Materials

Chemical compounds with their catalog number which used in this studied are DEAE- Cellulose (30477), Olive oil (75343), Sephadex G-50 column (S5897), Tween 20 (P9416), Tween-80 (P8074) and p-NPP (N2752), Sigma-Aldrich, Germany; Dialysis tubing cellulose membrane flat with 10 mm (D9277) and Rhodamine B (83689), Sigma, Germany; Millipore membrane filter 0.22 μm (GSWP02500) and 0.45 μm (HAWP04700), Merck Millipore, Germany; Acetone (100014), Ammonium sulphate (101217), Barium chloride (101716), Benzene (101783), Calcium chloride (102083), Chloroform (107024), Cobalt (II) chloride anhydrous (802540), Cyclohexane (102817), Ethanol (818760), Ethyl Acetate (100789), Hexane (104373), Isopropanol (113350), Magnesium Chloride (814733), Mercury(II) chloride (104417), Methanol (822283), Sodium deoxycholate (106504), Sodium dodecyl sulfate (817034), Toluene (108327), Triton X-100 (112298), Urea (108486) and Zinc chloride (108816), Merck, Germany. No human or animal was used in this research.

Sampling and media preparation

Sampling was conducted from Gheynarje Nir hot spring Ardebil, Iran (latitude 38° 2′8.98″N; longitude 47°59′0.39″E). Environmental conditions like temperature and pH was recorded as 60 °C and 8.6, respectively.

The medium used for lipase and biosurfactant production was a basal salt medium as described by Schlegel 1961 with some modification [13]. This medium comprised 5 g/l yeast extract, 10 ml of olive oil, 0.2 g/l of MgSO4.7H₂O, 0.01 g/l of FeSO₄.7H₂O, 0.01 g/l of CaCl₂.7H₂O, 1 g/l of NH4Cl, 0.5 g/l of K₂HPO4 and 0.1 ml of trace element solution (70 mg/l of ZnCl₂, 100 mg/l of MnCl₂.4H₂O, 200 mg/l of CoCl₂.6H₂O, 100 mg/l of NiCl₂.6H₂O, 20 mg/l of NaMoO₈.2H₂O, 26 mg/l of Na₂SeO₃.5H₂O and 1 ml of 25% HCl) [13]. We used 1% olive oil as source of carbon, energy and lipase and biosurfactant stimulator. First, 10 ml samples were grown in 250 ml sterile flasks containing 80 ml of basal medium supplemented with 1% of enzyme free detergent powder (Paksan Company, Iran), each culture was adjusted to pH 8, 9 and 10 and incubated in a rotary incubator at a temperature of 60 °C at 120 rpm for 72 h. In order to make solid plate, 20 g/l agar was added to basal medium. Isolated strain was cultured on agar plate incubated at 60 °C for 48 h. Finally, a loop full of isolated strain was added to 100 ml basal medium broth and incubated at 60 °C, pH 10 and agitation speed 180 rpm for 72 h.

Screening and isolation of lipolytic microorganism

Screening of lipase bacteria was obtained on solid plate assay for bacterial lipases according to Kouker and Jaeger’s method [16]. Based on the highest lipase specific activity, one colony was chosen for further investigations.

Screening for biosurfactant production microorganism

Antimicrobial activity

Several standard microorganisms strains like gram positive, gram negative bacteria and fungi were cultivated on nutrient agar plate at 37 °C, 24 h for bacterial and 48 h for fungi pathogens. The antimicrobial activity was investigated by disc diffusion method on MHA plate by using Bauer et al. [22] method. The diameter of inhibition zone was measured [22].

DNA extractions, PCR amplification, sequencing and analysis

Isolated bacterial were inoculated in a medium without olive oil containing 1 g/l sodium acetate, 1 g/l sucrose and incubated in a rotary shaker at 60 °C for 48 h. Furthermore, bacteria were cultivated on a solid plate containing 1 g/l sodium acetate, 1 g/l sucrose and 15 g/l agar in order to obtain single pure colonies, and incubated at 60 °C for 24 h. DNA extraction was carried out according to the Bust n’ Grab protocol [23]. The quality of extracted DNA was evaluated using nano drop spectrophotometer readings (Thermo Scientific, Wilmington, DE). The 16S rDNA gene sequencing amplification were conduct using universal primer forward 27F (5′-AGAGTTTGATCMTGGCTCAG-3′) and primer reverse 1492R (5′-GGCTACCTTGTTACGACTT-3′). Thereafter, PCR product was purified using high pure PCR purification kit according to the manufacturer’s instructions. Nucleotide sequencing of the amplified fragments was performed with dideoxy chain termination method (SEQLAB, Germany). DNA sequences were analyzed using BLAST program (https://blast.ncbi.nlm.nih.gov/Blast.cgi) [24]. The phylogenetic tree was drawn using molecular evolutionary genetic analysis version 5.0 software (MEGA, Germany). A phylogenetic tree was constructed by using the neighbor-joining method and it was analyzed based on 16S rDNA gene sequences compared to available sequences in the GenBank [25].

Effect of different factors on lipase and biosurfactant stability

The effect of different parameters on lipase activity was measured by incubating the extracellular enzyme with p-Nitrophenyl Palmitate as substrate for 4 h. In order to study biosurfactant emulsification index values were incubated free cellular biosurfactant in mixture of 2 ml of heavy petroleum for 24 h.

Solvent purification lipase and biosurfactant

Lipase and biosurfactant production was carried out in same basal medium and after 72 h incubation at 60 °C. The culture broth was centrifuged at 13300×g for 10 min at room temperature. In order to achieve the best result, various precipitation methods such as ammonium sulphate with acetone [27] chilled ethanol [28] and isopropanol were studied. At the final step of each method, pellets were filtered through a sterile 0.45 μm millipore membrane. The mentioned precipitation methods for both lipase and biosurfactant were the same.

The precipitate was resuspended in 2 ml of 0.1 M Tris-HCl buffer at pH 10 and dialyzed against 0.1 M Tris buffer at pH 10 for 2 h at room temperature. The concentrated enzyme was loaded on a sephadex G-50 column (2 cm × 150 cm) according to manufacture instructions. The fraction containing protein was determined by specific activity. Purity of protein was evaluated by using SDS-PAGE gel as describe by Laemmli [29].

Washing test

Wb = Weight of oil before washing.

Statistical analysis

Experimental data were repeated at least three times. Results are reported as the mean standard deviation. The obtained data were used analyzed using repeated measure ANOVA and significant differences were determined using mauchly’s test of sphericity. The results with a p-value less than <0.05 were considered as significant differences. Statistically analysis was performed using IBM SPSS statistics version 22 software (IBM, USA).

Screening and isolation of lipolytic and biosurfactant microorganism

Detergent formulations processes are performed under extremely high pH and temperature along with other addictive. The structure of thermophlic enzymes from living microorganism in hot springs are naturally stable and active at high temperatures [30]. Thermophilic enzymes from this microorganism are function at highly alkaline conditions, salinity and other harsh conditions [31]. Although all isolated bacteria are not capable to become commercially available [32]. So, screening of new lipase and biosurfactant producing bacteria are still required [33].

Our sampling site was a hot spring with 60 °C and pH 8.6. Such extreme ecosystem is one of the places in where resistant bacterial groups to harsh conditions could be found. These bacteria might produce metabolites that are suitable for industrial applications. As the favorable cleaning condition for washing powders is at high temperature and alkaline pH, therefore, having the components with high stability to temperature and pH could enhance the cleaning efficiency of detergents when applied as additive in the formulations.

Eighteen strains were isolated from culture with pH 10.In this study, we observed significant clear zone around bacteria colony. The hemolytic assay is not the specific method for screening biosurfactant producing microorganism and has limitation. Therefore, the hemolytic assay should be used as a primary method and drop collapse test, oil spreading assay and emulsification assay methods were studied to determine biosurfactant production [34].Only six strains were positive in lipase and biosurfactant. One strain was chosen for further investigations base on the fact that it had the highest emulsification index (E24 = 62.2%), and lipase specific activity of 1.8 U/mg Protein and antimicrobial activity. The isolated strains showed the positive result for drop collapse assay, oil spreading assay. Same results were reported by Nalini et al. (2013) from Serratia rubidaea SNAU02 with positive blood hemolysis, drop collapse assay and emulsification index of 52.2% [35].

DNA extractions, PCR amplification, sequencing and analysis

According to 16S rDNA sequencing, biochemical and morphological tests (Table 2), the isolated strain MZV101 was identified to be closely related to Ochrobactrum intermedium with 99% homology via DNA BLAST in NCBI GenBank and it was recorded with the number KX619441access in NCBI. A phylogenetic tree of strain MZV101 based on 16S rDNA using neighbor-joining method is shown in Fig. 1 [25]. Ochrobactrum is a gram-negative, strictly aerobic, catalase and oxidase positive and usually single-cell [36]. O. intermedium 2745–2 was the first environmental strain isolated from formation water in China [33]. The optimum growth temperature for O. intermedium strains are 20 to 37 °C and optimum are pH 7–7.3 [37]. In this study, O. intermedium strain MZV101 was isolated from a hot spring at a temperature of 60 °C and pH of 8.6 for detergent application. Lipase from Pseudomonas sp. and Bacillus sp. are commonly used in detergents, also Candida and Chromobacterium [38]. They are reports regarding the production of biosurfactant from Ochrobactrum anthropi strain, Ochrobactrum sp. 1C, Ochrobactrum anthropi strain 2/3, Ochrobactrum sp. strain BS-206 (MTCC 5720) [2, 39–41]. However, Mishra et al. reported biosurfactant and lipase production from O. intermedium strain P2 [42].

Test	O.intermedium Strain MZV101	O.intermedium strain LMG3301
Growth at 37 °C	+	+
H2S production/Utilization of:
Glycine	+	+
D-Alanine	+	+
L-Aspartate	+	+
D-Arabinose	+	+
L- Arabinose	+	+
Mannitol	+	+
Sorbitol	+	+
D-Lyxose	+	+
D-Fructose	+	+
Mannitol	–	–
D- Sorbbitol	+	+
Glycerol	–	–
Sucrose	+	+
Hydrolysis of:
Gelatin	–	–
Urease test after 24 h	–	–
Urease test after 48 h	–	–
Utilization of:
Citrate	+	+
Sensitivity to antibiotics
Amoxicillin (25 mg)	R	R
Colistin (50 mg)	R	R
Chloramphenico (30 mg)	R	R
Tetracycline (30 mg)	R	R

Morphological, physiological and biochemical characteristics of O. intermedium strain MZV101 had comparison with O. intermedium strain LMG3301. Results are scored as (+) positive, (−) negative and (R) resistance. Data were obtained from velasco et al. study [36]

Effect of different factors on lipase and biosurfactant stability

Effect of pH

High pH could modify protein structure and consequently changes the enzyme activity. The optimum lipase bacteria activity reported to be over ranges of pH 5–9 [43]. Generally, laundering performed at alkaline conditions, thus alkaline lipase were prefer for laundry detergent [33]. So, enzyme function at high pH is essential in detergent formulation [44]. O. intermedium strain MZV101 lipase maintains its activity and stability at pH 5–13 with maximum activity at pH 9 at 37 °C (Fig. 2). Lipase enzyme O. intermedium strain MZV101 has 80% stability at pH 10–13 and minimum stability between pH 6–8. The enzyme also remains stable after 24 h in all indicated pH range at room temperature. Chen et al. have declare that alkaline lipase from Achetobacter radioresistens was stable in optimum pH 10.0 at 30 °C and as a good potential for detergent application [45]. This report is in agreement with our results. Accordingly, other studies revealed high relative activity at lower pH from our obtained data in this study. For instance, Golaki et al. (2015) have been reported 70% relative activity of lipase 3646 from thermophilic indigenous Cohnella sp. A01 was stable in the pH range of 7 to 9 and maximal activity was obtained at pH 8.5 [46].

Biosurfactant with high stable ability in the presence of alkaline condition is required for the detergent additive because pH of laundry is generally between ranges of 9.0–12.0 [47, 48]. Many reports have been revealed the important role of bacteria biosurfactant as laundry detergents additives [12, 47, 49]. High stability was observed from pH 10–13 (E₂₄ average = 65.58%), on the other hand, emulsification index values decreased from pH 5–6 (E₂₄ average = 31.84%) (Fig. 2).These results showed that emulsification index values are accompanied with pH. These results are in agreement with those by Shavandi et al. who reported that emulsification activity was sensitive to pH variations [50]. From our same species, Ferhat et al. reported that biosurfactant from Ochrobactrum sp. 1C the highest (E24 average = 95%) at pH 7 to 11 was observed, while emulsification values decrease to (E24 = 70.58%) at pH 4 and temperature 37 °C [39]. Bhattacharya et al. have declaim that biosurfactant from Ochrobactrum sp. C1 the highest emulsification index values (E24 = 69.42%) were observed at low pH 7.3 and temperature 36.4 °C [51].

Effect of temperature

Lipase enzyme with activity and stability at the wide range of temperature is favoring characteristic for detergent applications, at high temperatures due to their responses provides fewer microbial contamination threats, high solubility of substrates and low viscosity of the reaction elements [52]. At low temperature can accomplish with synthetic unstable compounds and improves oil elimination from fabric; consequently, reduces energy consumption [52–54]. According to test results, lipase of O. intermedium strain MZV101 had remained active and stable at temperature range of 4–90 °C (Fig. 3). The lipase of O. intermedium strain MZV101 remains 80% active at 70–90 °C and with an optimal activity at 60 °C for 1 h at pH 10. In contrast to other lipases, lipase enzyme of O. intermedium strain MZV101 remained active even after 4 h at 60 °C [5]. Similarly, 60% relative activity for lipase 36,464 from thermophilic indigenous Cohnella sp. A01 at 60 °C and pH 10 for 180 min has been reported [46]. Lipases from Bacillus megaterium AKG-1 and Acinetobacter sp. have reported to be in the optimum temperatures range of 50–60 °C [55, 56]. Recently, García-Silvera et al. have described that lipase stability from Serratia marcescens wild type and three mutant strains in temperature over range of 5 to 55 °C with an optimum in temperature 50 °C and stable at pH 6 to 10 with optimum pH 8 for 1 h were obtained for detergent formulation and biodiesel [52]. Comparably with our results, Bora et al. have mentioned that the lipase from Bacillus sp. DH4 with optimum activity at temperature 60 °C and pH 9 as an additive for detergent formulation [57]. Cherif et al. have explained a high alkaline lipase produced by Staphylococcus sp. strain ESW the optimum activity was at temperature 60 °C and pH 12 and it was an ideal choice in detergent formulations [24].

Nevertheless, its emulsification index values increased with temperatures between 4 °C (E24 = 66.25%) to 60 °C (E24 = 70.41%), and this value remain stable between temperatures of 70–90 °C (E24 average = 69.34%) (Fig. 3). According to results, biosurfactant of O. intermedium strain MZV101 at wide range of temperature (4–90 °C) had remained stable and had no significant effect on emulsification index. Ferhat et al. reported that biosurfactant produced by Brevibacterium sp. 7G, it emulsification index values remain stable between 20 and 100 °C [39]. Mukherjee reported that biosurfactant produced by Bacillus subtilis DM-03 and DM-04 have thermal stability at pH 7–12 and 80 °C for 60 min, also compatibility and stability with commercial laundry detergents [48]. Comparable with our data, Sajna et al. mentions that biosurfactant from Pseudozyma sp. NII 08165 as an additive for laundry detergent at pH 8–12 and temperature 80 °C was stable for 2 h [47]. It has been described that biosurfactant from Bacillus subtilis PF1with good stability at pH 6–11 and temperature 30–60 °C and emulsification index (E24 = 100%) at pH 10–11 and 30 °C as laundry detergent additive [12]. In contrast to our data, other studies found stable biosurfactant as detergent additive in low pH and temperature. For instance, Hirata et al. [59] have reported that glycolipid biosurfactant from Candida bombicola ATCC22214 was active at temperature 20 °C and pH 8.94 as biodegradable low-foaming surfactants with high detergency ability [58].

Solvent purification lipase and biosurfactant

Even though these results differ from some earlier studies, Ameri et al. found that 80% pre-chilled ethanol with 39.8% yield was the best method to precipitate thermoalkalophilic lipase from bacterial strain Bacillus atrophaeus FSHM2 [74], and also Raza et al. (2017) found that 80% ammonium sulfate with 5.3 fold was the most suitable method to precipitate the lipase from Staphylococcus aureus for detergent industry [75], we found that this method can completely inactive the lipase from O. intermedium strain MZV101.

Isopropanol was unable to precipitate biosurfactant O. intermedium strain MZV101.The best result (E₂₄ = 70.99% and yield = 49.32%) was obtained from ethanol and also good results were obtained with ammonium sulphate (E₂₄ = 60.11% and yield = 48.04%) and acetone (E₂₄ = 62.87% and yield = 38.21%) (Table 5). It has been reported that biosurfactant from Nocardia sp. L-417 was stable by extraction of ammonium sulphate fractionation and chilled acetone in two primary purification steps [68]. The best results obtained by cold ethanol precipitation (E₂₄ = 70.99% and yield = 50.32%). Same result was reported by Ramasamy et al. for cold ethanol precipitation of the biosurfactant produced by biosurfactant Ochrobactrum anthropi MP3 [76]. Our experiments corroborate with previous results Salleh et al. who explain that organic solvent extraction was found as the best giving and the highest recovery at 89.70% (w/v) purification method for biosurfactant produced by Pseudomonas aeruginosa USM AR-2. Additionally, they were obtained good results with ammonium sulphate/acetone method precipitation [77].

Both enzyme and biosurfactant purification were continued by short dialysis against 0.1 M Tris buffer pH 9 and insignificant purity of biosurfactant and lipase was observed (Table 5). At the final step of lipase purification, the following results were obtained after the investigation of suitable column: DEAE- Cellulose column with a recovery of 5.241%, 2.649 fold, and G-10 Sephadex column with a recovery of 7.082%, 3.038 fold. Finally, the best result with a recovery of 48.452% and 19.095 fold was obtained from G-50 Sephadex column. The single protein band molecular mass was estimated on SDS-PAGE to be 99.42 kDa (Fig. 4). Comparable with our results, Kanwar et al. (2006) have mentioned high molecular mass of approximately 103 kDa on SDS-PAGE for lipase from Bacillus coagulans MTCC-6375 [60]. Furthermore, Dosanjh et al. have reported higher molecular mass 112 kDa from Bacillus GK 8 [78]. In contrast, Kumar et al. have describe lower molecular mass 31 kDa from B. coagulans BTS-3 [79] and Huang et al. have published protein mass 62-kDa lipase from Geotrichum marinum [80].

Antimicrobial activity

Higher antimicrobial activity with diameter of 10–14 mm clear zone was observed against the Gram-negative bacteria. The highest inhibition of zone were obtained with Escherichia coli (ATCC 25922) (14 mm), Proteus vulgaris (ATCC 13315) (12 mm) and Salmonella typhi (PTCC 1609) (11 mm). Biosurfactant of O. intermedium strain MZV101 demonstrated a weak antimicrobial activity with 2–4 mm diameter of clear zone against Gram-positive bacteria, and also the same result was observed against fungi. Noparat et al. have reported high antimicrobial activity against Gram-positive and lower activity against Gram-negative of biosurfactant produced by Ochrobactrum anthropi strain 2/3 against several pathogens and this is in contrast to the results achieved in our study from biosurfactant of O. intermedium strain MZV101 [41]. The inhibition of zone biosurfactant by isolated bacteria O. intermedium strain MZV101 against S. aureus (ATCC 25923 s) and Pseudomonas aeruginosa (ATCC 85327) were recorded to be in respect 2 mm and 15 mm. Biosurfactant from Ochrobactrum sp. 1C has reported to obtained (11.1937 mm) inhibition zone with S. aureus (ATCC 9144) and (15.9468 mm) with P. aeruginosa (ATCC) [39].

Washing test

The washing test of the lipase and biosurfactant O. intermedium strain MZV101 isolated in the present study was evaluated by their ability to remove olive oil stain from cloth. The solution of 1% detergent and Tris buffer could only remove 16.11% and buffer solution alone had insignificant removal ability. At alkaline condition, fatty acids were more easily eliminate from fabric but they stay on fabric because they are unable to saponify by the alkaline solution. Thus, by addition of lipase enzyme they could remove easily from fabric [15]. With the combination of lipase, detergent and buffer, high oil removal of 76.34% was observed and also the presence of lipase O. intermedium strain MZV101 without detergent shows 67.63% olive oil removal. Thirunavukarasu et al. (2008) had reported that the percentage of olive oil removal lipase produced by Cryptococcus sp. S-2 with other detergents from fabric was higher than 21.1% [65]. Grbavčić et al. reported good oil stain removal with the addition of lipase from P. aeruginosa in washing formulation [11]. It has been demonstrated that combination of extracelluar lipase from Micrococcus sp. ML-1 with different commonly used detergents could enhance the removal of greasy stains from textile [84]. Also, lipase from Ralstonia pickettii has been reported in the presence of detergent improves the removal of oil by 24–27% over treatment with detergent alone [15].

An oil removal about 60.11% was obtained in a solution containing buffer solution and isolated biosurfactant and also less olive oil removal of 23% was observed in solution without biosurfactant O. intermedium strain MZV101. Recently, it has been reported that by additional of CLP biosurfactants from B. subtilis strain DM-04 could enhance 9–12% elimination of oily stain from fabric [48]. Additionally, Jain et al. have mentioned that biosurfactant from an alkaliphilic bacterium Klebsiella sp. strain RJ-03, it alone removed 80% oil and with various laundry detergents have enhanced oil removal to (17–20%) which improved the washing performance [85].

Biosurfactant by reducing surface tension along with other enzymes is able to remove better oily stains [7]. The best result of 82.33% oil removal was obtained in combination of Lipase, biosurfactant, detergent and buffer. Hence, from the results of washing test, it can be concluded that lipase and biosurfactant O. intermedium strain MZV101 with good oil removal from white cotton can use as addictive washing component in laundry detergents. Same results have reported by Bhange et al. which biosurfactant and enzyme along with other detergent component could improve the stain removal efficiency [12].