Isolation and characterization of biosurfactant-producing microbes isolated from the gastrointestinal system of broiler birds fed a commercial diet

Figures & data

Table 1. Average colony forming units per milliliter for each GIT section.

GIT section	Mean cfu/mL
Crop	1.E + 10^a
Gizzard	1.E + 10^a
Small intestines	1.E + 09^b
Large intestines	5.E + 09^c
Grand mean	8.E + 09
p Value	<0.05

Rows with the same superscript are statistically insignificant while a different superscript means statistically different means at 5% significance level.

Figure 1. Isolation of potential biosurfactant producing microbes using hemolysis test.

Table 2. Distribution of microbes showing clearance zones in sheep blood agar.

GIT region	Number of microbes showing clear zones	Average clearance zone (mm)
Crop	4	3.1^a
Gizzard	7	2.6^a
Small intestines	24	5.6^b
Large intestines	32	4.7^b
Grand mean		4.0
p Value		0.001

Rows with the same superscript are statistically insignificant while a different superscript means statistically different means at 5% significance level.

Table 3. Phenotypic characteristics of bacterial colonies subcultured from broiler birds’ GIT extracted samples.

GIT region	Description
Crop	Colonies were creamish in color and of fine texture They had smooth edges
Gizzard	Colonies were light-yellowish in color Edges were smooth
Small intestines	Colonies were clustered with some showing slow and rapid growth patterns Whitish color with fine structure was noted
Large intestines	Colonies showed reduced growth rate, Filamentous edges were noted Some were whitish in color with some creamish colors observed

Figure 2. Appearance of clearance zones on oil spread technique.

Table 4. Oil spread test clearance zones and average emulsification index (E₂₄) for biosurfactants from different sections of broiler GIT.

GIT region	Average clearance zone (mm)	Av. E24 index (%)
Crop	6	26^a
Gizzard	0	0
Small intestines	8.4	39^b
Large intestines	6.5	26^a
Grand mean	5.2	30.3
p Value	<0.05	<0.05

Rows with the same superscript are statistically insignificant while a different superscript means statistically different means at 5% significance level.

Figure 3. Dendogram of community relatedness of forty-nine bacterial strains based on the Jaccard’s coefficient.

Table 5. Major phytochemical compounds identified in biosurfactant GCMS analysis.

Compound name	Formula	Retention time	Area	Match score	CAS#
Decane	C10H22	5.672	1,394,666	90.6	124-18-5
DECANE	C10H22	5.672	1,256,257	90.9	124-18-5
Octadecane	C18H38	7.910	1,169,565	97.4	593-45-3
(4′-Fluorophenyl) furane-2-carboxylate	C11H7FO3	30.675	106,593	92.0	2000216-10-6
1,7-Di(2′-methoxyphenyl)-4-nitroheptan-1,7-dione	C21H23NO6	33.151	111,992	91.3	2000730-87-9

Figure 4. A comparison of FTIR spectroscopy of Pseudomonas aeruginosa (bottom) and crude endogenous biosurfactant (top) extracted from E. coli strain. NB. Pseudomonas aeruginosa adapted from Khademolhosseini et al.²¹

Table 6. FTIR peaks identified in Experimental biosurfactant.

No.	Wavenumber cm^−1		Compounds responsible for the peak (Assignments)	Possible nutrient type
	Experiment	Literature
1	2069.28	2100–1800	Carbonyl compound, transition metal carbonyls	carbohydrate
		2150–1990	Isothiocyanate (-NCS) representing nitrogen multiple and accumulated double bond compound
2	1647.00	1680–1620	Olefinic (alkene) (Alkenyl C = C stretch),	Protein (Amide I)
		1650–1590/1650–1550	Ether and oxy compound (primary/secondary amine, NH bend)
		1680–1630/1650–1600	Carbonyl compound (amide, quinone or conjugated ketone)
		1690–1590	Nitrogen multiple and cumulated double bond compound (open-chain imino (–C = N–))
3	1542.24	1560–1540/1555–1485	Nitrogen-oxy compound (aliphatic nitro compounds or aromatic nitro compounds)	Protein (Amide II)
4	1403.52	1420–1370	Sulfur-oxy compounds (organic sulfates)	Protein and collagen
		1420–1300	Carbonyl compound (carboxylate –carboxylic acid salt)
		1410–1310	Alcohol and hydroxy compound (phenol or tertiary alcohol, OH bend)
5	1225.50	1300–700	Saturated aliphatic (alkene/alkyl) (skeletal C-C vibrations)	Alkene
		1240–1190	Simple hetero-oxy compounds (phosphorus-oxy compounds –Aromatic phosphates (P-O-C stretch))
6	1076.08	1150–1000	Aliphatic organohalogen compound (aliphatic fluoro compounds, C–F stretch)	Organic compound
		1150–1050/1140–1070	Ether and oxy compound (alkyl-substituted ether, C–O stretch or cyclic ethers, large rings, C–O stretch)
		1090–1020	Primary amine, C–N stretch
		1100–1000/1100–900	Common inorganic ions (phosphate ion or silicate ion)
7	604.61	600–608	CH out of plane bending vibrations	Organic material

Synthesized from Nandiyanto et al.²⁹

Table 7. Small intestine extracted biosurfactant sample radio receptor assay output.

Antibiotic test	Concentration (cpm)	Control point (cpm)
Tetracyclines^a	274	1023
Beta lactams	1542	1036
Sulfa drugs^a	189	1163
Amphenicols	584	406

^aPositive result. Result reading less than control point result is positive whereas result reading greater than control point, result is negative.

Table 8. Observed broiler GIT derived biosurfactants antimicrobial activity at 5% (m/v).

Biosurfactants’ sample source	Clearance effect
Crop	+
Small intestines	+++
Large intestines	++
Antibiotic (Terranox)	+++
Distilled water	–

Key: +: reduced growth of microbes; ++ growth reduction evident; +++ inhibition zone clearly visible; – no inhibition.

Khademolhosseini R, Jafari A, Mousavi SM, Hajfarajollah H, Noghabi KA, Manteghian M. Physicochemical characterization and optimization of glycolipid biosurfactant production by a native strain of Pseudomonas aeruginosa HAK01 and its performance evaluation for the MEOR process. RSC Adv. 2019;9(14):7932–7947.

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Data availability statement

Data are presented in-text, and a link has been provided for data in repository.