ORIGINAL RESEARCH

Microbes, Infection and Chemotherapy ISSN: 2789 - 4274
https://doi.org/10.54034/mic.e1586

Characterization of biosurfactant-producing bacterial strains isolated from agro-industrial wastes in southwestern, Nigeria

Ismail B. Onajobi¹, Jamiu O. Adeyemi¹, Frank A. Orji³, Oyindamola J. Samson^1,*, Haneefat O. Egberongbe¹, Suliamon A. Aina², Oluwabukola T. Afolabi¹, Lawrence O. Adebajo¹, Obasola E. Fagade⁴

¹Department of Microbiology, Faculty of Science, Olabisi Onabanjo University, Ago-Iwoye, Nigeria.

²Departamento de Zoología y Biología Ambiental, Facultad de Ciencias, Universidad Olabisi Onabanjo, Ago-Iwoye, Nigeria.

³Departamento de Biotecnología, Instituto Federal de Investigación Industrial, Oshodi, Nigeria.

⁴Departamento de Microbiología, Facultad de Ciencias, Universidad de Ibadan, Ibadan, Nigeria.

Corresponding author: E-mail:oyindamolasamson1997@gmail.com

Orcid ID: ^*https://orcid.org/0000-0003-0404-7634

Received on november 24, 2022

Accepted for publication: december 20, 2022

Abstract

Introduction. The difficulty of managing trash and cleaning up the environment prompted interest in biosurfactants and surface-active proteins made by microbes. The study aims to augment bacterial isolates from agro-industrial wastes targeted for possible mass production of biosurfactants. Methods. Six agro-industrial wastes from Cassava, Palm kernel, and Sawdust from six agro-industrial sites within Ijebu area in Ogun State were collected for standard laboratory analyses in the Biotechnology Unit of the Federal Industrial Institute for Research, Oshodi (FIIRO). Five screening methods; blood hemolysis, lipase activity, blue agar hydrolysis, oil spreading, and emulsification index (EI24) were carried out to confirm biosurfactant production. Isolates with the highest hyper-production were subjected to 16rRNA molecular identification. Results. The study justified efficient biosurfactant production from 4 bacterial isolates out of 26 screened bacterial isolates from hydrocarbon degraders and 29 heterotrophic screened bacterial isolates, making a total of 55 screened bacterial isolates. Screening results reveal the emulsification capacities of identified Pseudomonas putida strain SG1, Acinetobacter baumanii strain MS14413, Bacillus zhangzhouensis strain cdsV18, and Burkholderia cepacia strain 717. Conclusion. Biosurfactant bacteria produced in all agricultural and industrial wastes considered in this study are capable of mass production.

Keyword: agro-industrial wastes, biosurfactants, bacteria, optimization, screening.

Introduction

Wastes management has continued to be a serious concern as a substantial amount of wastes, pollutants, and contaminants are released into our environment daily from domestic and industrial sources(1-3). Thus, exploring safer, cheaper, less toxic, and eco-friendly means to reduce, reuse and recycle unwanted materials becomes imperative(4,5). Biosurfactants (BS) are amphipathic biomolecules with hydrophobic and hydrophilic activities obtained from microorganisms (bacteria, yeasts, and filamentous fungi) that have the ability to form micelles (active surface molecules) and break the surface tension of liquid surfaces extra-cellularly(5,6).

Their hydrophobic and hydrophilic nature enables these characteristics and has been vastly reported for several applications in many fields, including food processing, pharmaceutics, bioremediation, biodegradation, and enhanced oil recovery(7). Considering the characteristics above and the fact that they can be recovered from cheaper substrates, which include agro-industrial wastes, which are readily available, they are better than chemical surfactants(8). Due to their high production costs and ongoing struggles with optimization, these biosurfactant-producing microorganisms face a significant economic hurdle compared to their chemical counterparts(2,9).

On these bases, the present study focused on the identification of biosurfactant production potential in bacterial strains from agricultural and industrial wastes from southwestern Nigeria, with the following specific objectives(10,11) isolation and characterization of bacterial strains isolated from agro-industrial wastes, screening of isolated bacterial strains for biosurfactant production by blood hemolysis, hydrolysis on blue agar, oil spreading, lipolytic capacity and emulsification index and molecular characterization of successfully isolated bacterial strains to the strain level by adopting molecular characterization techniques(12). The study aims to augment bacterial isolates from agro-industrial wastes targeted for possible mass production of biosurfactants and biosorption of heavy metals.

Materials and methods

Studyarea

The microbiological analysis of the study was carried out at the Federal Institute of Industrial Research Oshodi (FIIRO) in the biotechnology department. Agro-industrial waste samples were collected from the following sites, Agricultural Farm Settlement of the Olabisi Onabanjo University, Ago-Iwoye, Apoje Farms Ijebu-Igbo, Mini campus Sawmill Ago-Iwoye, Oke-Oru Sawmill Oru, Abusi Edumare Sawmill Ijebu-Igbo and Oke-Eri Sawmill, Odogbolu all within South-Western Nigeria.

Figure 1
Map of the study area

Source: www.viamichellin.com

Isolation of Bacteria

Each sample collected was serially diluted, total bacterial count present in the samples was determined by the pour plate method on nutrient agar. Sample suspensions were prepared by 10-fold serial dilutions with 1 gram of sample, using 10ml peptone water as diluents. Aliquots of the fourth and sixth dilutions were spread with an Eppendorf pipette on triplicates of sterile 12 ml of nutrient agar. The plates were incubated in the incubator for 24 hours in an inverted orientation at 37 °C(13). Colonies that formed during this incubation period were counted using this formula:

The enumeration of bacteria was carried out using the method reported by Chikere et al.(14) and Nwachukwu et al.(15). After incubation, morphologically and different colonial colonies were observed and sub-cultured on a nutrient agar by streak method to obtain pure cultures. They were subsequently seeded into 6 ml nutrient agar slants. The slants were kept in the bio-freezer at 4 °C as stock cultures.

Total Heterotrophic Bacterial Count

The total heterotrophic bacterial counts (THBC) were carried out by measuring one gram of each of the samples and serial diluting them nine-fold in sterile distilled water. One(1) ml of the diluents was aseptically poured into sterile Petri dishes. The dilutions were dispensed aseptically on sterile Petri dishes. The Petri dishes were incubated at 37°C for 24 h, after which the colonies were counted. These were done in triplicates. The colonies were further sub-cultured to obtain pure colonies(13,15).

Total Hydrocarbon Degrading Bacterial Count

Hydrocarbon using bacterial count was finished utilizing Mineral Salt Medium (MSM) agar on which 1% Double Reason Lamp oil (DPK) was added as the significant carbon source; before this, the DPK was sifted utilizing a Whatman channel paper No 1. Two percent (2%) agar was added to empower the hardening of the medium, as indicated by Venty et al.(16).

Colonial and morphological characterization of Bacterial isolates

Colonial and morphological characterization of the isolates was done using a standard microbiological method. Shape, pigmentation, elevation, size, appearance, and motility were used for morphological characteristics according to Onajobi et al.(5) and Venty et al.(16).

Blue agar hydrolysis

Mineral Salts Agar (MSA) was supplemented with cetyltrimethylammonium bromide (CTAB: 0.5 mg/ml-methylene blue (MB: 0.2 mg/ml) and was prepared as reported by Nordiyana et al.(17,28). Carbon sources that were tested are glucose, sodium acetate, mannitol, glycerol, sodium acetate, and peptone. The dark blue halo coloration on the plate was considered positive for biosurfactant production.

Emulsification index(EI₂₄)

The emulsification record for biosurfactant-delivering microscopic organisms was completed utilizing the technique of Raza et al.(18). Mannitol Salt Media was enhanced with 1% Lamp fuel for seven days in an orbital hatchery at 180 cycles each moment (rpm) at 28ºC. Without cells, the supernatant was acquired by centrifuging the stock culture at 15,000 rpm for 15 min. Two milliliters of the supernatant of the organic entity inside the response cylinder and 2 ml of lamp fuel were added as hydrocarbon substrate test. The combination was vortexed at high velocity for 2 minutes and noticed for rate emulsification at stretches four h through 24 h. The emulsification record (EI24) was determined by estimation of the level of the emulsion layer (a) partitioned by the complete level (b), duplicated by 100 (〖EI〗_24=a/b×100). This measure was acted in the same size glass test tubes as per Meenakshisundaram et al.(19).

Oil spreading test

The oil impregnation test was completed in polystyrene Petri dishes (100 mm × 15 mm) containing 20 μL of unrefined oil that was thoroughly stratified over 20 mL of refined water. A drop (∼10 μL) of separated supernatant was cautiously pipette onto the focal point of the oil layer. The distance across the unmistakable zone on the outer layer of the oil layer was estimated and contrasted with the negative controls, as indicated by Ibrahim et al.(20).

Lipolytic test

The assay medium was composed of (g/l); Tributyrin, 2 ml; Gum Arabic, 4; Agar, 15; Phosphate buffer at pH 4.6, up to 1L. The lipase assay medium was prepared, and the cell-free filtrate of the biosurfactant producer was used as a source of lipase enzyme, as elucidated by Sidkey et al.(21). Lipolytic activity was detected by clearing zones around the hole in comparison to the turbid background of the assay plates.

Molecular characterization of Bacterial isolates

Bacterial isolates with very high potentials as hyper-producers of biosurfactant were identified further using Molecular Biology tools such as polymerase chain reactions, sequencing, and blast programs through the extraction of the genomic DNA of bacterial isolates, amplification by Polymerase Chain Reaction (PCR) using 16s-rRNA primer, sequencing of the isolate DNA and blast programs were used to reveal the name of the isolate according to the method of Joshi and Deshpande(22).

Statistical analyses

Data obtained were subjected to statistical analyses using the Statistical Package for Social Sciences (SPSS) version 20.0. Mean values were compared using Analysis of Variance (ANOVA) according to Zhang and Liang(23). Screening results were presented as Mean±Standard deviation. Post hoc test was adopted using the Student-Newman-Keuls (SNK). A probability value (p – value) less than 0.05 was considered statistically significant.

Results

Total Heterotrophic Bacterial counts

The results of the Total Heterotrophic bacterial counts on Cassava flake, Palm kernel, Sawdust, and Cassava peel showed no significant difference in the heterotrophic bacterial counts recorded with Cassava flakes and Sawdust. However, the highest heterotrophic bacterial count was recorded with Sawdust. On the other hand, the lowest heterotrophic bacterial count was recorded with Cassava peel, as presented in Figure 2.

Figure 2
Total Heterotrophic Bacterial counts; Bars with similar superscripts are not significantly different at p < 0.05

Total Hydrocarbon Degrading Bacteria count

The results of the hydrocarbon-degrading bacteria count are shown in Figure 3. Palm kernel had the highest hydrocarbon-degrading bacteria count (7.10 x 106 CFU/g). This was not significantly different from the Cassava peel (6.80 x 106 CFU/g). However, the hydrocarbon-degrading bacteria count recorded with the Palm kernel and Cassava peel were significantly higher than those of Sawdust and Cassava flake, respectively.

Figure 3
Total Hydrocarbon Degrading Bacteria count; Bars with similar superscripts are not significantly different at p < 0.05

Colonial and morphological characterization

The gram reaction and morphological appearance of the heterotrophic samples are presented in Table 1. Most of the samples are gram-positive rods though some have short rods, some appear to have single colonies, some are clustered, and some occur in chains. Likewise, most are mucoidal in colonial appearance, while some are milkfish in color.

Table 1
Morphological appearances and Gram reactions of Heterotrophic bacteria

S/N Isolate code Gram reactions and Microscopy

1 CF 124 gram-positive rods in singles.

2 CF 125 gram-positive rods in singles.

3 CF 126 gram-positive rods in clusters.

4 CF 121 gram-positive rods in chains and clusters.

5 CF 122 gram-positive rods in clusters.

6 CF 123 gram-positive rods in chains.

7 PKB 104 gram-positive rods in clusters.

8 PKB 105 gram-positive ellipsoidal cells in clusters.

9 PKB 106 gram-positive rods in singles.

10 PKB 107 gram-positive slender rods in clusters.

11 PKB 108 gram-positive rods in chains and clusters.

12 SD 101 gram-positive rods in long chains.

13 SD 102 gram-positive short rods in chains.

14 SD 103 gram-positive short rods in clusters.

15 S₂33 gram-positive fat rods in singles.

16 S₃Q gram-positive long rods in singles.

17 CS 113 gram-positive rods in chains and clusters.

18 PKB 114 gram-positive rods in chains.

19 CS 111 gram-positive rods in chains and clusters.

20 CS 112 gram-negative rods in singles and clusters.

21 CP 101 gram-positive cocci in singles.

22 CP 102 gram-positive rods in clusters.

23 CP 103 gram-positive rods in singles.

24 CP 104 gram-negative rods in singles.

25 CF 131 gram-negative rods in singles.

26 CF 132 gram-positive rods in clusters.

27 CF 133 gram-negative rods in clusters.

28 CSP₃MFR gram-positive rods in singles.

29 S33 gram-positive rods in singles.

PK, Palm kernel bargasse; CF, Cassava flake; SD, Sawdust; CP, Cassava peel;
CS, Cassava Shaft

S/N	Isolate code	Gram reactions and Microscopy
1	CF 124	gram-positive rods in singles.
2	CF 125	gram-positive rods in singles.
3	CF 126	gram-positive rods in clusters.
4	CF 121	gram-positive rods in chains and clusters.
5	CF 122	gram-positive rods in clusters.
6	CF 123	gram-positive rods in chains.
7	PKB 104	gram-positive rods in clusters.
8	PKB 105	gram-positive ellipsoidal cells in clusters.
9	PKB 106	gram-positive rods in singles.
10	PKB 107	gram-positive slender rods in clusters.
11	PKB 108	gram-positive rods in chains and clusters.
12	SD 101	gram-positive rods in long chains.
13	SD 102	gram-positive short rods in chains.
14	SD 103	gram-positive short rods in clusters.
15	S₂33	gram-positive fat rods in singles.
16	S₃Q	gram-positive long rods in singles.
17	CS 113	gram-positive rods in chains and clusters.
18	PKB 114	gram-positive rods in chains.
19	CS 111	gram-positive rods in chains and clusters.
20	CS 112	gram-negative rods in singles and clusters.
21	CP 101	gram-positive cocci in singles.
22	CP 102	gram-positive rods in clusters.
23	CP 103	gram-positive rods in singles.
24	CP 104	gram-negative rods in singles.
25	CF 131	gram-negative rods in singles.
26	CF 132	gram-positive rods in clusters.
27	CF 133	gram-negative rods in clusters.
28	CSP₃MFR	gram-positive rods in singles.
29	S33	gram-positive rods in singles.

The gram reaction and morphological appearance of the hydrocarbon samples are presented in Table 2. Most of the samples are gram-positive rods though some have short rods, some appear to have single colonies, while some are clustered, and some occur in chains. Also, most have a mucoidal colonial appearance, while some are milky.

Table 2
Colonial appearances and microscopy of hydrocarbon utilizing pure bacterial isolates

S/N Isolate code Coloneal appearance Microscopy and Morphology

1 CSP₁ Mucoidal and convex shape and entire with 10mm size in diameter Gram-positive rods in single

2 S₂3 Milkish and translucent column shape is oval and about 4mm in size Gram-positive rods in single

3 PKBJ Mucoidal and convex with an entire shape of about 10mm size in diameter Gram-positive rods in clusters

4 S₃(2) Mucoidal and convex, with a wavy edge and about 12mm size Gram-positive long rods in clusters

5 S₂Q Milkish and raised colonies, with an entire shape 5mm in size Gram-negative rods in clusters

6 S₂31 Milkish and raised colonies, with an entire shape 5mm in size Gram-positive long rod in singles

7 S3 Mucoidal and convex with an entire shape size equal to 12mm Gram-positive long rods in singles

8 S₃Q1 Convex shape with light yellow pigment shape equal to 2-4mm Gram-negative long rods in clusters and single

9 PKBMY2 Translucent colonies Gram-negative rods in singles

10 PKBMYQ Pinkish colony with a convex structure size is 4mm Gram-positive rods in clusters

11 PKBMR(2) Mucoidal with wavy edge and size is 12mm Gram-positive rods in clusters

12 PKBJ2 Mucoidal with wavy edge and size is 12mm Gram-negative rods in single

13 CSP₃MT(1) Mucoidal with entire edge and size is 6mm Gram-negative rods in singles

14 CJP2 Mucoidal and convex shape 6-8mm Gram-positive rods in clusters

15 CSPMAR₃ Translucent and convex structure size equal to 8-10mm Gram-negative rods in singles

16 PKBMY(2) Milky and translucent colonies in size 10mm Gram-positive cocci in singles

17 PKBJ22 Mucoidal and oval in shape, translucent size equal to 10mm Gram-positive rods in singles and cluster

18 S2 Mucoidal and with wavy edge size 12mm Gram-positive long and slender rods in singles

19 S₁Q Mucoidal and milky in color Gram-negative rods in singles

20 CSF2 Mucoidal and milky in color Gram-negative rods in singles

21 CSP₃ Mucoidal and milky in color Gram-positive rods in single

22 S₂4 Mucoidal and milky in color Gram-negative short and fat rods in single

23 PKBJ₃ Mucoidal, translucent, 10mm in size Gram-positive rod in singles and long chain

24 S₃(2)1 Mucoidal, translucent, 10mm in size Gram-positive rods in singles

25 S₂Q1 Mucoidal, light yellow pigment size equal to 6m Gram-positive rod in singles

26 S₂32 Wavy edge, Milkish and 8-10mm size in diameter Gram-positive rod in short chain

PK, Palm kernel bargasse; CF, Cassava flake; S, Sawdust; CP, Cassava peel; CS, Cassava Shaft

S/N	Isolate code	Coloneal appearance	Microscopy and Morphology
1	CSP₁	Mucoidal and convex shape and entire with 10mm size in diameter	Gram-positive rods in single
2	S₂3	Milkish and translucent column shape is oval and about 4mm in size	Gram-positive rods in single
3	PKBJ	Mucoidal and convex with an entire shape of about 10mm size in diameter	Gram-positive rods in clusters
4	S₃(2)	Mucoidal and convex, with a wavy edge and about 12mm size	Gram-positive long rods in clusters
5	S₂Q	Milkish and raised colonies, with an entire shape 5mm in size	Gram-negative rods in clusters
6	S₂31	Milkish and raised colonies, with an entire shape 5mm in size	Gram-positive long rod in singles
7	S3	Mucoidal and convex with an entire shape size equal to 12mm	Gram-positive long rods in singles
8	S₃Q1	Convex shape with light yellow pigment shape equal to 2-4mm	Gram-negative long rods in clusters and single
9	PKBMY2	Translucent colonies	Gram-negative rods in singles
10	PKBMYQ	Pinkish colony with a convex structure size is 4mm	Gram-positive rods in clusters
11	PKBMR(2)	Mucoidal with wavy edge and size is 12mm	Gram-positive rods in clusters
12	PKBJ2	Mucoidal with wavy edge and size is 12mm	Gram-negative rods in single
13	CSP₃MT(1)	Mucoidal with entire edge and size is 6mm	Gram-negative rods in singles
14	CJP2	Mucoidal and convex shape 6-8mm	Gram-positive rods in clusters
15	CSPMAR₃	Translucent and convex structure size equal to 8-10mm	Gram-negative rods in singles
16	PKBMY(2)	Milky and translucent colonies in size 10mm	Gram-positive cocci in singles
17	PKBJ22	Mucoidal and oval in shape, translucent size equal to 10mm	Gram-positive rods in singles and cluster
18	S2	Mucoidal and with wavy edge size 12mm	Gram-positive long and slender rods in singles
19	S₁Q	Mucoidal and milky in color	Gram-negative rods in singles
20	CSF2	Mucoidal and milky in color	Gram-negative rods in singles
21	CSP₃	Mucoidal and milky in color	Gram-positive rods in single
22	S₂4	Mucoidal and milky in color	Gram-negative short and fat rods in single
23	PKBJ₃	Mucoidal, translucent, 10mm in size	Gram-positive rod in singles and long chain
24	S₃(2)1	Mucoidal, translucent, 10mm in size	Gram-positive rods in singles
25	S₂Q1	Mucoidal, light yellow pigment size equal to 6m	Gram-positive rod in singles
26	S₂32	Wavy edge, Milkish and 8-10mm size in diameter	Gram-positive rod in short chain

Substrate Screening results for Heterotrophic degraders

The sample screening results for heterotrophic degraders. Results showed no significant difference (p > 0.05) in the reactions of both the Cassava peel and Sawdust to Lipase test and Blood hemolysis. Also, Cassava peel and Sawdust showed no reaction to the oil spreading test. The emulsification index was highest with the Sawdust. This was, however, not significantly different from that of the Cassava peel. Similarly, Sawdust showed a positive response (66.7%) to the Blue agar test, as presented in Figure 4.

Figure 4
Substrates screening results for heterotrophic degraders

Table 3
Screening result for heterotrophic degraders

Screening test
Agro-industrial wastes Lipase test Blood hemolysis Oil spreading Emulsification index Blue agar

Cassava peel 15.13±3.12^b 0.00±0.00^c 44.50±12.02^a 9.67±5.51^b Negative (100%)

Cassava flake 0.00±0.00^c 32.00±2.00^a 11.00±1.00^c 13.00±3.00^b Negative (100%)

Palm kernel 25.50±5.66^a 33.75±14.05^a 0.00±0.00^d 31.43±8.12^a Positive (14.3%)

Sawdust 18.48±8.19^b 22.13±6.74^b 20.57±8.08^b 18.43±2.62^b Positive (50.0%)

abcdMeans (±Standard deviation) in the same column having similar superscript are not significantly different at p < 0.05.

Screening test Agro-industrial wastes	Lipase test	Blood hemolysis	Oil spreading	Emulsification index	Blue agar
Cassava peel	15.13±3.12^b	0.00±0.00^c	44.50±12.02^a	9.67±5.51^b	Negative (100%)
Cassava flake	0.00±0.00^c	32.00±2.00^a	11.00±1.00^c	13.00±3.00^b	Negative (100%)
Palm kernel	25.50±5.66^a	33.75±14.05^a	0.00±0.00^d	31.43±8.12^a	Positive (14.3%)
Sawdust	18.48±8.19^b	22.13±6.74^b	20.57±8.08^b	18.43±2.62^b	Positive (50.0%)

The results for screening for the hydrocarbon degraders are presented in Table 5, with isolate S2Q from Sawdust, with 35.7 having the highest average, and isolate CSP3MT(1) from cassava shaft having the lowest average of 3.7.

Table 4
Colonial appearances and microscopy of hydrocarbon utilizing pure bacterial isolates

S/N Isolate code Lipase test B
H OS Blue Agar Avarage

01 S33 13 31 o + 14.9

02 CSP₃MFR 11 31 o - 14

03 S₂33 20 35 o - 18.3

04 S₃Q 9 26.5 o + 11.8

S/N	Isolate code	Lipase test	B H	OS	Blue Agar	Avarage
01	S33	13	31	o	+	14.9
02	CSP₃MFR	11	31	o	-	14
03	S₂33	20	35	o	-	18.3
04	S₃Q	9	26.5	o	+	11.8

The substrate screening results for hydrocarbon degraders are presented in Table 3. Cassava flake showed no reaction to the Lipase test. However, this reaction to the Lipase test was significantly higher with the Palm kernel than with the Sawdust and Cassava peel, respectively. Similarly, Cassava peel showed no reaction to the Blood hemolysis test. Reaction to Blood hemolysis test was also highest with the Palm kernel and lowest with Sawdust. On the other hand, the Palm kernel showed no reaction to the oil spreading test.

Reactions to the oil spreading test were significantly higher with Cassava peel. This was followed by Sawdust and Cassava flake, respectively. Results, however, showed a significantly higher Emulsification index with the Palm kernel. However, the Emulsification index recorded with Sawdust, Cassava flake, and Cassava peel were not significantly different. This was lowest with cassava peel. Meanwhile, Cassava peel and Cassava flake showed a negative response (100%) to the Blue agar test. However, Sawdust (50.0%) and Palm kernel (14.3%) showed some positive responses to the Blue agar test.

Bacterial isolates screening results for heterotrophic degraders

The results for screening for the heterotrophic degraders are presented in Table 4.4, with isolate S233 with 18.3 having the highest average.

Molecular identification of the four most successful biosurfactants hyper-producing strains of Bacteria

The molecular identities of the four most successfully screened isolates are represented in Table 6 with their different ascension numbers. The isolates are completely from Sawdust and Palm kernel.

The gel electrophoresis result is represented in Plate 1, revealing positions of DNA molecular markers, DNA bands of four isolates Pseudomonas putida strain SG1, Acinetobacter baumanii strain MS14413, Bacillus zhangzhouensis strain cdsV18 and Burkholderia cepacia strain 717 respectively.

Table 5
Screening results for hydrocarbon degraders

S/N Isolate code Lipase test Blood hemolysis Oil spreading Blue Agar Avarage

1 CSP₁ 16 - 36 - 17,3

2 S₂3 31,5 17,5 14 +` 21

3 PKBJ 12,5 25,5 0 + 12,7

4 S₃(2) 26 27 14 + 22,3

5 S₂Q 25 21 61 - 35,7

6 S₂3 18,3 35 17 - 23,5

7 S3 12,5 19,5 13 - 15

8 S₃Q 8 17,5 17 + 14,2

9 PKBMY2 0 13,5 0 - 4,5

10 PKBMYQ 17,5 26 0 - 14,5

11 PKBMR(2) 30 44,5 0 - 24,2

12 PKBJ21 31 - 0 - 10,3

13 CSP₃MT(1) 11 - 0 - 3,7

14 CJP2 18,5 - 53 - 23,8

15 CSPMAR3 15 - 0 - 5

16 PKBMY(2) 10 44,5 0 - 18,2

17 PKBJ2 52 48,5 0 - 33,5

18 S2 14 25,5 0 - 13,2

19 S₁Q 12,5 14 8 + 11,5

20 CSF1 - 32 11 - 14,3

PK, Palm kernel bargasse; CF, Cassava flake; S, Sawdust; CP, Cassava peel; CS, Cassava Shaft

S/N	Isolate code	Lipase test	Blood hemolysis	Oil spreading	Blue Agar	Avarage
1	CSP₁	16	-	36	-	17,3
2	S₂3	31,5	17,5	14	+`	21
3	PKBJ	12,5	25,5	0	+	12,7
4	S₃(2)	26	27	14	+	22,3
5	S₂Q	25	21	61	-	35,7
6	S₂3	18,3	35	17	-	23,5
7	S3	12,5	19,5	13	-	15
8	S₃Q	8	17,5	17	+	14,2
9	PKBMY2	0	13,5	0	-	4,5
10	PKBMYQ	17,5	26	0	-	14,5
11	PKBMR(2)	30	44,5	0	-	24,2
12	PKBJ21	31	-	0	-	10,3
13	CSP₃MT(1)	11	-	0	-	3,7
14	CJP2	18,5	-	53	-	23,8
15	CSPMAR3	15	-	0	-	5
16	PKBMY(2)	10	44,5	0	-	18,2
17	PKBJ2	52	48,5	0	-	33,5
18	S2	14	25,5	0	-	13,2
19	S₁Q	12,5	14	8	+	11,5
20	CSF1	-	32	11	-	14,3

Table 6
Molecular identities of the four most successful biosurfactants hyper-producing strains of Bacteria

S/N Isolate code Lipase testIdentity % Similarity Accession Number

1 S₂Q Pseudomonas putida SG1 100 MN318320.1

2 S₂3 Acinetobacter baumanii MS14413 100 CP054302.1

3 PKBMR(2) Bacillus zhangzhouensis cdsV18 100 MN826587.1

4 PKBJ2 Burkholderia cepacia 717 100 NR_029209.1

S/N	Isolate code	Lipase testIdentity	% Similarity	Accession Number
1	S₂Q	Pseudomonas putida SG1	100	MN318320.1
2	S₂3	Acinetobacter baumanii MS14413	100	CP054302.1
3	PKBMR(2)	Bacillus zhangzhouensis cdsV18	100	MN826587.1
4	PKBJ2	Burkholderia cepacia 717	100	NR_029209.1

Plate 1
Gel electrophoresis showing DNA molecular markers, DNA bands of Pseudomonas putida strain SG1, Acinetobacter baumanii strain MS14413, Bacillus zhangzhouensis strain cdsV18 and Burkholderia cepacia strain 717 on lanes 2,3,4 and 5 respectively

Figure 5
Phylogenetic tree of bacterial strains

Discussion

The outcomes got in the current study showed that cassava squanders recorded the biggest number of province counts, as heterotrophic microscopic organisms have more state counts over the hydrocarbon utilizers since they are critical, as recently detailed by Banat et al.(24). In the meantime, Bacillus species were disengaged from every one of the squanders, despite the fact that more are from cassava piece like Gharaei-Fathabad(25) that chipped away at bacterial biosurfactants in the drug industry.

Acinetobacter baumanii MS14413 was isolated from Sawdust which is similar to Lee et al. (26), who reported systems biotechnology for strain improvement using mollases. Pseudomonas putida strain SG1 was isolated from Sawdust, similar to Meenakshisundaram et al.(19), which studied future microbial surfactants. Burkholderia cepacia strain 717 was isolated from palm kernel baggasse, another likely novel biosurfactant producer.

Bustamante et al.(27) screened 68 bacterial disconnects from soil and saw just 6% of secludes with great emulsification action of up to 61%, which is in line with the high emulsification of Pseudomonas putida SG1 and Burkholderia cepacia strain 717 in the current review. Estimating emulsification units helps pick the carbon and energy hotspots for evaluating biosurfactant creation. Adeyemi et al. (2) utilized a cassava flour handling emanating as a substrate surfactant delivered by B. subtilis, which concurred with the current concentrate where Bacillus zhangzhouensis was separated been a comparative strain.

The ability of the bacterial isolates from these wastes to produce biosurfactants is important, considering the level of pollution and contamination in the said areas and the need to use indigenous and ecologically friendly products in the remediation process. Moreover, since interest in biosurfactants as a novel research area continue to gain the attention of intending scientist, this demand the development of many methods for the screening of biosurfactant-producing strains. Emulsification capacities of Pseudomonas putida strain SG1, Acinetobacter baumanii strain MS14413, Bacillus zhangzhouensis strain cdsV18 and Burkholderia cepacia strain 717 make them a new potential candidate for biosurfactant production. It is noted to state that more of the most outstanding biosurfactant-producing bacteria came from Palm kernel bargasse and Sawdust. Although the most producing isolate Pseudomonas putida strain SG1 was isolated from Sawdust. This investigation supports Kalvandi et al. (3) theory that surfactants generated by all three isolates are responsible for the release of TPH from the soil. The SHA302 isolate was chosen as an effective isolate for molecular identification owing to its increased efficiency in TPH release. The findings of the 16S rRNA gene sequencing showed the highest degree of affinity (93.98%) between our strain and Bacillus pumilus strain ATCC 7061 (T), accession number NR 043242. The strain has the entry number OK285074 and was listed in the gene bank as Bacillus sp. strain SHA302 (T).

It was referred to from Sidkey et al. (21) that it is critical to take note that a large portion of the scientists has involved most extreme few evaluating techniques for the choice of biosurfactants makers; they proposed that a solitary strategy isn't reasonable to distinguish all sort of biosurfactants. Thusly, a mix of different techniques is expected for powerful screening. This examination concurred with the crafted by Nordiyana et al. (28) that likewise detached Bacillus subtilis. They involved Potato substrate as a carbon hotspot for biosurfactant creation.

The screening results of the present study are also in alignment with the work of Femi-Ola et al.(29), which screened and characterized biosurfactant-producing bacteria from soil samples in Ogun State, Nigeria. Silva et al.(30) and Makkar et al.(31) reported the application of a modified drop-collapse technique for surfactant quantization and screening of biosurfactant-producing microorganisms, which is another complimentary screening method but was not considered in the current study.

Conclusión

Cassava peel from the study reveals to have a potential production level than Sawdust and Cassava flake to the oil spreading test, while Palm kernel has a very high emulsification index. All the Agro-industrial wastes considered in this work harbor biosurfactant strains producers with the capacity of mass production. More work should be carried out on these novel biosurfactant production strains.

Author Contribution Statement

The authors confirm their contribution to the paper as follows: Study conception and design:
Ismail B. Onajobi, Jamiu O. Adeyemi. Data collection: Ismail B. Onajobi, Jamiu O. Adeyemi, Frank A. Orji, Oyindamola J. Samson.
Analysis and interpretation of results: Haneefat O. Egberongbe, Suliamon A. Aina, Oluwabukola T. Afolabi, Lawrence O. Adebajo and Obasola E. Fagade.
Draft manuscript preparation: Ismail B. Onajobi, Jamiu O. Adeyemi, Oyindamola J. Samson.
All authors reviewed the results and approved the final version of the manuscript. All authors agreed to be responsible for all aspects of the work to ensure the accuracy and integrity of the published manuscript.

Conflicts of interest

The authors declare no conflict of interest.

Funding

The authors declare no source of funding was received from any bodies or institutes.

Ethics statement

This work was authorized by the Research Ethics Committee (OOU-REC) of Faculty of Science, Olabisi Onabanjo University, Ago Iwoye.

Availability of data

The authors declare no source of funding was received from any bodies or institutes.

Acknowledgments

The authors hereby appreciate members of Microbiology Laboratory, Department of Microbiology, Olabisi Onabanjo University, Ago Iwoye, Nigeria.

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