Production of Single-Cell Oil from a Local Isolate Bacillus subtilis Using Palm Fronds

This study, conducted at the Department of Biology, University of Anbar, Iraq, focuses on addressing the escalating issue of contamination and aims to acquire microbial oils to alleviate the global shortage in plant and animal oil production, utilizing environmental waste as a carbon source to reduce global pollution and select efficient local bacterial isolates of Bacillus subtilis for the production of single-cell oil (SCO) using local soil and environmental waste as a carbon source. Four isolates were selected as the best in producing single-cell oil, with the isolate with code C4 standing out as it recorded the highest production. It is worth noting that all these isolates belong to the bacteria type Bacillus subtilis. Palm fronds were found to be the most suitable environmental residue for SCO production compared to other waste materials (wheat straw and wheat bran). Submerged cultures were used to improve SCO production, with optimal conditions determined as pH 7, a temperature of 30°C, carbon source concentration of 3 g/100 ml, inoculum volume of 3 ml/100 ml, inoculum density of 20 × 107 cells, and an incubation period of 72 hours. The Soxhlet extraction method was used to obtain the oil, which was found to contain high percentages of unsaturated fatty acids, particularly linoleic acid (46.030%) and palmitoleic acid (16.579%). The oil was highly soluble in chloroform and ethanol but insoluble in water. The saponification test indicated the potential for soap production from the oil. This comprehensive research addresses the need for locally sourced and sustainable SCO production, offering insights into the selection of efficient bacterial isolates, the optimization of cultivation conditions, and the valuable properties of the resulting SCO. The significance of this study lies in the production of single-cell oil from soil-isolated Bacillus subtilis bacteria for use in food applications.


Introduction
Te industrial revolution and the rapid development of agricultural technologies led to the introduction of a multitude of pollutants into our daily lives [1], which, over time, began to create environmental problems.It was believed that nature could overcome these environmental problems by itself, but the increasing amount of waste has slowly begun to exceed the limits that can be naturally absorbed [2].
Currently, environmental research focuses on mitigating the environmental damage caused by waste build-up [3] with regard to the by-products of agricultural industries, considering them as intermediates to the biological production of high-value products, which represents a viable alternative to deal with the environmental impacts of waste [4][5][6].
Te use of microorganisms in ecosystems can help reduce waste and create a pollution-and toxin-free environment [7].
Oily microorganisms can produce single-cell oil (SCO) through the use of diferent carbon sources in their growth media [8].Industrial applications that make use of these organisms are somewhat limited in number, such as ingredients added to cosmetics and using cooking oil to produce biofuel; however, due to the high cost of production, but the combination of SCO production and other biotechnology applications, including waste and byproducts, has overcome this particular difculty [9].Oils derived from microbial sources are called microbial oils or SCO oils [10].
SCOs are intracellular lipids composed of TAGs, free fatty acids, polar lipids, sterols, hydrocarbons, and pigments [11,12].Te term SCO is used as a parallel to SCP to refer to oils of microbial origin [13].
All microorganisms contain lipids, usually at about 6 to 8% (w/w) of the dry cell weight [14].Microorganisms that accumulate more than 20% of their weight as fat on a cell dry weight basis are considered oily microorganisms [15], a defnition that includes various types of microalgae, bacteria, yeasts, and molds capable of accumulating fat.
Oily microorganisms can accumulate lipids and predominant tri-glycerol and fatty acids more efciently than plants in a shorter production cycle that gave yields ranging from 20 to 87% of their dry weight [16].
Microorganism-derived oils, also known as single-cell oils (SCOs), are similar in composition to vegetable oils [17] and animal fats [11]; however, SCOs are preferred over oils derived from plants and animals because it is easy to increase their production.
Also, SCO production is unafected by factors such as seasonal changes, geographic location, harvest time, and transportation, which are a concern when relying on plant and animal materials [18].
Te propensity of microorganisms to accumulate lipids is mainly determined by their genetic characteristics [19], and this may difer between species or even between strains of a particular species [20].
B. subtilis bacteria, also known as grass bacillus or hay bacillus, are mostly found in soil [22] and are not pathogenic to humans [23].B. subtilis bacteria can be isolated from many terrestrial and aquatic environments, which make this species apparently omnipresent; however, this type of bacteria is the same as all other bacilli that can form inactive spores with high durability that enables them to resist nutritional defciency and unfavorable environmental conditions [24].
Te study aims to produce single-cell oils SCO from local bacterial B. subtilis isolates using available plant waste in the Iraqi local environment.Additionally, it aims to identify the fatty acid content of these plant oils.

Methodology
2.1.B. subtilis Isolate.Twenty samples were collected from the soil of the rhizosphere of diferent plants that were selected from separate areas in Anbar Governorate for the purpose of isolating B. subtilis bacteria.
A series of decimal dilutions of the selected soil samples was prepared by adding 10 grams of each soil sample after cleaning out impurities to 90 milliliters of sterile water in a glass fask with a capacity of 250 milliliters and then placed in a water bath at 80 °C for 20-30 minutes to degrade any vegetative cells accompanying the B. subtilis spores, after which a series of decimal incisions were made.
Te method of pouring plates was used, as 1 ml of the fourth dilutions, 10 − 4 , and the ffth, 10 − 5 , were taken, each of which was placed in a sterilized Petri dish along with the isolation medium, consisting of 10 g tryptone, 5 g yeast extract powder, 10 g NaCl, and 15 g Agar in 1000 ml of distilled water.Te plates were incubated at 30 °C for 24 hours, after which the growing isolates were purifed by subculturing them on the same used culture media.Te process was then repeated several times to obtain pure cultures and single colonies.To ensure that the developing isolates belonged to the genus B. subtilis, they were plotted on the selective medium (HiCrome TM Bacillus Agar Base) prepared according to the manufacturer's instructions.Te dishes were incubated at 30 °C for 24 hours, after which the appearance of bacterial colonies in a light green color on this medium indicates the presence of B. subtilis.

Identifcation of B. subtilis Isolates.
Te obtained bacterial colonies were identifed based on their morphological characteristics including colony shape, texture, height, color, and edges.Te characteristics of the bacterial cells were then studied microscopically after staining them with Gram's stain according to Holt et al. [25].To ensure the formation of spores, they were stained with Malachite green.Biochemical tests were also conducted for the bacterial isolates in order to diagnose them.Tese tests included the following: Oxidase test, as per Baron et al. [26]; catalase test, as per Baron et al. [26]; motility test, as per Murray et al. [27] and Forbes et al. [28]; Voges-Proskauer test, as per Colle et al. [29] and MacFaddin [30]; methyl red test, as per Colle et al. [29] and MacFaddin [30]; indole test, as per Cruickshank et al. [31]; urease test, as per MacFaddin [30] and Forbes et al. [32]; citrate utilization test, as per Atlas et al. [33] and MacFadden [30]; and starch analysis, as per Benson [34].
Testing the growth of isolates in a saline medium concentration of 6.5% NaCl: the growth efciency of bacterial isolates was tested in a saline medium with a concentration of 6.5% by replacing the percentage concentration in the medium of a previously prepared B. subtilis isolate with a concentration of 6.5%.

Testing the Ability of the Diagnosed Bacterial Isolates to
Produce Oil (Primary Screening) 2.3.1.Te First Stage.Te selected bacterial isolates were cultured on a laboratory-prepared oil agar medium consisting of 40 grams peptone, 5 grams sucrose, 5 milliliters sunfower oil, and 15 grams agar in 1000 milliliters of distilled water, by making a circle with a diameter of 1 cm in the center of the dish and incubating at a temperature of 30 °C for 48 hours, after which the diameters of the bacterial colonies growing on the medium were measured.

Te Second Stage.
Te growing bacterial colonies were reseeded on the oil agar medium by making a circle with a diameter of 1 cm in the center of the plate and 2 International Journal of Biomaterials incubating at a temperature of 30 °C for 24 hours only.After that, the most efcient isolates were selected, and the diameters of the colonies growing in the center of the plate were measured.

Testing the Efciency of the Selected Isolates on Collecting
Oil Using Sudan Black Dye B (Secondary Screening).Te selected oil-producing isolates were planted on a nutrient agar medium in the form of a circle with a diameter of 1 cm in the center of the plate and incubated at a temperature of 30 °C for 24 hours.Te surface of the plate was then immersed in a solution of Sudan black dye B, prepared by dissolving 0.3 grams of dye powder black Sudan in 100 ml of 70% ethanol and then being left for 30 minutes followed by gently washing the plate with 96% ethanol [35].

Diagnostics with Vitek 2 Compact
System.Te Vitec device, produced and developed by the French company bioMerieux located in the laboratory of Al-Ramadi Teaching Hospital, was used to diagnose the selected bacterial isolate after confrming it by means of biochemical tests.
2.6.Preparation of the Bacterial Inoculum.Te selected B. subtilis inoculum was prepared by culturing it in a 250 ml glass beaker containing 100 ml of nutrient broth culture medium and incubating for 24 hours, after which a white thin tissue appeared on the top of the surface and a turbidity of the medium was observed, indicating the readiness of the vaccine [36]; the bacterial density in the inoculum was then estimated.

Election of the Best Environmental Waste as a Carbon
Source for Oil Production.According to the amount of oil produced from the bacterial isolate in the media of the three liquid wastes, palm fronds and wheat straw wastes were prepared by washing to remove dirt and impurities.Ten, they were air-dried in an electric oven at 65 degrees Celsius for 48 hours (except for the bran, which was already dry and clean).Te three wastes were ground to pass through a 40micron sieve.Te liquid culture method was used to determine the amount of oil produced from the bacterial isolate in each of the three media used.Te three liquid media were prepared by dissolving 0.1 grams of peptone, 0.24 grams of KH 2 PO 4 , 0.24 grams of K 2 HPO 4 , and 2 grams of palm fronds, wheat bran, or wheat straw.
After that, 1 ml of the bacterial inoculum was added to the liquid fronds medium, 1 ml of the bacterial inoculum was added to the liquid hay medium, and 1 ml of the bacterial inoculum was added to the bran liquid medium, each using conical fasks with a capacity of 250 ml.Te fasks were incubated at a temperature of 30 °C for 24 hours.After being fltered and dried in an electric oven at a temperature of 60 °C for 24 hours in a Soxhlet device using petroleum ether as a solvent to extract the oil, the percentage of oil produced from the bacterial isolate was extracted in each medium.

Determination of Optimal Conditions for Single-Cell Oil
Production from B. subtilis.Te liquid medium of palm fronds (single-cell oil production medium) was used to determine the factors afecting the production of single-cell oil, as will be explained in the following sections, using conical fasks of 250 ml capacity, where 100 ml of liquid medium were placed in each.

pH.
To determine the optimal pH for single-cell oil production, the pH of the medium was set at 10, 9, 8, 7, 6, and 5.

Temperature.
To determine the optimal temperature for the production of single-cell oil in the production medium, diferent temperatures (45,40,35,30,25, and 20 °C) were used.

Inoculum Volume.
To determine the optimal inoculum size for single-cell oil production, diferent inoculum volumes were used (5, 4, 3, 2, 1, and 0.5 ml/100 ml of the liquid production medium).

Concentration of Carbon
Source.Palm fronds were used as a carbon source to produce single-cell oil at diferent concentrations (1, 2, 3, 4, 5 g/100 ml) in the medium to determine the optimal concentration for oil production.

Incubating Time.
To study the efect of the incubation period on the production of single-cell oil, the selected isolates were grown for diferent time periods (24,48,72, and 96 hours), as the isolate was grown in 100 ml of liquid medium, each separately, at the optimum temperature, pH, carbon source concentration, and inoculum size identifed in the previous steps.
Te percentage of oil produced in each step was estimated using the Soxhlet device according to the method described in A.O.A.C. (1990).

Developing the Selected Bacterial Isolate According to the
Optimal Conditions for Production.Te selected oilproducing bacterial isolates were developed according to the optimum conditions obtained for single-cell oil production, as determined in the previous sections.

Solubility Test.
A certain volume of water, ethanol, and chloroform was placed in three separate glass test tubes, and the oil produced was then added in an amount of 1 ml to each tube, after which the tubes were then stirred.Tereafter, the melting nature of the oil produced was observed for each tube [35].

Saponifcation
Test. 2 ml of NaOH sodium hydroxide solution (at a concentration of 2%) was added to 3 ml of the oil produced in a test tube to observe the formation of a soapy solution [35].
International Journal of Biomaterials

Estimation of Fatty Acids in the Oil Produced.
Long-chain fatty acids were estimated via the GC device (Perkin Elmer) according to the standard method of Raslan [37].Te fow rate was 1 ml/min and 105 °C for 1 minute and then the temperature was fxed to 220 °C with 4 psi, whilst short-chain fatty acids were estimated according to the standard method of Torii et al. [38].Using 1 gram of the oil, 3 ml of hexane was added, and the mixture was shaken for three minutes.Afterward, it was centrifuged for ten minutes at a speed of 10,000 revolutions per minute.Te supernatant layer was withdrawn and dried with nitrogen gas.Ten, 1 ml of methanol was added, and it was injected into an HPLC system at a fow rate of 0.8 ml per minute and a temperature of 30 °C.

Results and Discussion
3.1.B. subtilis Isolate.In our study, B. subtilis was isolated from the soil of the rhizosphere of diferent plants, as the bacteria belonging to the genus Bacillus are classifed as rhizobacteria that promote plant growth and are found in the rhizosphere [39].
Al-Rawi and Qadir [40] and Al-Attar et al. [41] indicated the possibility of isolating B. subtilis from soil.15 bacterial isolates were obtained that grew with high efciency on the isolation medium (Figure 1(a) out of 20 soil samples brought to the laboratory, where the isolation percentage was 75%.Also, all bacterial isolates grew on the prepared selective medium (HiCrome TM Bacillus Agar Base) as indicated by their light green color Figure 1(b).

Culturing and Microscopic
Characteristics.Te cultivation characteristics of the bacterial isolates grown on the selective medium were studied, from which it was found that most of the colonies of these bacterial isolates are circular in shape with diferent regular and irregular edges (some of them have zigzag or lobed edges) and are white-yellow in color and medium to large in size.
Te cells' shapes and arrangement after being stained with Gram's stain showed that they are sticky, straight, or oblong and positive for Gram's stain, often in the form of pairs or chains Figure 2(a), while staining them with Malachite green showed that they are spore-forming Figure 2(b).From these characteristics, they were found to match the characteristics of the B. subtilis bacterium [42].

Biochemical Tests.
Te results of the biochemical tests adopted in the diagnosis of bacterial isolates are shown in Table 1.

Testing the Ability of the Diagnosed Bacterial Isolates to
Produce Oil

Primary Screening (First Stage).
Te results of the initial screening reported in Table 2 indicate the growth of all bacterial isolates (15) from diferent places with the same condition on a medium agar of oil that was used as a source of carbon and energy.Te same table shows a discrepancy in the diameters of the growing colonies.Tis could be due to the variation in the ability of these isolates to consume oil as their only source of carbon and energy, as well as the nature of the source of isolation for bacteria.Te same table also shows that the largest diameter of the growing colonies was 8.5 cm, whilst the lowest was 1.5 cm.
Trough these results, the eight most efcient bacterial isolates in growth were selected for the purpose of screening them during the second stage, which are K3-L3-K3/1-C 4-E 4-M 4-F4/1-D 4/1.

Primary Screening (Second Stage).
Table 3 shows the results of the second stage of the initial screening.Te results showed that four highly efcient isolates were obtained.Te diameter of the colonies for these isolates was 8.5 cm after 24 hours of incubation on average.Tese isolates are E 4-C 4-D4/1-F 4/1.

Testing the Efciency of the Selected Isolates to Collect Oil Using Sudan Black Dye B (Secondary Screening). Te results
showed that the isolate with the symbol C4 was the most efcient in its ability to accumulate oil (Figure 3).
Osman et al. [43] used Sudan black dye B to examine the ability of Rhodotorula diobovata yeast strains to produce oil.

Diagnostics Vitek 2 Compact
System.Te results of the diagnostic tests on the Vitec device confrmed that the isolate with the local code C4 belongs to the type B. subtilis, with a probability of 91%.

Choosing the Optimal Environmental Waste as a Carbon
Source for Oil Production.Te results in Figure 4 regarding the proportions of oil obtained using the three previously mentioned agricultural wastes and by the method of liquid farms showed that there is a discrepancy in oil production, as the highest production was 2.698% using palm fronds, while the lowest was 1.670% using wheat bran.
Te discrepancy in the oil production according to the source used may be due to the fact that palm fronds contain a high proportion of fat in the form of lignin, which helps to increase the percentage oil collection by the selected bacterial isolate; the high ratio of C : N and the low proportion of nitrogen in palm fronds are important reasons for the increased proportion of oil so produced.Te C : N ratio is considered one of the most important factors in the production and collection of microbial fats [16,44].
It should be noted that to the best of our knowledge, there is no previous study in this feld with which direct comparison can be made.23.92%; oil productivity decreased signifcantly at other pH values.Tese results are consistent with those of et al. [45] who also noted that the best SCO production from B. subtilis was at pH 7.0 and with those of Rasouli et al. [21] who also reported that pH 7.0 was the optimum in production for the bacteria Kocuria sp.; by contrast, Khaleel and Al-Rawi [46] indicated that pH 10 was the optimal pH for the production of oil from the bacterium Pseudomonas fuorescens.

Efect of Optimum Conditions on SCO Production
In this regard, Mironov et al. [47] indicated that pH has a clear efect on oil production rates as it is a stress factor for cell growth and afects the aggregation of oils and the formation of fatty acids.pH also afects various metabolic processes, including nutrient solubility and absorption, and the activity of enzymes responsible for oil production.
3.7.2.Temperature.Figure 6 shows that the highest oil production for the selected B. subtilis bacteria was at a temperature of 30 °C, for which the proportion of oil produced was found to be 42.13% of the biomass, followed by a temperature of 35 °C, where oil production was 22.52%; production was found to decrease signifcantly at other temperatures.Tese results are consistent with those of Rasouli et al. [21], who reported that the optimal temperature for single-cell oil production from Kocuria sp. was 30 °C, and Khaleel and Al-Rawi [46] who indicated that the highest SCO production from Pseudomonas fuorescens was, again, at a temperature of 30 °C.
Temperature is an essential and infuencing factor in the growth and vital activities of microorganisms, as well as an essential means for controlling metabolic activities, both structural and destructive, especially in fermentation processes [48].
A majority of studies indicate that there is a clear efect of temperature on the production of unicellular oils and the synthesis of unsaturated fatty acids through its efect on the cellular membranes and their fuidity, since the latter afects the work of membrane-bound enzymes and transport mechanisms to and from the cell [47].6 International Journal of Biomaterials 3.7.3.Inoculum Volume.Te results in Figure 7 show that the highest oil productivity was when the bacterial inoculum added at a volume of 3 ml/100 ml to a production medium with a density of 20 × 10 7 cell/ml, for which production was 43.93%, after which the production rate decreased when the inoculum volume was increased or decreased.Te results we obtained agree with those of Khaleel and Al-Rawi [46] who showed that an inoculum volume of 3 ml/100 ml medium was optimal for the production of SCO from Pseudomonas fuorescens.
Te concentration of inoculum is one of the more important factors in production, as the production process is controlled in its conditions, including the size of the inoculum used to reach the highest production [49].
Te size and type of the production process determine the amount of inoculum used.Tis amount must be in a quantity commensurate with the amount of culture medium prepared for production.Te aim is to reduce the fermentation period at the start of production [50].Low pollen size leads to a longer imprinting phase, which in turn delays the onset of lipid accumulation and thus leads to a decrease in production [51].Also, a low pollen size leads to a decrease in biomass production, which reduces lipid productivity [52].As for increasing the amount of inoculum, this leads to rapid growth and depletion of nutrients, which afects the fat yield.Also, an increase in the amount of the inoculum leads to an increase in competition for nutrients in the culture medium, which causes a decrease in growth [53].

Carbon Source Concentration.
Figure 8 shows that the highest oil yield was at a concentration of 3 g/100 ml medium, as the proportion of oil produced was 49.05% after an incubation period of 48 hours and at a temperature of 30 °C and a pH of 7. Tis decreased at other concentrations to its lowest of 28.58% at a concentration of 5 g palm fronds/ 100 ml medium.Te important factor on which single-cell lipid formation depends is on the availability of nutrients that lead to a large amount of carbon source to stunted growth and oil production even if nitrogen is present in less quantity.
Tere is a need for further knowledge and experience in selecting an appropriate carbon source and determining an appropriate concentration.Te relationship between the rate at which the carbon source is added and fat accumulation is positively linked.However, high concentrations of carbon source actually inhibit production of oil from microorganisms.
Te C : N ratio is also of great importance in the production of microbial oils [44].

Incubation Period.
In order to determine the best incubation period for the fermentation medium, time periods ranging from 24 hours to 96 hours were adopted.Te results shown in Figure 9 show that the highest single-cell oil production by the selected bacterial isolate was after 72 hours of incubation, where the production rate reached 50.58% at a temperature of 30 °C and a pH of 7, while oil production notably decreased when the incubation period was reduced to less than 72 hours.
It was also noted that production decreased after 92 hours of incubation to 48.99%.Te incubation period that we obtained difers from that reported by Zhang et al. [54] who indicated that the incubation period of 48 hours was optimal in the production of SCO from B. subtilis bacteria after growing them on cotton stalks as the only source of carbon and from that reported by Khaleel and Al-Rawi [49] who indicated that the best unicellular oil production from Pseudomonas fuorescens using corn cobs as a source of carbon source was also after 48 hours of incubation.
Te duration required for incubation is related to a number of factors such as the type of isolate selected, pH, temperature, amount of inoculum, and composition of the  International Journal of Biomaterials production medium [55].By increasing the incubation period, oily organisms can accumulate additional biomass.Tis leads to an increase in fat productivity that dependent on the availability of nutrients and the specifc conditions of cultivation [56].Productivity can decrease with increasing incubation period due to nutrient depletion, changes in the medium such as pH, autolysis of cells, and the release of toxins into the medium [57].
Figure 9 shows that the optimal incubation period for production is 72 hours, which is sometimes considered quite a long period.Tis may be due to the nature of the carbon source used (palm fronds), since this source contains a high proportion of lignin.Bacteria need a longer period and the ability to secrete specialized enzymes for the purpose of its analysis.Note that this source led to a higher productivity than SCO.

Developing the Selected Bacterial Isolate According to
Optimal Conditions for Production.Te selected bacterial isolate was grown according to all optimal conditions that were obtained from pH, temperature, volume of inoculum, carbon source concentration, and incubation period, as shown in Table 4. Te proportion of oil production after setting all conditions to optimal was 51.32%.
Tis result difers from that of Rasouli et al. [21] who achieved a 24.57% production of unicellular oil from Kocuria sp. while being similar to that achieved by Khaleel and Al-Rawi [46] who obtained a 55.47% production of SCO from Pseudomonas fuorescens.
3.10.Confrmation Tests for the Oil Produced.Te oil produced from B. subtilis was liquid at room temperature, with a brownish-yellow color, as shown in Figure 10.Confrmation tests were conducted, including the following.

Solubility Test.
Te results of the solubility test conducted on the oil produced shown in Figure 11(a) indicated that the oil had a high solubility in chloroform solution and a lower solubility in ethanol, while it did not show any solubility in water.
Oils have been identifed as a variety of hydrophobic compounds that are soluble in nonpolar organic solvents such as ether and chloroform, which are insoluble in water [58].3.11.Te Fatty Acid Content of the Oil Produced.Te results of analysis of long-chain fatty acids using the GC device and short-chain fatty acids using the HPLC device reported in Table 5 A and B, respectively, indicated that the oil contained a high proportion of unsaturated linoleic acid, at 46.03%.Cianchetta et al. [16] concluded that linoleic acid and stearic acid are among the most important fatty acid constituents of the unicellular oil produced from Cutaneotrichosporon oleaginosum, Rhodosporidiobolus azoricus, and Lipomyces starkeyi.Khaleel and Fakhri [60] obtained SCO with high linoleic acid and stearic acid content, while Burgstaller et al. [61] noted that the main fatty acid present in the oils produced from Apiotrichum brassicae and Pichia kudriavzevii was oleic acid.

Conclusion
Isolating strains of Bacillus subtilis from local agricultural soil efciently produced SCO.Te utilization of local environmental waste shows the potential for obtaining high yields of SCO.Te produced oil composition contains a high proportion of unsaturated fatty acids.Tese fndings highlight the promising potential of using local resources for sustainable single-cell oil production.
4.1.Recommendations.Te recommendations are exploring new methods for extracting monounsaturated oil, increasing the extracted oil's percentage, and conducting further studies to isolate fatty acids according to their types from the produced oils.

3. 7 Figure 2 :
Figure 2: Microscope photos of B. substiles cells.(a) B. subtilis cells under a compound light microscope (100x magnifcation), after staining them with Gram stain.(b) Formation of blackboards after 30 days, from keeping B. subtilis in the refrigerator.

Figure 1 :
Figure 1: Bacteria growth.(a) Growth of bacterial isolates on the selective medium prepared in the laboratory.(b) Growth of bacterial isolates on the prepared selective medium.

Figure 4 :
Figure 4: Percentage oil production according to type of plant waste used.

Figure 5 :
Figure 5: Te efect of pH on the oil produced from B. subtilis isolate.

Figure 8 :
Figure8: Te efect of the concentration of the carbon source (palm fronds) in g/100 ml medium on the oil produced from the bacterial isolate B. subtilis at pH 7, a temperature of 30 °C, and an inoculum volume of 3 ml/100 ml medium.

3. 10 . 2 .
Soaping Test.Te results of the saponifcation test conducted on the oil produced from B. subtilis illustrated in Figure11(b) showed that a soapy solution was formed.

Figure 9 :
Figure9: Te efect of the incubation period (hours) on the oil produced (%) from an isolate of B. subtilis at pH 7, a temperature of 30 °C, an inoculum volume of 3 ml/100 ml medium, and a carbon source concentration of 3 g/100 ml medium.

Table 1 :
Te results of the biochemical tests adopted in the diagnosis of bacterial isolates.
Order Isolate code Oxidase Catalase Motility Simmons citrate Urease MR VP Indole Starch analysis Growth in 6.5% NaCl 1 K 3

Table 3 :
Te average diameter of the colonies growing on the oil agar medium after 24 hours.

Table 2 :
Average diameters of colonies growing on oil agar media after 48 hours.Figure 3: Isolate C4 stained with Sudan black dye.
Figure 7: Efect of amount of inoculum (ml) on oil produced by B. subtilis isolate at pH 7 and a temperature of 30 °C.