Lipolytic potential of gut isolates in silkworm Bombyx mori. L

doi:10.21203/rs.3.rs-7536610/v1

The current study describes that the bacteria isolated from the midgut of the silkworm Bombyx mori had the ability to degrade the oil form of lipids and could produce the lipolytic enzymes that hydrolyse the lipids into simpler compound form that could be uptake by the bacteria as their energy source (carbon source), which had been evidenced primarily by the Tributyrin and TWEEN 20 Agar plate assay method. Further, the six isolates were selected from the primary screening process and so the growth curve assays of Olive oil and Tributyrin Medium were performed, which showed that the isolates utilised the lipid substrate; therefore, the cell densities were increased gradually from 12 hours, 24 hours and 48 hours of Tributyrin Medium and quiet difference in the Olive oil Medium. The FTIR analysis and the HPLC analysis reported that the six isolates utilized and lysed the lipid substrate in the tributyrin and olive oil medium, which was confirmed by the peaks obtained from the six isolates compared with their respective controls. From all the reports, isolate Serratia spp was very active compared with the other five isolates, so the effect of the pH of isolate Serratia spp was tested on the phosphate buffer and tris buffer where the isolate Serratia spp were very active on the phosphate buffer. Among the six isolates, the most potent lipolytic isolate was Serratia spp.

Lipid degradation

Tributyrin

silkworm gut

Bombyx mori gut

Gut Serratia spp.

Gut Bacillus spp

Enzymes such as cellulolytic, proteolytic, and lipolytic enzymes that play a variety of roles in digestion are secreted by the microbiome, which has an impact on the growth and development of the host. In addition to producing antimicrobial chemicals that provide colonization resistance against invasive infections, the gut microflora of B. mori is linked to other physiological functions. Furthermore, the silkworm gut microbiota experienced significant alterations after becoming infected with a disease. The B. mori cytoplasmic polyhedrosis virus (BmCPV)-infected silkworms displayed an increase in Enterococcus and Staphylococcus abundances but a decrease in Delftia abundance (Sun et al. 2016). Compared with plant- or animal-derived enzymes, microbial enzymes are more valuable due to their wide range of catalytic activities, potential for large yields, genetic modification, constant supply, and quick growth on minimal media (Soma Prabha 2017). Lipase passes through many important processes, including trans-esterification, aminolysis, and esterification. (Javed et al. 2017). Lipase produced by the silkworm gut microbiota exhibits potent antiviral properties against BmNPV, demonstrating that digestive juice plays a significant role in BmNPV peroral infection. (Feng et al. 2019). Because of their greater activity and neutral or alkaline pH optimal values, bacterial lipase is believed to be more wide spread and suited to tolerate adverse industrial environments than fungal lipase. (Patel et al. 2016). Enzymes that catalyses the hydrolysis of triglyceryl into diacylglycerol, monoacylglycerol, free fatty acids, and glycerol are known as lipolytic enzymes or lipases. The most significant class of biocatalysts utilised in several biotechnological applications are microbial lipases. (Feder et al. 1992). However, only few investigations have demonstrated that Lepidoptera carry intestinal microbes. Over the years, the primary focus of rigorous scientific research has been on microorganisms' ability to synthesize useful metabolites. The presence of lipase-producing bacteria was checked, and culture conditions, including temperature, pH, agitation speed, incubation period, and substrate specificity, were optimised to produce extracellular lipase. To determine the maximal lipase activity, the effects of metal ions, carbon sources, and nitrogen sources were evaluated in further detail. Culture in Tributyrin agar was used to select the bacterial isolates (Sagar et al. 2013).The isolates used the lipid substrate, as established by growth curve tests of the Tributyrin and Olive Oil Medium. As a result, the cell densities were progressively raised from the 12 hours, 24 hours, and 48 hours of Tributyrin Medium with little variation in the Olive Oil Medium. The peaks identified in the isolated cultures supported the FTIR and HPLC analyses’ findings that the isolates absorbed and lysed the lipid substrate in the Tributyrin and Olive Oil medium. Long-chain acylglycerol hydrolysis and synthesis are catalysed by microbial lipases (triacylglycerol acyl-hydrolases, EC 3.1.1.3). They are now receiving a massive interest due to the quick advancement of enzyme technology (Andualema and Gessesse 2012).

Sample Collection

Fifth instar silkworm larvae (Bombyx mori) were collected from the Central Silk Board [CSB], Extension Centre, Vaniyampatti, Srivilliputtur, Virudhunagar District, Tamil Nadu. Freshly collected fifth instar larvae were fed sterile young tender mulberry leaves. Silkworms were surface-sterilized with 75% ethanol for 1 min and rinsed three times with distilled water before dissection. The dissection was performed over a sterile wax-moulded plate using a dissection kit, such as a sterile needle, forceps, and scissors. The dissection of silkworm was performed to collect the midgut aseptically and store it in phosphate buffer saline (PBS) with a pH range between 7.4 and 8, as the midgut of Bombyx mori was in a similar range. PBS was used to maintain the microflora in the midgut of silkworms, and the collected midgut were stored in a freezer for further processing.

Isolation of the Migut Microflora

The stored midgut was thawed and gently vortexed. Then, the supernatant or liquid presented above the midgut is transferred. Then, using the supernatant, serial dilutions were prepared (from 10^− 1 to10^− 7) using PBS (Phosphate Buffer Saline). Using the spread plate, patch plate, and streak plate techniques, dominant colonies were isolated and used for further analysis of lipase screening and oil degradation

Screening of Lipase and Lipolytic Microorganisms

The isolated dominant colonies were then screened for lipolytic activity. Thus, the primary screening method involved plating dominant isolates on media enriched with lipid substrate. Thus, a sensitive plate assay technique was followed. Therefore, three different lipid substrate-supplemented media were used: Tween 20 agar medium and Tributyrin agar medium. The compositions of the media whereas follows: (i) Tween 20 agar medium: nutrient broth, 1.3 g/100 mL; Tween 20- 1-2% and agar- 2g/100mL; (ii) trichlorid agar medium (Himedia TBA medium were used, where Tributyrin oil was supplemented at the range of 1–2%).

Growth Curve Assay of Lipase and Oil using Bacteria

The growth curve assay was performed for the dominant and primarily screened lipolytic colonies, where the colonies were inoculated into two different Erlenmeyer flasks containing inoculum medium consisting of 0.5% beef extract, 0.5% peptone, 0.5% sucrose, 0.3% NaCl, 0.2% K₂HPO₄ and incubated at 30^oC for 24 h on a rotary shaker at 200 rpm. After 24 h of incubation, the two-inoculum media were transferred into two other Erlenmeyer flasks containing production media consisting of 2.0% peptone, 0.5% sucrose, 0.1% (NH₄)₂SO₄, 0.1% MgSO_4, 0.2% K₂HPO_4, 1.0% olive oil and 2.0% peptone, 0.5% sucrose, 0.1% (NH₄)₂SO₄, 0.1% MgSO_4, 0.2% K₂HPO_4, 1.0% tributyrin oil; were incubated at 30^oC on a rotary shaker at 200 rpm. The samples were collected after 12, 24, and 48 h, and the cell density was measured against the cell-free control by optical density at 600 nm using UV-Visible spectrophotometry.

Effect of pH

A single colony of gut isolates was inoculated in two different buffers, namely 100 mM phosphate buffer (Na₂HPO₄, NaH₂PO₄, NaCl & Distilled water) and 100 mM Tris HCl buffer (Tris HCl and distilled water), at different pH levels, such as pH 4,5,6,7,8,9 to understand the growth of lipolytic colonies under adverse conditions. Therefore, its efficiency in degrading lipid substrates, such as oil, can be studied.

Lipolytic activity analysis by FTIR spectroscopy

Fourier transform infrared spectroscopy (FTIR) is a technique used to obtain the absorption or emission spectrum of a solid, liquid, or gas. In FTIR analysis, the isolated bacterial colonies were studied for the lipolytic activity on olive oil and tributyrin oil at different time periods of incubation, such as 12 h, 24 h, and 48 h.

HPLC Analysis

The isolated colonies were incubated in olive oil production medium. After 24 h of incubation, the production medium with the culture was centrifuged at 10,000 rpm for 15–20 min to settle down the cells at the bottom. After centrifugation, the supernatant was transferred, dissolved in an equal volume of methanol, and centrifuged as necessary. The solvent was then allowed to undergo high-performance liquid chromatography (HPLC).

Molecular Identification of Potent Lipolytic Isolates

Genomic DNA preparation

The sonicated gut suspension was subjected to serial plating. Microbial genomic DNA was extracted following the protocol described by Ausubel et al. 1994. The gut susppension was subjected to chemical and enzymatic rupture by adding 90µl of 10% SDS and 90µl lysosyme (20 mg/ml) (gently mixed). The tubes were incubated at 37°C for 1.5 h each tube was mixed with 150µl of 5M NaCl before the addition of 100µl of 5M NaCl containing 10% cetyltrimethyl-ammonium bromide. The samples were thoroughly mixed and incubated at 65°C for 3 min. DNA was extracted using phenol: chloroform: isoamyl alcohol (25:24:1[vol/vol/vol]). DNA was precipitated using 70% ethanol and recovered by centrifugation (Broderick et al. 2004). Pellets were resuspended in 20µl of TE buffer.

Template preparation for microbial fingerprinting analysis

The bacterial colonies picked up from the subcultured plates were inoculated into 1 ml of TSB broth and shaken for 3 h at 200 rpm and 37°C. After turbidity development, the tubes were centrifuged at 10,000 rpm for 10 min. The pellet obtained was subjected to treatment with 50 µl of colony lysis buffer (CLB) (1% Triton X-100, 20mM Tris HCl pH 8.0, 2mM EDTA pH 8.0). The mix was vortexed for a few seconds and heated at 65°C for 15mins, then 5 µl from each tube was transferred to fresh tubes as the template for microbial fingerprinting analysis.

BOX PCR amplification

BOX PCR was performed to identify the microbial footprints of BOX elements in specific strains. The reaction mixture contained 5 µl of 10X PCR buffer, 10 mM dNTP mixture (2.5mM each), 1.0 µM primer, 1.5 unit Taq DNA polymerase, and 50 ng template DNA. Amplification was performed with a final volume of 50 µl using a thermal cycler (ABI). The template was subjected to initial denaturation at 94°C for 4 min, final denaturation at 94°C for 1 min, primer annealing at 60°C for 2 min, and extension of the reaction primer was extended at 74°C for 2 min. The steps were repeated for 40 cycles, and the final extension was performed at 74°C for 5 min. PCR-amplified fragments were electrophoresed on 2% agarose gel and examined under UV light after staining with ethidium bromide.

ERIC PCR amplification

Enterobacterial repetitive intergenic consensus sequence (ERIC) primer amplification was used to identify significant diversity and compare the fingerprints of 16S rRNA genes in the gut community. A 25 µl reaction mixture comprises of 1.0 µM upstream primer Eric F and downstream primer Eric R, 10 PCR buffer, 2.0 µl 10 mM dNTP mixture (2.5 mM each), 1.5-unit thermostable Taq DNA polymerase, and 50 ng of template DNA. The template was subjected to initial denaturation at 94°C for 4 min and final denaturation at 94°C for 1 min, followed by primer annealing at 60°C for 2 min, and the reaction primer was extended at 74°C for 2 min. The steps were repeated for 40 cycles, and the final extension was performed at 74°C for 5 min. The amplified products were electrophoresed on 2% agarose gel and examined under UV light after staining with ethidium bromide.

16S rRNA amplification

To amplify 16S rRNA, the universal eubacterial primers 27F and 1492R were used. The reaction was performed under the following conditions using a 27F forward primer and 1492R reserve. The final volume of the mix was 25 µl contains 5 µl of 10X reaction buffer, 2.5 µl of 10 mM dNTP mixture, 1 µl of 10 µM forward and reverse primers, 2.5 units Taq polymerase, and 50mg template DNA. The initial denaturation at 94^ºC for 3 min was cycled for 35 reactions with denaturing the template for 30 sec at 94^ºC, annealing at 55^ºC for 1.5minute, the reaction was extended for 2.5 min at 72^ºC and the reaction was finally extended for 10 min at 72^ºC.

Isolation of the midgut microflora

From the steak plate, patch plate, and pure plate techniques, 16 dominant colonies were isolated based on their morphological identification noted as A,B,C,D,E,F,G,H,I,J,K,L,M,N, O. and X (Fig. 1).

Screening of Lipase and Lipolytic Microorganisms

Among the 16 isolated colonies, most of the colonies showed positive results in lipolytic activity in primary screening i.e., sensitive plate assay method.

(i) Tween 20 Agar Plate Method

In this plate method, colonies A, E, Staphylococcus spp, Pseudomonas spp, I, Serratia spp, L, M, and Burkholderia spp showed positive results by producing zone of clearance around the colonies on the tween 20 agar plate (Fig. 2.0). Other colonies, such as B, C, D, G, J, O, and X (Table.1) were showed negative results by neither producing any zone of clearance nor the zone of inhibition around the colonies (Table.2; Fig. 2.1).

(ii) Tributyrin Agar Plate Method

In this plate method, colonies A, B, C, D, E, Staphylococcus spp, Bacillus cereus, Pseudomonas spp, I, Bacillus spp, Serratia spp, M, Burkholderia spp, and X showed positive results by producing zone of clearance around the colonies on the tributyrin agar plate, and other remaining colonies showed negative results by neither producing any zone of clearance nor the zone of inhibition around the colonies. By comparing the results of the Tween 20 and tributyrin agar plate methods, the isolates Staphylococcus spp, Bacillus cereus, Pseudomonas spp, Bacillus spp, Serratia spp, and Burkholderia spp were selected for further processing, and their zone of clearance were measured (Table. 2; Fig. 2.2). The tween 20 plate pertaining the efficient isolates were treated with Rhodomine B, which also ensured the lipolytic activity; notably, isolate 10 Serratia spp showed a remarkable notification in the plate assay, and same was compared with the control (Fig. 2.3)

Growth Curve Assay of Lipase and Oil using Bacteria

In the olive oil medium, cell populations were examined by measuring the bacterial density through UV-Vis Spectrophotometer. The maximum cell population was observed on the 48 hours of incubation, except for the isolate Bacillus spp. The Isolates Serratia spp, Burkholderia spp, and Bacillus cereus showed a gradual increase in cell density from 12 hours to 48 hours, whereas the isolates Staphylococcus spp and Pseudomonas spp revealed that the bacterial cell density had a fluctuation at 24th hours but the cell density reached maximum at 48th hours. Thus, the cell density had some fluctuation at 24th hours as on comparing with 12th and 24th hours (cell densities were decreased). The Isolate Bacillus spp showed maximum density at 24 hours and decreased after 24 hours (Table. 3; Fig. 3.0 & 3.1).

Effect of pH

The isolate Serratia spp showed the maximum growth at 12 hours and then their activity and growth were retarded after 12 hours and became null at pH 6 during 48 hours in phosphate buffer; which represented that it had maximum functional value at pH 5, 7, 8, and 9 (Table.4; Fig. 4). Similarly, in Tris buffer, the isolate Serratia spp showed maximum activity of growth at 12 hours and started to retard after 24 hours. The activity became null at pH 7. The results indicated that the isolate Serratia spp had an optimum pH range of 4, 5, and 8 (Table.4; Fig. 4.1).

(Table.4; Fig. 4.1).

The potent isolates belonged to Serratia spp., and Bacillus spp. The lipolytic activities of these compounds were observed by FTIR and HPLC analysis. These species were dominant among the isolated bacteria from the midgut of the lepidopteran silkworm B. mori. FTIR analysis was utilised to detect the characteristic peaks and functional groups. FTIR was a vibrational spectroscopic technique where the spectral bands in the FTIR were molecule specific and contributed direct information about the biochemical composition. FTIR peaks were relatively narrow and in many cases were associated with vibration of particular chemical compound (or a single functional group) in the molecule from control 1 (Tributyrin medium), the functional groups such as amine, hydrate, aromatic acid, and alcohol were detected through FTIR. The functional groups amine, alcohol, aromatic acid, and hydrate were observed from the FTIR analysis of the six isolates Staphylococcus spp, Bacillus cereus, Pseudomonas spp, Bacillus spp, Serratia spp, and Burkholderia spp, which explained that the functional group on the control and the six isolate samples showed the same functional group that may insist, that the tributyrin lipid substrate may dissociate into simpler compounds. The functional groups observed on the control and sample were the same, so it may not be insist that the six isolate didn’t utilise or lysed by the lipid substrate as the control composed of other nutrient content, and the peaks observed from the samples and the control also differed. Thus, the six isolates may have lysed the lipid substrate supplemented in the nutrient medium. From control 2 (Olive oil medium free of bacteria), functional groups such as hydrate, alcohol, and aromatic compounds were observed by FTIR analysis. The six isolate samples had the following functional groups: hydrate, amine, alcohol, esters, aromatic compounds, Benzene derivatives, Carboxylic Acid, Alkane, Alkyne, Alkene and Oxime. This explains why the six isolates were actively used the lipid substrate olive oil. This denoted that the six isolates had potential for lipolytic activity and were potent lipolytic microorganisms. From the control and samples of 1 (Tributyrin medium) and 2 (Olive Oil Medium), the isolates Serratia spp, Pseudomonas spp, Staphylococcus spp, and Burkholderia spp were effective isolates that could able to utilise the lipid substrate (Table. 5).

In this current study the lipolytic bacteria were isolated from the gut of the silkworm Bombyx mori and the lipolytic bacteria were isolated through the primary screening using the Tributyrin and Tween 20 Agar plate assay methods. The zone of clearance observed using the tributyrin and tween20 agar plate assay method indicated that the bacteria utilised the lipid substrates tributyrin and tween20 as their nutritional source. The isolated bacteria Staphylococcus spp, Pseudomonas spp, Staphylococcus spp, and Burkholderia spp showed that they could utilize both the substrate, whereas Bacillus cereus and Bacillus spp showed that they could uptake the substrate tributyrin alone. Serratia spp was a potent lipolytic isolate that demonstrated high efficiency in lipid degradation in the profile of all kinetics studies performed.

This study discusses the potential of various bacterial isolates found in the stomach of B. mori to produce lipolytic enzymes. Swathiga et al. (2019) found that bacterial isolates from the same family of silkworms, Bacillus and Pseudomonas, had strong lipolytic activity in the stomach of the silkworm, another wild silkworm that is common in the same habitat. In addition, a metadata study by Pinto-Tomás et al. (2018) revealed that bacterial symbionts from the Bacillaceae and Pseudomonadaceae families predominate the screened lepidopteran species by > 60%, with bacteria from the genus level dominating (Pseudomonas spp, Bacillus spp). Bacterial associations in insect guts have been shown to have a significant impact on the physiology of their hosts (Wong et al. 2011). It was discovered that a lipolytic Bacillus spp. isolated from the silkworm B. mori has antiviral activity against the nucleopolyhedrovirus (Liu et al. 2018).These studies imply that, in addition to helping their hosts with food digestion and nutritional provisioning, bacterial symbionts from these genera may be performing distinct basic roles in their hosts. The previous investigations suggests that 71% of the lipolytic gut bacterial isolates belonged to the genus Bacillus, which dominated the group. According to Grillo et al. (2007), triacylglycerides are hydrolysed in the midgut, and the byproducts of this digestion are absorbed in the gut lumen and used for the subsequent production of additional complex lipids. Long-chain lipids, such as linoleic and linoleic acid, are necessary for lepidopteran insects. These lipids are obtained from the diet by lipases secreted in the midgut lumen, and their absence may result in incomplete pupa development, failure to close, and wing deformities (Christeller et al. 2011).

According to the hypothesis of microecology, insects do not have a full enzyme system and therefore require numerous types of enzymes that gut bacteria supply for food digestion, nutrient absorption, and biological metabolism. (Feng et al. 2011).The lipase-producing bacteria medium had a pH of almost 10.0, which is comparable to that of the silkworm stomach. This implied that the isolates had similar intestinal environments in which lipase was generated. This shows that silkworm cells do not produce all of the gut lipase; recent research has found that the gut bacteria of velvet bean caterpillars release some of their gut proteases (Visôtto et al. 2009). The characteristics of these bacteria still need to be investigated in vivo, even though the maximal bacterial density and lipase activity were assessed at approximately 48 h in vitro.

Lipase plays a potential role in the degradation of fats, oils, and other forms of lipids (Patel et al. 2016). In this study, lipolytic bacteria were isolated from the gut of the silkworm Bombyx mori and the lipolytic bacteria were isolated through primary screening using the Tributyrin and Tween 20 Agar plate assay methods. The zone of clearance observed using the tributyrin and Tween20 agar plate assay method indicated that the bacteria used the lipid substrates Tributyrin and Tween20 as their nutritional sources. The isolated bacteria Staphylococcus spp, Pseudomonas spp, Staphylococcus spp, and Burkholderia spp showed that they could utilise both substrates, whereas Bacillus cereus and Bacillus spp showed that they could uptake the substrate tributyrin alone.

Funding

All authors declare that no funding was obtained from any sources.

Declaration of Competing Interests

All the authors declare that there is no Competing Interest and conflicts.

Author Contribution

Dr. JP and Dr. MK designed the study, Dr. JP, Dr. SM and Dr. KM executed the experiments, analysed the results and drafted the MS, Dr. JP and Dr. MK reviewed the MS and submitted the MS.

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Table.1 Lipase producing Microorganism collected from the gut of Silkworm

S.No.	Colonies	Tributyrin agar plate	Tween 20 agar plates
1.	A	Positive	Positive
2.	B	Positive	Negative
3.	C	Positive	Negative
4.	D	Negative	Negative
5.	E	Positive	Positive
6.	Staphylococcus spp	Positive	Negative
7.	Bacillus cereus	Positive	Positive
8.	Pseudomonas spp	Positive	Positive
9.	I	Negative	Negative
10.	Bacillus spp	Positive	Negative
11.	Serratia spp	Positive	Positive
12.	L	Negative	Positive
13.	M	Positive	Positive
14.	Burkholderia spp	Positive	Positive
15.	O	Negative	Negative
16.	X	Positive	Negative

Table.2 Lipolytic activity of selected isolates on TBA and Tween 20

S. No.	Name of the Bacterial Isolates	TBA (Zone of Clearance in mm)	Tween 20 (Zone of Clearance in mm)
1.	Staphylococcus spp	12 mm	-
2.	Bacillus cereus	13 mm	22 mm
3.	Pseudomonas spp	10.5 mm	5 mm
4.	Bacillus spp	16 mm	-
5.	Serratia spp	10 mm	11 mm
6.	Burkholderia spp	12 mm	10 mm

Table.3 Growth Curve Assay of Tributyrin Oil and Olive Oil

Tributyrin Oil
Name	12 Hours	24 Hours	48 Hours
Control (Uninoculated)	0	0	0
Staphylococcus spp	0.6013	0.6722	0.7726
Bacillus cereus	0.2828	0.3559	0.4418
Pseudomonas spp	0.6957	0.8085	0.8715
Bacillus spp	0.4502	0.52	0.6778
Serratia spp	0.6987	0.751	0.9648
Burkholderia spp	0.4116	0.4667	0.6047
Olive Oil
Control (Uninoculated)	0	0	0
Staphylococcus spp	2.052	1.886	2.488
Bacillus cereus	1.864	1.968	2.315
Pseudomonas spp	1.77	1.742	2.024
Bacillus spp	1.728	1.735	1.705
Serratia spp	1.864	1.981	2.12
Burkholderia spp	1.725	1.907	2.318

Table.4 Effect of pH- Phosphate Buffer and Tris Buffer

*Isolate Serratia spp* in phosphate buffer**
S.No.	Different pH ranges	12 Hours	24 Hours	48 Hours
1.	4	0.1	0.1072	0.003
2.	5	0.124	0.119	0.029
3.	6	0.154	0.0824	0
4.	7	0.157	0.0916	0.011
5.	8	0.128	0.0834	0.008
6.	9	0.155	0.0843	0.022
Isolate Serratia spp in Tris Buffer
1.	4	0.128	0.09	0.013
2.	5	0.093	0.0739	0.006
3.	6	0.107	0.0741	0.002
4.	7	0.111	0.0784	0
5.	8	0.1	0.0815	0.009
6.	9	0.112	0.0788	0.003

Lipolytic potential of gut isolates in silkworm Bombyx mori. L

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Abstract

Figures

INTRODUCTION

MATERIALS AND METHODS

Sample Collection

Isolation of the Migut Microflora

Screening of Lipase and Lipolytic Microorganisms

Growth Curve Assay of Lipase and Oil using Bacteria

Effect of pH

Lipolytic activity analysis by FTIR spectroscopy

HPLC Analysis

Molecular Identification of Potent Lipolytic Isolates

Genomic DNA preparation

Template preparation for microbial fingerprinting analysis

BOX PCR amplification

ERIC PCR amplification

RESULTS

Isolation of the midgut microflora

Screening of Lipase and Lipolytic Microorganisms

Growth Curve Assay of Lipase and Oil using Bacteria

Effect of pH

DISCUSSION

Declarations

References

Tables

Supplementary Files

Status:

Version 1