Screening of Microorganisms from Wastes and Identification of the Optimal Substrate for Biosurfactant Production

Abstract

The production of biosurfactants from organic wastes has received significant attention due to its potential cost savings. This study involved the isolation of biosurfactant-producing microorganisms from waste sources. The surfactant properties of the 37 studied isolates were assessed by reducing surface tension and their emulsifying properties, determined by the emulsification index E24. We assessed the ability of these isolated strains to produce biosurfactants using various waste substrates, namely potato peelings, waste cooking oil and sunflower cake. Our results showed that sunflower cake exhibited better growth and biosurfactant production for most of the strains studied. This highlights that sunflower cake is a potentially effective and economical substrate for the production of biosurfactants. The most effective strains allowing to achieve an emulsification index above 50% and reduce surface tension below 40 mN m⁻¹ were Enterobacter sp. 2pp, strain 2wfo, Peribacillus sp. 1mo, Sphingomonas sp. 2mo, Ochrobactrum sp. 5mo, Shouchella sp. 6mo, Bacillus sp. 1os, Bacillus sp. 2os. Among these strains, both previously known strains as biosurfactant producers and previously unknown strains were found. Thus, we found that among representatives of the genus Sphingomonas there are effective producers of biosurfactants. The highest yield of biosurfactant on a medium with glycerol and glucose was shown by the Bacillus sp. 2os strain of 0.501 and 0.636 g L⁻¹, respectively.

1. Introduction

Rising environmental concerns have propelled a quest for novel, eco-friendly methodologies across diverse domains. One such area of interest involves the investigation of substituting synthetic surfactants with biologically derived alternatives [1,2]. These biologically derived surfactants, termed biosurfactants, are currently under exploration for their potential applications in agriculture, the bioremediation of oil-contaminated soils, and enhanced oil recovery.

However, despite the burgeoning interest in biosurfactants, their practical applications remain restricted due to the high production costs associated with them. Potential resolutions to this challenge encompass the development and scaling of technological processes and reduction in the expenses tied to raw materials utilized. An emerging strategy involves the use of organic waste as a primary raw material, presenting a promising avenue to significantly diminish the cost of biosurfactants while simultaneously fostering the integration of waste materials into recycling cycles [3,4,5].

Organic wastes from food and agricultural industries, industrial byproducts such as wastewater, raw glycerol, and waste generated from meat production, as well as oil-contaminated soil, are all considered viable substrates for biosurfactant production. These waste materials are rich in nutrients, encompassing a wide array of sources such as sugarcane molasses, crop cakes, banana, orange, and potato peels, waste frying oils, coconut oils, rapeseed oils, moringa and cassava residues, distillery waste, effluents from food and vegetable production, coffee wastewater, among others [6]

The selection of the substrate for biosurfactant production is pivotal as various carbon sources exert differing effects on product yield. For instance, Aparna et al. (2012) discovered that Pseudomonas sp. 2B yielded varying amounts of rhamnolipid, producing 4.14, 4.38, 3.24, and 4.09 g L⁻¹ when cultivated on glycerol, coconut oil cake, orange peel, and whey, respectively [7]. Similarly, Moussa (2014) observed that the rhamnolipid yield of Pseudomonas aeruginosa TMN was 2.9 ± 0.02 g L⁻¹ on a glucose substrate and 1.35 ± 0.01 g L⁻¹ on glycerol, while sucrose resulted in a notably lower yield of 0.91 g L⁻¹ [8].

The metabolic pathways and resultant homologues of biosurfactants display significant variability contingent upon the strain used, as well as the substrate employed [9,10,11,12]. Ndlovu et al. (2017) studied surfactin analogues obtained from Bacillus amyloliquefasciens ST34, noting the dominance of surfactins C13–C15 across samples. Similarly, P. aeruginosa ST5 generated six rhamnolipid congeners, with Rha–C10–C10 being the most abundant [13]. Mouafo et al. (2017) highlighted the ability of three Lactobacillus strains to produce biosurfactants with higher lipid content on a glycerol substrate compared to sugarcane molasses [12]. This difference was attributed to the mechanism of glycerol consumption primarily directed toward the lipolytic pathway and gluconeogenesis, allowing for the production of fatty acids and sugars [14].

Moreover, the properties of resulting surfactants are contingent upon the substrate used. Surfactant biomolecules, classified into surfactants and emulsifiers, exhibit distinct roles: surfactants reduce surface tension while emulsifiers partake in the formation and stabilization of emulsions [15]. Some biomolecules, however, possess both surfactant and emulsifying properties. Distinguishing these properties necessitates the application of diverse evaluation methodologies [16]. Biosurfactants are identified through methods assessing the reduction in surface and interfacial tension, while bioemulsifiers form stable emulsions without significant alterations in surface/interfacial tension across phases. For instance, Stoimenova (2014) reported the ability of an indigenous strain of industrial wastewater Pseudomonas fluorescens to produce glycolipid biosurfactants in a medium containing hexadecane, mineral oil, vegetable oil, and glycerol, with the highest emulsifying capability observed in the vegetable oil medium [17].

The influence of substrate on biosurfactant biosynthesis highlights the need for careful substrate selection. The use of organic wastes as substrates for the synthesis of biosurfactants has not been sufficiently studied, since only a small number of wastes have been studied as substrates. In particular, the study has not previously been carried out on the isolation of microorganisms and the build-up of biosurfactants on different types of organic municipal and agricultural wastes. In the present study, isolates were obtained and tested for their ability to synthesize biosurfactants on various organic wastes. Subsequently, the most effective waste-microorganism pair was selected, giving the highest biosurfactant productivity.

2. Materials and Methods

2.1. Waste Sampling

To isolate microorganisms, the following organic wastes were selected: waste from grease traps and oil traps of a water utility (Chelnyvodokanal LLC, Naberezhnye Chelny, Russia), potato peelings and used frying oil (catering restaurants, Kazan, Russia), rapeseed and sunflower cake (JSC Kazan Oil Extraction Plant, Kazan, Russia), oil-contaminated soil; soil contaminated with motor oil (car service areas, Kazan, Russia).

2.2. Isolation of Microorganisms from Wastes

Isolates of microorganisms from wastes were cultivated using a minimal medium (g L⁻¹): NaNO₃ 2.0, KH₂PO₄ 0.5, K₂HPO₄ 1.0, MgSO₄ 7H₂O 0.5, KCl 0.1, FeSO₄ 7H₂O 0.01. The pH was adjusted to 7.0 with 1 N HCl/NaOH. The investigated wastes were added as the sole carbon source to the autoclaved medium. Various carbon sources were separately introduced into the medium. The initial waste materials, such as potato peelings, rapeseed and sunflower cake, oil-contaminated soil, soil contaminated with motor oils, and waste from water utility grease traps and oily sludge, as well as waste frying oil, were included in the mineral medium at a volume of 2% (v/v) [18].

Bacterial strains were inoculated in 250 mL flasks containing 100 mL of medium and incubated at 120 rpm at 28 °C for 72 h. Following incubation, the strains were subjected to the limiting dilution method and subsequently sown on a solid MPA medium. Individual isolates of microorganisms were obtained by evaluating the morphological characteristics of colonies on a solid medium in a Petri dish and through microscopic analysis utilizing an Axio Lab A1 light microscope (Carl Zeiss, Jena, Germany).

2.3. Assessment of Biosurfactant Production Ability

The assessment of emulsifying ability was conducted using the E24 method [19]. All measurements were carried out in triplicate. This procedure involved combining an equal volume of cell-free strain culture supernatant with crude oil. The cell-free supernatant was obtained via centrifugation of the liquid cell culture (10 min, 5000 rpm). The resulting mixture was vortexed and allowed to stand for 24 h at room temperature. Subsequently, the height of the emulsified column was measured, and E24 was calculated using the formula:

$$\mathrm{E}24=\frac{emulsion layer height}{total height of the liquid column in the test tube}\times 100\%$$

(1)

The cell-free strain culture supernatant was analyzed for the reduction in water surface tension (ST) using the Du Nouy ring method with a K20 tensiometer (KRUSS, Hamburg, Germany) at room temperature [20].

2.4. Determination of Strain Species

The total genomic DNA of the isolates was extracted utilizing a K-Sorb reagent kit for DNA extraction on microcolumns (SINTHOL Company, Moscow, Russia). Subsequently, the nucleotide sequence of the samples was determined through the Sanger sequencing method employing an ABI 3730 DNA Analyzer (Life Technologies, Carlsbad, CA, USA). Genomic libraries were constructed using general bacterial primers 27f-1492r [21]. The sequences obtained for each strain were matched with the sequences from the database using the BLAST NCBI. The sequences were aligned using MEGA 10.0 software.

2.5. Cultivation of Isolates on Different Waste Types

The chosen isolates were cultured on four distinct substrates: pure glycerol, potato peelings, waste frying oil, and sunflower cake. For the preparation of potato peelings and sunflower cake, both were dried at 55 °C for 4 days, crushed into fine powder, and subsequently, a 10% (w/v) solution of the powder in distilled water was autoclaved. The solution was filtered through gauze to obtain a clear filtrate, which was then added sterilely at a 4% (v/v) concentration to the previously described mineral medium. Waste frying oil and glycerol were directly added to the medium at a 2% (v/v) concentration before autoclaving. Biosurfactant producers were grown in 500 mL flasks containing 200 mL of medium at 120 rpm at 28 °C for 72 h.

2.6. Extraction of Biosurfactants

The cell culture was centrifuged at 5000 rpm for 10 min at room temperature to obtain cell-free supernatant. Extraction of biosurfactants was carried out using the acid precipitation method—bringing the cell-free supernatant to pH = 2 using 5 N HCl. The acidified supernatant was kept at 4 °C overnight and then centrifuged at 4 °C for 40 min at 3700 rpm. The biosurfactant precipitate was dissolved in a mixture of methanol and chloroform in a ratio of 2:1 and filtered using a 0.22 µm filter (Sartorius, Gottingen, Germany). The biosurfactant was obtained by evaporating the solvent on a rotary evaporator (IKA, Staufen im Breisgau, Germany) and evaluated gravimetrically.

2.7. Statistical Analysis

The error bars depicted in the figures indicate the standard error of means derived from the replicates. To evaluate the data within groups of values (characterizing each substrate type and individual strain), weights were assigned, and the weighted arithmetic means were computed. The statistical analysis was carried out using Statistica 10.0 software (StatSoft Inc., Tulsa, OK, USA). Graphs were generated using Microsoft Excel 2016 MSO (Microsoft, Redmond, WA, USA).

3. Results and Discussion

3.1. Isolation of Biosurfactant-Producing Microorganisms from Waste

Microorganisms capable of biosurfactant synthesis were isolated from various categories of wastes, including agricultural sources (potato peelings, rapeseed, and sunflower cake), industrial sources (oil-contaminated soil and soil contaminated with motor oils), and municipal wastes (waste from grease traps and water utility oily sludge, waste frying oil).

A total of 37 strains were isolated from these waste materials, and their genera were identified through Sanger sequencing (

Table 1. Microorganisms isolated from waste.

Wastes	Strains	The Closest-Related Strain	Identity, %	Identified as
potato peelings	1pp	Enterobacter sp. strain XN81	98.5	Enterobacter sp. 1pp
	2pp	Enterobacter ludwigii strain E8-13	97.6	Enterobacter sp. 2pp
	3pp	Bacterium strain BS0657	93.4	3pp
	4pp	Uncultured bacterium clone RBL10-19	94.9	4pp
	5pp	Enterobacter ludwigii strain 160-a blue	98.7	Enterobacter sp. 5pp
rapeseed cake	1rc	Pantoea sp. LL69	83.6	Pantoea sp. 1rc
grease trap	1gt	Proteus mirabilis, isolate AHI-2	99.5	Proteus sp. 1gt
	2gt	Bacillus aerophilus strain 0125	97.4	Bacillus sp. 2gt
oily sludge	1s	Citrobacter freundii strain E51	99.4	Citrobacter sp. 1s
	2s	Sphingomonas echinoides strain B18	94.7	Sphingomonas sp. 2s
waste frying oil	1wfo	Sphingomonas sp. strain FKP374	97.5	Sphingomonas sp. 1wfo
	2wfo	Uncultured Sphingomonas sp., clone: LR564B-24	89.8	2wfo
	3wfo	Pseudomonas stutzeri DSM 10701	99.6	Pseudomonas sp. 3wfo
	4wfo	Uncultured Sphingomonas sp., clone: LR564B-24	95.4	Sphingomonas sp. 4wfo
	5wfo	Sphingomonas echinoides strain KCOM 3301 (=JS364)	91.6	Sphingomonas sp. 5wfo
sunflower cake	1sc	Sphingomonas sp. strain FKP374	94.6	Sphingomonas sp. 1sc
	2sc	Sphingomonas sp. Hc_01N 16S	90.4	Sphingomonas sp. 2sc
	3sc	Bacterium strain BLEC3	89.5	3sc
	4sc	Uncultured Sphingomonas sp., clone: LR564B-24	87.4	Sphingomonas sp. 4sc
	5sc	Sphingomonas sp. PP-2 16S	83.9	Sphingomonas sp. 5sc
soil contaminated with motor oil	1mo	Peribacillus frigoritolerans strain TG15	94.6	Peribacillus sp. 1mo
	2mo	Sphingomonas echinoides strain MERYL5-24	94.1	Sphingomonas sp. 2mo
	3mo	Nocardiopsis sp. XLI-8	93.9	Nocardiopsis sp. 3mo
	4mo	Bacillus gibsonii strain S-2	95.0	Bacillus sp. 4mo
	5mo	Ochrobactrum sp. strain S2n90	81.2	Ochrobactrum sp. 5mo
	6mo	Shouchella gibsonii strain LMITABS00983	95.0	Shouchella sp. 6mo
	7mo	Sphingomonas sp. strain MEREH12	83.6	Sphingomonas sp. 7mo
oil contaminated soil	1os	Bacillus tequilensis strain RS53	99.6	Bacillus sp. 1os
	2os	Bacillus toyonensis strain FORT 102	99.1	Bacillus sp. 2os
	3os	Bacillus amyloliquefaciens strain Sihong_838_1	81.7	Bacillus sp. 3os
	4os	Staphylococcus sp. strain FKR3-1	90.8	Staphylococcus sp. 4os
	5os	Bacillus cereus strain 2-2 16S	76.6	Bacillus sp. 5os
	6os	Sphingomonas sp. strain SA4_1	83.6	Sphingomonas sp. 6os
	7os	Sphingomonas sp. strain BWLP17	87.6	Sphingomonas sp. 7os
	8os	Sphingomonas sp. strain MERYL1-1	87.3	Sphingomonas sp. 8os
	9os	Uncultured Sphingomonas sp., clone: LR564B-24	83.6	Sphingomonas sp. 9os
	10os	Uncultured bacterium, clone SIP12-RT-12	86.8	10os

).

The investigation revealed a significant variance in the number of strains isolated from different waste sources. The largest number of strains was retrieved from oil-contaminated soils, while the smallest count originated from rapeseed cake. Among the total isolated strains, 33 were identified at the genus level, and four strains remained unidentified.

The dominant genus observed among these isolates was Sphingomonas sp., with ten strains, followed by six strains attributed to the genus Bacillus sp. Notably, five strains were isolated from potato peelings, predominantly affiliated with the genus Enterobacter sp., a common genus found in organic wastes [22]. The prevalence of Sphingomonas sp. in natural environments, such as soils, is noteworthy as they demonstrate the ability to degrade hydrocarbons [23].

The majority of the strains from oil-contaminated soils (10 strains) were divided between Sphingomonas and Bacillus, with each genus accounting for four strains. Conversely, the minimum number of strains emerged from rapeseed cake, predominantly featuring Pantoea sp. 1rc. The Pantoea genus encompasses a diverse array of bacteria isolated from various environments and is commonly found in the rhizosphere of rapeseed [24].

3.2. Evaluation of Isolates for Their Surface Tension Reduction Abilities

The isolates were assessed for their emulsifying and water surface tension reduction abilities, primarily on a conventional medium containing glycerol (

Table 2. Surface tension on culture media with different carbon substrates.

Type of Wastes	Strains	ST, mN m−1
		Glycerol	Potato Peelings	Waste Frying Oil	Sunflower Cake
Potato peelings	Enterobacter sp. 1pp	58.05 ± 0.681	54.75 ± 0.415	63.28 ± 0.453	55.66± 0.269
	Enterobacter sp. 2pp	43.84 ± 0.206	56.73 ± 0.486	43.41± 0.136	51.96± 0.269
	3pp	52.86 ± 0.599	56.42 ± 0.442	47.99± 0.198	59.03± 0.234
	4pp	45.06 ± 0.198	55.99 ± 0.441	47.74± 0.187	46.53± 0.190
	Enterobacter sp. 5pp	49.58 ± 0.069	61.93 ± 0.305	53.96± 0.354	51.22± 0.56
rapeseed oil	Pantoea sp. 1rc	43.41 ± 0.263	53.81 ± 0.328	52.57± 0.512	49.8± 0.345
grease trap	Proteus sp. 1gt	65.14 ± 0.695	61.58 ± 0.269	48.37± 0.353	58.61± 0.61
	Bacillus sp. 2gt	65.95 ± 0.438	60.27 ± 0.361	56.25± 0.553	62.09± 0.655
oily sludge	Citrobacter sp. 1s	58.59 ± 1.419	52.52 ± 0.225	48.37± 0.335	53.36± 0.489
	Sphingomonas sp. 2s	66.53 ± 0.545	52.11 ± 0.315	56.25± 0.256	58.99± 0.605
waste frying oil	Sphingomonas sp. 1wfo	53.53 ± 0.433	47.33 ± 0.120	49.82± 0.487	50.45± 0.180
	Sphingomonas sp. 2wfo	54.27 ± 0.508	27.83 ± 0.033	47.87± 0.303	29.78± 0.03
	Pseudomonas sp. 2wfo	55.64 ± 0.488	49.70 ± 0.169	43.04± 0.233	58.81± 0.61
	Sphingomonas sp. 4wfo	60.34 ± 0.578	53.60 ± 0.598	54.13± 0.321	55.75± 0.614
	Sphingomonas sp. 5wfo	55.93 ± 0.596	57.91 ± 0.501	54.8± 0.567	58.32± 0.576
Sunflower cake	Sphingomonas sp. 1sc	69.4 ± 0.288	62.02 ± 1.073	63.56± 0.679	65.34± 0.678
	Sphingomonas sp. 2sc	69.29 ± 0.284	64.62 ± 0.761	66.45± 0.665	65.12± 0.608
	3sc	27.83 ± 0.034	59.09 ± 1.147	60.34± 0.64	65.73± 0.603
	Sphingomonas sp. 4sc	68.14 ± 0.351	63.89 ± 0.914	64.87± 0.677	65.99± 0.555
	Sphingomonas sp. 5sc	35.78 ± 0.352	67.87 ± 0.604	65.45± 0.501	63.39± 0.679
soil contaminated with motor oil	Peribacillus sp. 1mo	21.64± 0.018	45.76 ± 0.174	43.56± 0.344	57.57± 0.456
	Sphingomonas sp. 2mo	39.71± 0.03	50.34 ± 0.379	36.77± 0.09	28.32± 0.05
	Nocardiopsis sp. 3mo	48.61 ± 0.097	29.98 ± 0.098	45.34± 0.185	3.45± 0.025
	Bacillus sp. 4mo	47.9 ± 0.648	64.75 ± 0.654	45.1± 0.205	52.11± 0.432
	Ochrobactrum sp. 5mo	29.15 ± 0.077	50.85 ± 0.125	33.89± 0.1	34.49± 0.061
	Shouchella sp. 6mo	59.15 ± 0.389	29.15 ± 0.082	42.58± 0.186	3.14± 0.02
	Sphingomonas sp. 7mo	47.88 ± 0.355	55.95 ± 0.820	48.91± 0.307	53.98± 0.399
oil contaminated soil	Bacillus sp. 1os	53.45 ± 0.484	33.07 ± 0.480	45.13± 0.205	24.19± 0.056
	Bacillus sp. 2os	60.35 ± 0.564	30.86 ± 0.257	32.56± 0.11	3.99± 0.01
	Bacillus sp. 3os	50.11 ± 0.494	31.02 ± 0.234	35.76± 0.101	29.17± 0.055
	Staphylococcus sp. 4os	45.76 ± 0.284	53.30 ± 0.384	44.55± 0.234	49.75± 0.155
	Bacillus sp. 5os	29.42 ± 0.042	28.8 ± 0.090	58.63± 0.566	28.66± 0.04
	Sphingomonas sp. 6os	63.43 ± 0.355	62.11 ± 0.588	61.23± 0.453	63.54± 0.666
	Sphingomonas sp. 7os	64.91 ± 0.566	43.37 ± 0.948	50.08± 0.334	51.1± 0.456
	Sphingomonas sp. 8os	59.78 ± 0.67	49.91 ± 0.142	53.87± 0.788	53.08± 0.489
	Sphingomonas sp. 9os	68.08 ± 0.561	45.12 ± 0.074	45.31± 0.305	47.55± 0.311
	10os	57.51 ± 0.475	50.34 ± 0.086	53.45± 0.398	55.67± 0.499

reduction of surface tension to 40 mN m⁻¹ is highlighted in red.

). Research indicates that a crucial criterion for substantial surface-active properties is the reduction of surface tension to around 40 mN m⁻¹ [25]. Consequently, among the isolates tested, six strains demonstrated notable surface tension reduction capabilities. Notable strains encompassed 3sc, Sphingomonas sp. 5sc (derived from sunflower cake), Peribacillus sp. 1mo, Sphingomonas sp. 2mo, Ochrobactrum sp. 5mo (isolated from soil contaminated with motor oil), as well as Bacillus sp. 5os (from oil-contaminated soil). Among these, there are well-recognized biosurfactant producers, such as representatives of the Peribacillus genus (previously known as Brevibacterium) [26], reducing surface activity to levels of 25.9–27.6 mN m⁻¹ [27,28]. Additionally, the Ochrobactrum genus, isolated from motor oil-contaminated soil, managed to reduce the surface tension of the growth medium from 70 to 30.8 mN m⁻¹ [29]. An intriguing aspect of this study is that the three members of the genus Sphingomonas we isolated exhibited significant emulsifying activity. In particular, strain 2mo, identified as Sphingomonas sp., showed a remarkable surface activity of 39.7 mN m⁻¹. Whereas previously, other studies noted that most Sphingomonas strains do not have the ability to produce biosurfactants [30].

The subsequent phase involved selecting more cost-effective substrates to augment biosurfactant production. To this end, three substrate types—potato peelings, used frying oil, and sunflower cake—were chosen. The efficacy of all isolated strains on these selected waste materials was evaluated by measuring the degree of surface tension reduction (Table 2). These particular wastes were identified as the most high-volume waste sources in the city of Kazan, Republic of Tatarstan, Russia.

An analysis of these waste sources for cultivating biosurfactant producers revealed certain trends (Table 2). Notably, sunflower cake emerged as the most effective substrate, facilitating significant biosurfactant production with noteworthy surface activity in nine strains spanning various genera: 2wfo, Sphingomonas sp. 2mo, Nocardiopsis sp. 3mo, Ochrobactrum sp. 5mo, Shouchella sp. 6mo, Bacillus sp. 1os, Bacillus sp. 2os, Bacillus sp. 3os, Bacillus sp. 5os.

Conversely, used frying oil was the least effective substrate for biosurfactant production, leading to an effective reduction in surface tension in only four strains of genus—Sphingomonas sp. 2mo, Ochrobactrum sp. 5mo, Bacillus sp. 2os, Bacillus sp. 3os.

Among representatives of the genus Bacillus, for example, the species Bacillus toyonensis is known, which was previously isolated from oil-contaminated areas and demonstrated the ability to reduce surface tension to 47 mN m⁻¹ [31]. Similarly, Chaurasia et al. (2020) isolated Bacillus tequilensis LK5.4 from soybean, displaying the capacity to reduce the surface tension of the culture medium by up to 40% [32]. Nocardiopsis sp. B4, isolated from seawater, exhibited a surface tension decrease to 29 mN m⁻¹ during cultivation, with an E24 emulsification index of 80% [3].

3.3. Evaluation of Emulsifying Properties of Isolates

In the subsequent stage, all isolates were examined for the presence of emulsifying properties in their produced metabolites. Emulsification is considered significant when the emulsification index exceeds 50% [33,34] (

Table 3. E24 values in culture media with different carbon substrates.

Type of Wastes	Strains	E24, %
		Glycerol	Potato Peelings	Waste Frying Oil	Sunflower Cake
potato peelings	Enterobacter sp. 1pp	20 ± 1	7 ± 3	20 ± 5	15 ± 5
	Enterobacter sp. 2pp	50 ± 5	36 ± 5	35 ± 5	50 ± 3
	3pp	20 ± 2	29 ± 3	20 ± 2	20 ± 1
	4pp	50 ± 5	14 ± 2	20 ± 1	21 ± 5
	Enterobacter sp. 5pp	30 ± 5	9 ± 1	10 ± 3	5 ± 3
rapeseed oil	Pantoea sp. 1rc	50 ± 5	4 ± 0	4 ± 0	30 ± 3
grease trap	Proteus sp. 1gt	5 ± 0	21 ± 0	10 ± 1	10 ± 2
	Bacillus sp. 2gt	0 ± 0	7 ± 3	5 ± 0	5 ± 0
oily sludge	Citrobacter sp. 1s	5 ± 0	7 ± 4	5 ± 0	5 ± 0
	Sphingomonas sp. 2s	10 ± 2	7 ± 4	10 ± 1	10 ± 2
waste frying oil	Sphingomonas sp. 1wfo	0 ± 0	14 ± 5	7 ± 2	0 ± 0
	Sphingomonas sp. 2wfo	30 ± 2	29 ± 5	30 ± 2	7 ± 4
	Pseudomonas sp. 2wfo	20 ± 1	50 ± 5	30 ± 3	20 ± 2
	Sphingomonas sp. 4wfo	5 ± 0	14 ± 3	10 ± 1	5 ± 0
	Sphingomonas sp. 5wfo	5 ± 0	14 ± 2	10 ± 2	10 ± 3
sunflower cake	Sphingomonas sp. 1sc	20 ± 3	14 ± 2	14 ± 4	14 ± 5
	Sphingomonas sp. 2sc	30 ± 5	0 ± 0	5 ± 0	5 ± 0
	3sc	50 ± 5	14 ± 2	30 ± 2	21 ± 4
	Sphingomonas sp. 4sc	10 ± 2	14 ± 3	10 ± 1	14 ± 5
	Sphingomonas sp. 5sc	20 ± 1	14 ± 4	14 ± 2	29 ± 6
soil contaminated with motor oil	Peribacillus sp. 1mo	50 ± 3	14 ± 0	50 ± 3	50 ± 2
	Sphingomonas sp. 2mo	50 ± 2	14 ± 0	50 ± 2	71 ± 4
	Nocardiopsis sp. 3mo	30 ± 1	14 ± 1	7 ± 1	7 ± 2
	Bacillus sp. 4mo	0 ± 0	0 ± 0	0 ± 0	0 ± 0
	Ochrobactrum sp. 5mo	50 ± 5	14 ± 2	20 ± 2	21 ± 1
	Shouchella sp. 6mo	30 ± 5	29 ± 3	29 ± 2	7 ± 3
	Sphingomonas sp. 7mo	10 ± 2	50 ± 3	30 ± 1	20 ± 5
oil contaminated soil	Bacillus sp. 1os	50 ± 2	29 ± 5	50 ± 1	57 ± 5
	Bacillus sp. 2os	5 ± 0	50 ± 1	30 ± 4	7 ± 0
	Bacillus sp. 3os	20 ± 0	21 ± 5	20 ± 1	14 ± 2
	Staphylococcus sp. 4os	5 ± 0	14 ± 3	5 ± 0	5 ± 0
	Bacillus sp. 5os	20 ± 0	0 ± 0	10 ± 0	14 ± 3
	Sphingomonas sp. 6os	30 ± 2	14 ± 2	10 ± 0	10 ± 3
	Sphingomonas sp. 7os	10 ± 1	14 ± 1	10 ± 2	10 ± 4
	Sphingomonas sp. 8os	30 ± 3	14 ± 3	20 ± 3	20 ± 2
	Sphingomonas sp. 9os	20 ± 1	29 ± 5	20 ± 5	20 ± 1
	10os	20 ± 1	50 ± 2	20 ± 1	20 ± 4

E24 > 50% are highlighted in red.

).

On traditional glycerol-based sources, eight strains—identified as genus Enterobacter sp. 2pp, 4pp (from potato peelings), Pantoea sp. 1rc (from rapeseed cake), 3sc (from sunflower cake), Peribacillus sp. 1mo, Sphingomonas sp. 2mo, Ochrobactrum sp. 5mo (from soil contaminated with motor oil), and Bacillus sp. 1os (from oil-contaminated soil)—demonstrated substantial emulsifying activity (over 50%).

Several of these genus have been previously identified for their emulsification capabilities. For instance, Rabiei (2013) noted that a consortium of Enterobacter sp. genus representatives could produce biosurfactants with an emulsification index exceeding 70%, thereby reducing the surface tension of the nutrient medium from 72 to 31 mN m⁻¹ [35]. Additionally, Essghaier et al. (2023) demonstrated that endophytes of Pantoea alhagi species are capable of producing biosurfactants with an emulsifying activity of 82% [36]. The species representative of B. tequilensis LK5.4 exhibited a maximum emulsification index of 52% [32].

Upon assessing new waste substrates, it was observed that the selected waste materials yielded substances with comparatively lower emulsifying activity than glycerol. Specifically, while glycerol facilitated the synthesis of bioemulsifiers in 8 strains, potato peelings and cake showed 4, and used frying oil demonstrated this trait in 3 strains.

These results indicated that metabolites from strains exhibiting high surface activity may not always exhibit high emulsifying activity. However, metabolites of such strains as 4pp, Peribacillus sp. 1mo, Sphingomonas sp. 2mo, and Ochrobactrum sp. 5mo have both of these activities. It is known from the literature that many bacterial strains produce a diverse mixture of analogues and congeners of biosurfactants under the influence of a single carbon source present in the nutrient medium [37]. Moreover, studies show that surface tension values and emulsification indices may not be uniformly correlated for the same strains [16]. Microorganisms are capable of synthesizing various mixtures of heteropolysaccharides, lipopolysaccharides, lipoproteins and proteins, potentially indicating varying degrees of biosurfactant and bioemulsifying properties. It is known that substances with low molecular weight have mainly surface activity, while substances with high molecular weight are effective as emulsion stabilizers [38]. The combination of polysaccharides, fatty acids and protein components in bioemulsifiers allows achieving greater emulsifying potential [16]. Some studies note the influence of the substrate C/N ratio on surface and emulsifying activity, which is not detected at the same C/N values [39]. Notably, other studies also note the ability of microorganisms of the genera Peribacillus sp., Sphingomonas sp. and Ochrobactrum sp. to produce both biosurfactants and bioemulsifiers, while representatives of the genera Enterobacter sp. and Pantoea sp. produce substances that are mainly biosurfactants.

It’s evident that different strains exhibited varied performance across different substrates. Notably, strains such as Bacillus sp. 2os, Bacillus sp. 3os и Sphingomonas sp. 2mo displayed activity across multiple substrates, whereas Peribacillus sp. 1mo solely showed effectiveness in the glycerol substrate. This variability might be attributed to the individual metabolic capabilities of each specific strain.

The process of identifying the most effective substrate involved the assignment of weight to each parameter (Supplement Table S1). Weight scores were designated to the lowest surface tension (ST) values and the highest E24 values, which were then used to calculate the weighted arithmetic mean (Supplement Table S2). Based on these assessments, it was determined that sunflower cake served as the most effective medium for cultivating microorganisms with surface-active properties. In contrast, glycerol displayed greater suitability for emulsifying properties. Sunflower cake not only supported the growth of microorganisms with emulsifying properties but also secured the second-highest position in the ranking. Considering our objective to select a cost-effective substrate for further investigations, sunflower cake was selected, while potato peelings were identified as the least effective substrate. The efficacy of sunflower cake as a substrate for biosurfactant production has been corroborated by numerous studies [40,41]. For instance, Ciurko et al. (2022) reported that sunflower cake enabled the enhancement of Bacillus subtilis surfactin at a concentration of 1.19 ± 0.03 g L⁻¹ [39].

During the evaluation of the most effective strains, the individual weights for each value and their corresponding weighted arithmetic mean were analyzed. Remarkably, the most effective strains, those with weight scores surpassing 20, included eight specific strains: Enterobacter sp. 2pp, strain 2wfo, Peribacillus sp. 1mo, Sphingomonas sp. 2mo, Ochrobactrum sp. 5mo, Shouchella sp. 6mo, Bacillus sp. 1os, Bacillus sp. 2os.

3.4. Biosurfactants Yield

The yield of biosurfactants for proficient strains in a medium based on sunflower cake was evaluated (Figure 1). Additionally, the yield of biosurfactants in a conventional glycerol-containing medium was also assessed for comparison.

In the glycerol-based medium, the biosurfactant yield ranged from 126–636 mg L⁻¹, while in the medium with sunflower cake, it was between 250–502 mg L⁻¹. Notably, the strain of Sphingomonas sp. 2mo exhibited the lowest yield in the glycerol-based medium, while a strain of the genus Ochrobactrum sp. 5mo showed the lowest yield in the sunflower cake medium. These yields align with existing literature data [8,42]. Rane et al. (2017) reported biosurfactant production in minimal media, noting 0.207 g L⁻¹ when glucose served as the sole carbon source, and 0.241 g L⁻¹ with molasses [18]. However, biosurfactant production wasn’t feasible using whey as the carbon source. Additionally, Rane found that agro-wastes, bagasse, and orange peel extracts yielded 0.127 and 0.089 g L⁻¹ of biosurfactant, respectively. Das (2018) discovered that Pseudomonas azotoformans AJ15 yielded rhamnolipids in the range of 0.6–0.76 g L⁻¹ while cultivating on potato peelings ranging from 5–15 g L⁻¹ [43]. Similar concentrations of sugarcane waste resulted in yields of 0.6–0.97 g L⁻¹.

Notably, the strain Bacillus sp. 2os achieved the highest yield in both glycerol and sunflower cake mediums. Other studies confirm the potential of this genus to attain biosurfactant yields within the range of 1–2.5 g L⁻¹ [30]. However, these results indicate the need for further optimization of the nutrient medium. Despite the slightly superior yield observed based on glycerol, sunflower cake exhibited comparable results and proved to be more economically advantageous.

The manifestation of surface-active and emulsifying properties in biosurfactants of the same microorganisms on different substrates can be explained by the different hydrophilic-lipophilic balances of these substrates. The different hydrophilic-lipophilic balance of sunflower cake and glycerol ensures the synthesis of biosurfactants of different molecular weights with different properties [44].

4. Conclusions

The findings of this study revealed that strains isolated from various sources, including potato peelings, rapeseed and sunflower cake, oil-contaminated soil, soil contaminated with motor oils, waste from water utility grease traps and oily sludge, as well as used frying oil, possess the capacity for biosurfactant production. From these isolates, 8 microorganisms were identified as displaying the greatest potential in biosurfactant synthesis with surfactant and emulsifying properties (Enterobacter sp. 2pp, strain 2wfo, Peribacillus sp. 1mo, Sphingomonas sp. 2mo, Ochrobactrum sp. 5mo, Shouchella sp. 6mo, Bacillus sp. 1os, Bacillus sp. 2os), which will be utilized in further investigations. In this study, for the first time, representatives of the genus Sphingomonas were isolated that have the ability to synthesize biosurfactants with significant emulsifying and surfactant properties. Based on the biosurfactant yields, the Bacillus sp. 2os strain demonstrated the highest biosurfactant production capability—0.501 and 0.636 g L⁻¹ for glycerol and glucose, respectively. The study investigated the feasibility of utilizing three types of waste as carbon sources: potato peelings, rapeseed and sunflower cake, and waste frying oil. It was determined that among all substrates, sunflower cake exhibited the most promising potential for biosurfactant production. This substrate led to the most substantial reduction in surface tension and the highest emulsification index among the majority of the strains tested.