The Production Of Biodetergents By Bacteria

Published Date: 02 Nov 2017

Jayashree,C. Ravi Chandra, K.V.Osborne W.J.*

School of Biosciences and Technology, VIT University, Vellore-632014, Tamil Nadu, India

Corresponding Author: [email protected] Contact: +919894204309

Abstract

Biosurfactants are amphiphillic compounds produced by various bacteria and fungi which reduce surface and interfacial tension. These microorganisms are capable of using different substrates such as glycerol, mannitol, fructose, glucose, n-paraffins and vegetable oils to produce biosurfactants. The various bacterial biosurfactants includes glycolipids, cellobiose lipids, polyol lipids, sophorolipids, rhamnolipids. In our study, bacterial cultures were isolated from sewage water and were screened for its ability to produce biosurfactants. The isolate VIT-JR1 and VIT-JR2 were found to be effective in removing stain. Further, the biosurfactants were extracted from the broth and was identified using GCMS and the compound was identified as fucose and erythritol which showed that the strain is an effective resource for the production mannosyl erythritol. The effective biosurfactant producing microbe isolated was characterized by morphological and biochemical methods. The isolate had 99% similarity to Enterobacter cloacae. This study proves that the surfactants produced possess greater commercial value.

Keywords: Biosurfactants, Glycolipids, GCMS, Fucose, Erythritol.

Introduction

Biosurfactants are compounds that are produced extracellularly or as part of the cell membrane by microorganisms like bacteria, yeast and fungi and contain glycosidic linkage with the reducing end of the hydrophobic and hydrophilic moieties that reduce surface tension and interfacial tension between individual acid is involved in ester formation (Karnath et al 1999).

Some of the bacteria capable of producing biosurfactants are Candida tropicalis, Pseudomonas auerginosa, Pseudomonas fluorescence, Brevibacterium casei, Flavobacterium aquatile (Cassidy and Hudak 2000). The bacterial genus Pseudomonas is capable of using different substrates, such as C11 and C12 alkanes, pyruvate, citrate, glucose, maltose, sucrose (Robert et al 1989). These biosurfactants have the properties of reducing the surface tension, act as stabilizers and emulsifiers, and are generally biodegradable (Mulligan and Gibbs 1993). They have some distinct advantages over the commercially available detergents including high specificity, biodegradability and biocompatibility (Cooper 1986).

In recent years, extensive studies have been carried out in the area of biosurfactants due to their potential use in various areas, such as the pharmaceutical industry, food industry, agriculture, petroleum industry and the paper and pulp industry. Research in this area is of foremost importance, keeping in mind the present concern regarding protection of the environment (Garcia 1992).

Biosurfactants can be divided into two classes: low-molecular-weight molecules, which are used to lower the surface tension, and high-molecular-weight polymers, that are more effective as emulsifying and stabilizing agents. The classes of low-weight surfactants include glycolipids, lipopeptides and phospholipids, whereas high-molecular-weight ones include particulate and polymeric surfactants (Nitschke 2007). They can be grouped as glycolipids, phospholipids, fatty acids, lipopeptides, neutral lipids (Bierman et al 1987). These compounds are either anionic or neutral. Only those containing amine groups are cationic. The hydrophobic part of the molecule can be long-chain fatty acids, α- alkyl-α-hydroxy fatty acids. The hydrophilic part can be an amino acid, carbohydrate, phosphate or alcohol (Lang and Wagner 1987).

The main drawback with commercial application of biosurfactants is that their large-scale production is not economical. To overcome this problem and to compete with the commercially available synthetic surfactants, an effective microorganism and an inexpensive substrate have to be developed for biosurfactant production. Agro-industrial wastes can be considered as promising and cheap source of substrates for biosurfactant production, which could alleviate the industrial waste management problems (Maneerat and Songklanakarin 2005).

The aim of this study was to isolate bacterial cultures from sewage water for their ability to produce biosurfactants. The isolate VIT-JR1 and VIT-JR2 were found to be effective cultures capable of producing biosurfactants. Further, the biosurfactants produced were characterized by GC-MS analysis. The culture VIT-JR2 was identified by 16S rRNA method.

Materials and methods

Sample location and sample collection

The waste water sample was collected from the secondary treatment plant at VIT University. The sample was collected in a sterile screw capped bottle and was processed immediately in the laboratory for the isolation of biosurfactant producing bacteria.

Isolation of the microorganism

The sample obtained was serially diluted and was plated onto nutrient agar plates. The inoculated plates were incubated at 30°C for 48 h. After incubation the colonies obtained were purified and was maintained in glycerol stock (Borjana et al 2002).

Inoculum preparation for screening of biosurfactant

The isolate was cultivated in an erlenmeyer flask containing 50 ml of nutrient broth and was incubated at 30°C and at 150 rpm for 24 h. Cells were harvested by centrifugation at 5000 rpm for 20 minutes. The centrifuged microbial mass was suspended in a Minimal salt medium (MSM) with the following composition (g/L): NH4Cl, 1.0; KH2PO4, 3.0; MgSO4.7H20, 0.2. The pH was adjusted to 7.0 with a solution of KOH (1N). Finally, 1% v/v of glycerol was added to the medium as a carbon source. The flask was incubated at 30 °C on a rotary incubator shaker at 200 rpm, for 24 h (Venkata and Karanth 1989).

Screening of bacterial cultures for biosurfactant production

For screening of the bacterial cultures for biosurfactant production, the above bacterial culture medium was centrifuged at 7000 rpm for 20 min and the supernatant was collected. Small pieces of cloth stained with 50µl of crystal violet were dipped into the supernatant and centrifuged again at 800 rpm for 15 min. Among the three isolates, stain removal activity was seen for two of the strains. These two strains were selected for further biosurfactant production process.

Morphological and biochemical characterization

Effective isolates were characterized morphologically by gram staining and hanging drop and biochemically by oxidase test, catalase test, indole test, methyl red test, Voges Proskauer test and citrate utilization test (Cappucino and Sherman 1992).

Production of biosurfactants

Cells of two effective cultures strains were harvested by centrifugation and were taken as inoculum for further fermentation process for the production of biosurfactants. The production of biosurfactants was performed in 500 ml Erlenmeyer flasks containing 100 ml MSM (with same composition as described above) and 5 % v/v glycerol as the sole carbon source. The pH was adjusted to 7.0 with a solution of KOH (1N). The medium was inoculated with the cells that were harvested from the MSM medium with 1% v/v glycerol. The flasks were incubated in a rotary incubator shaker at 200 rpm and 30 °C for 7 days (Venkata and Karanth 1989).

Extraction and purification of biosurfactants

For the extraction of biosurfactants produced by the isolates, the 7 day fermentation culture medium was taken and centrifuged for 20 min at 7000 rpm. The supernatant was collected. Two volumes of diethyl ether : methanol (1:1, v/v) was added to the supernatant and shaken for 30 min for extraction of biosurfactants. The mixture was evaporated to dryness. Finally the residue was dissolved in methanol (Venkata and Karanth 1989).

GC-MS analysis of biosurfactants

The purified biosurfactant was separated and subjected to GC-MS analysis. GC-MS analysis was performed using Perkin Elmer GC model (30 m × 0.25 mm ×0.25 µm) Clarus 680 (Mass spectrometer Clarus 600 EI). The Clarus 680 GC used purified helium as the carrier gas, at a constant flow rate of 1 mL/min. One microliter of samples were injected and oven temperature was programmed from 60°C to 300°C for 2 mins at the rate of 10°C/min and then isothermally held for 6 min until the analysis was completed (Noh et al 2012).

16S rRNA sequencing

The bacterial strains were characterized using the primers 27 F (5’-AGAGTTTGATCCTGGCTCAG-3’) and 1492 R (5’-GGTTAACCTTGTTACGACTT-3’). DNA was extracted from the cells and 16S rRNA sequence was determined by the fluorescent dye terminator method using the sequencing kit (ABI Prism big dye terminator cycle sequencing ready reaction kit v3.1). Products were run on ABI-13730 in to L-capillary DNA sequencer (ABI-13730 XL). Capillary DNA sequencer (ABI Prism 310 genetic analyser Tokyo, Japan). The aligned sequences were computed using CLUSTAL W software and sequence homologies were determined using BLAST search to create and evolutionary distance matrix (Poongumylali et al 2008)

Results and Discussions:

Isolation of microorganisms

After incubation, three different distinguished colonies were isolated based on their variation in colony morphology. These isolates were streaked to obtain pure colonies (Fig.1).

Fig.1: Three distinguished colonies isolated.

Screening for biosurfactant production

The cloth pieces were stained with crystal violet and were treated with the supernatant of the bacterial culture medium (Fig.2. (a) & Fig.2.(b)). Among the three bacterial isolates obtained VIT-JR1 & VIT-JR2 were found to be efficient in removal of stain from cloth. These were taken for further biosurfactant production. Thavasi et al 2008, Priya et al 2009, Vandana et al 2012, Anandaraj et al 2010, previously has reported that the cultures obtained from oil spilled soil were also capable of producing biosurfactants.

IMG_20130307_145836 IMG_20130307_152243Fig.2(a): Crystal Violet stained cloth Fig.2(b): Stain removed from cloth

Morphological and biochemical characterization

The results of the morphological and biochemical tests are shown in Table 1. From the results of the biochemical tests, VIT-JR1 was found to be Pseudomonas sp (Table.1). Further 16S rRNA sequencing was performed for VIT-JR2 and it was found to be Enterobacter cloacae.

Table.1: Biochemical characterization of screened isolates

Tests

VIT-JR1

VIT-JR2

Indole

Negative

Negative

Methyl Red

Negative

Negative

Voges-Proskauer

Negative

Positive

Catalase

Negative

Positive

Oxidase

Positive

Negative

Triple Sugar Iron

K/K

K/A

Simmon Citrate Agar

Positive

Positive

Production of biosurfactants

The viscous yellowish product and the white powder product obtained from the extraction and purification processes were defined as a crude biosurfactant and crude bioemulsifier, respectively. These were further analysed with GC-MS to identify the components present. Sarin et al., 2008, has reported production of biosurfactant/bioemulsifier by the isolate Enterobacter cloacae LK5 obtained from oil contaminated soil.

Confirmation of isolates as biodetergents

The cloth pieces were again stained with crystal violet and were treated with the supernatant of the bacterial culture medium VIT-JR1 and VIT-JR2. The stains on the clothes were found to be completely removed after this treatment which confirmed that the bacterial isolates were efficient in removal of stain from cloth.

GC-MS analysis

GC-MS analysis was used to identify the biosurfactant in the samples. Many peaks were obtained in the chromatogram. The chromatogram for VIT-JR2 is shown in Fig.3 and the components that have been identified in the sample are listed in Table 4. Referring to that, it can be seen that in the sample fucose and erythritol were present in the highest concentration.

Fig.3. Chromatogram for VIT-JR2

Table 4. Compounds detected in the sample by GC-MS

No.

Molecular weight

Compound

Molecular formula

1.

164.16

Fucose

C6H12O5

2.

122.00

Erythritol

C4H10O4

3.

164.00

Beta L- arabinopyranoside

C6H12O5

4.

146.00

Allyl 2-sulfanaylpropanoate

C6H10O2S

5.

152.00

Arabinitol

C5H12O5

6.

182.00

Mannitol

C6H14O6

Enterobacter cloacae is a Gram-negative, motile, facultative bacteria that produces EPS that are uncommon in most bacteria (Iyer et al 2005). Previously, it was reported that in a nutrient medium containing glycerol by product as the sole carbon source, an Enterobacter strain produced an exopolysaccharide (EPS) composed of glucose, galactose and fucose (Alves et al 2010). Fucose-containing polysaccharides have thickening, emulsifying and/or film-forming properties. They have increased market value because fucose is one of the rare sugars, which is difficult to obtain and has many applications that include active components in cosmetics and pharmaceuticals (Vanhooren & Vandamme 1999).

Erythritol was accumulated in the medium which confirmed that the strain was an effective resource for the production of Mannosyl erythritol. Mannosyl erythritol has been reported as an active biosurfactant by Morita et al 2011. It is a glycolipid type biosurfactant which shows extraordinary interfacial properties and efficient biochemical actions. It has erythritol as a hydrophilic headgroup and fatty acids as the hydrophobic chain (Morita et al 2011). They are highly biodegradable and have mild production conditions. Mannosyl erythritol finds a wide range of applications in the food, cosmetic, and pharmaceutical industries, etc. (Tomotake et al 2009)

Identification by 16S rRNA technique:

G:\JR.bmp

Fig. 4 Phylogenetic tree based on 16S rRNA gene sequence comparison showing the position of strain VIT-JR2 and related species.

The biosurfactant producing bacteria was identified using 16S rRNA technique. Molecular characterization of the biosurfactant producing bacteria showed 99% similarity towards Enterobacter cloacae (Fig. 4).

Conclusion:

The strain VIT-JR1 was identified by morphological and biochemical tests as Pseudomonas sp. The strain VIT-JR2 was identified by 16S rRNA technique as Enterobacter cloacae. The strains VIT-JR1 and VIT-JR2 were found to use cheap sources like glycerol to produce biosurfactants like fucose and mannosyl erythreitol. Fucose and mannosyl erythritol find wide range of applications in the food, cosmetics and pharmaceutical industries. Further, the biosurfactants produced efficently removed the stain from cloth. Hence, the present study proves that the bacterial strain obtained could be effectively used commercially for the removal of stains.

Acknowledgement:

The authors wish to thank the lab members of VIT for their contribution.