonas psychrophila.Further studies to purify and completely characterize their lipolytic enzymes, will help us to understand all the potential offered by these "novel" lipases/esterases, that could be used for future applications.Our results demonstrate the vast opportunities offered by extreme environments for exploration and bioprospecting of different metabolic activities of interest.

Figure 3
Figure 3 Lipolytic activities from the isolates USBA 843E, USBA 844A and USBA 846E at different temperatures and pH.

(
Bardgett et al., 2005).The phyla Proteobacteria and Actinobacteria (with Dermacoccus nishinomiyaensis as the only isolate) identified in the glacier enrichment cultures have been reported from other cold environments (Cheng & Foght, 2007; Steven et al., 2007; Neufeld et al., 2004; Shivaji et al., 2006).However, some studies also indicate the presence of different taxa such as Bacteroidetes and Firmicutes.They also report the presence of psychrophilic and lipolytic bacteria including Pseudomonas, Aeromonas, Burkholderia and Ralstonia, (Yuan et al., 2010; Joseph et al., 2008; Hemachander & Puvanakrishnan, 2000) which were also found in our enrichment cultures.One particular finding in our study was the isolation of bacteria with different colony morphotypes.This polymorphism complicated the macroscopic description of some of the isolates, but after finishing the bacterial identification, this change was observed only for the Pseudomonas isolates.Workentine et al. (2010) described that Pseudomonas can change its colony morphotype depending on the culture conditions.In terms of lipolytic activity, this potential for Pseudomonas fragi and Ralstonia picketti was confirmed by the large hydrolysis halos produced in the fatty substrates (Fig 1 and 3).These species have been previously reported for their lipolytic activity and stability at low temperatures (Joseph et al., 2008; Hemachander & Puvanakrishnan, 2000).The other two isolates with interesting activity were Pseudomonas psychrophila and Stenotrophomonas rhizophila.They were chosen to perform enzymatic activity assays, not only for the production of hydrolysis halos, but also for their "novelty" value.Bai & Rai Vittal.(2014) studied the relationship between protease and lipase production with quorum sensing molecules in P. psychrophila PSPF19.Their objective was to inhibit the exoenzyme production.Therefore, they never characterized the lipases or proteases produced by the strain PSPF19.The closest report of the lipolytic activity in P. psychrophila, was performed by Yumoto et al. (2001) with Pseudomonas fragi lipase, which has a close phylogenetic association with P. psychrophila.In conclusion, our study is the first to date to perform the preliminary characterization of its lipolytic activity.In 2002 Wolf et al. did the whole characterization of Stenotrophomonas rhizophila, a novel plant-associated bacterium.
rhizophila at low temperatures.After this publication, Hasan-Beikdashti et al. (2008) optimized the culture conditions to produce the S. maltophila lipase to hydrolyze capsaicin and produce vanillylamine, which has antibacterial and anti-inflammatory properties.Li et al. in 2013 and 2016 cloned and characterized the lipases: CGMCC 4254 (novel cold-active and organic solvent-tolerant lipase), and the GS11 (cold-active, solvent-tolerant and alkaline) also from S. maltophila.Nevertheless, this is the first study that reports the lipolytic activity in S. rhizophila.The optimal conditions for the three evaluated strains were 50ºC and pH 8 (Fig 8).Those temperatures and pH conditions have been already evaluated in Pseudomonas aeruginosa (Dharmsthiti et al., 1998), Pseudomonas fluorescens (Kulkarni et al., 2002), Pseudomonas sp.INK1 isolated in the Antarctic (Park et al., 2012).

Table 1
Taxonomic assignment of lipolytic bacterial isolates using BLASTN and accession numbers.
The distance tree from glacier enrichment culture includes 4 clades from the phylum Proteobacteria, which are associated with the genera Pseudomonas, Stenotrophomonas, Ralstonia and Burkholderia.The fifth clade belongs to the phylum Actinobacteria, represented by the genus Dermacoccus (Fig 2a).The paramo enrichment culture tree showed only 3 clades.The isolates belong to the phylum Proteobacteria represented by the genera Aeromonas, Pseudomonas and Stenotrophomonas (Fig 2b).