The potential of Bacillus species isolated from Cinnamomum camphora for biofuel production

Background Increasing concerns about climate change and global petroleum supply draw attention to the urgent need for the development of alternative methods to produce fuels. Consequently, the scientific community must devise novel ways to obtain fuels that are both sustainable and eco-friendly. Bacterial alkanes have numerous potential applications in the industry sector. One significant application is biofuel production, where bacterial alkanes can serve as a sustainable eco-friendly alternative to fossil fuels. This study represents the first report on the production of alkanes by endophytic bacteria. Results In this study, three Bacillus species, namely Bacillus atrophaeus Camph.1 (OR343176.1), Bacillus spizizenii Camph.2 (OR343177.1), and Bacillus aerophilus Camph.3 (OR343178.1), were isolated from the leaves of C. camphora. The isolates were then screened to determine their ability to produce alkanes in different culture media including nutrient broth (NB), Luria–Bertani (LB) broth, and tryptic soy broth (TSB). Depending on the bacterial isolate and the culture media used, different profiles of alkanes ranging from C8 to C31 were detected. Conclusions The endophytic B. atrophaeus Camph.1 (OR343176.1), B. spizizenii Camph.2 (OR343177.1), and B. aerophilus Camph.3 (OR343178.1), associated with C. camphora leaves, represent new eco-friendly approaches for biofuel production, aiming towards a sustainable future. Further research is needed to optimize the fermentation process and scale up alkane production by these bacterial isolates.


Background
Fossil fuels have been used for decades to produce liquid fuels such as diesel, gasoline, and kerosene.However, it is predicted that petroleum reserves will be depleted within 40 years [1].This has raised concerns about the global petroleum supply, and environmental issues such as global warming and climate change.As a result, there is growing interest in exploring alternative fuel sources [2].Consequently, a significant focus has been on developing alternative biosynthesis methods for sustainable and ecofriendly biofuel.
Alkanes are hydrocarbons that are essential for biofuel production.They are the key building blocks of renewable biodiesel.Alkanes offer a sustainable alternative to fossil fuels, aligning with global efforts to reduce climate change and improve environmental sustainability [3].The stable chemical structure of alkanes also helps biofuels retain their quality and performance in long-term storage.This stability is critical for meeting the requirements of individual consumers as well as commercial and industrial sectors [4].The traditional commercial production of alkanes constantly increases production costs, non-renewable energy consumption, and gaseous pollutants [5].The microbial biosynthesis of alkanes can be a promising sustainable alternative for chemical production [6].Incorporating microorganisms into the global future of green energy can achieve a distributed and sustainable supply chain that is safe, reliable, and responsive to ever-changing global demand [7,8].
The biosynthesis of alkanes by bacteria has attracted significant attention in recent years due to its potential for biofuel production [9].Bacterial alkanes possess desirable properties, including high energy content and low freezing points compared to other biofuel sources, making them well-suited for specific applications in the aviation or automotive industries [10].
Cinnamomum camphora (L.)J. Presl., popularly known as the camphor tree, is a member of the Lauraceae family.It is native to China, Korea, and Japan and is extensively cultivated in Asia, Africa, North America, and Australia [11].Every part of the plant contains volatile organic compounds that have medicinal properties [12].Previous studies have shown that it is possible to utilize endophytes that inhabit plant tissues to synthesize compounds similar to those produced naturally by the host plants [13].This approach avoids the risk of over-harvesting or the negative effects of climate change on plants, which can affect the production of these compounds [14].Although alkane biosynthesis has been recognized in various microorganisms, including cyanobacteria, genetically modified bacteria, yeasts, and fungi, no studies have reported the production of alkanes by endophytic bacteria [15].Therefore, this study aims to isolate endophytic bacteria from C. camphora leaves and screen the production of alkanes by the isolates in different culture media.The study is an attempt to find renewable sources for bio-alkanes that may be promising for sustainable biofuel production.

Plant material
Leaves from healthy trees of C. camphora were collected from Aswan City, Egypt (24° 5′ 20.1768'' N, 32° 53′ 59.3880'' E) and brought directly to the Aswan University bacteriology laboratory for the isolation of endophytic bacteria.

Isolation and identification of endophytic bacteria
The surfaces of the leaves were sterilized using 5% NaClO, 70% CH 3 CH 2 OH, and autoclaved distilled water, respectively [16].In a 9 mL sterile saline solution, 1 g of the leaves was mashed well.One milliliter of the resulting suspension was then inoculated in trypticase soy and nutrient agar plates.Plates were incubated for 72 h at 37 ℃.Three isolates coded as Camph.1,Camph.2, and Camph.3 were subjected to molecular identification by partially sequencing their 16S rRNA genes.The amplification primers 27F and 1492R were used [17].The separation of PCR products was performed using 1% (w/v) agarose gel.The sequencing of the obtained bands was commercially performed at SolGent Co., Korea.The sequence similarity and identity percentages were determined using the NCBI website (https:// www.ncbi.nlm.nih.gov/).An accession number was gained for each isolate after submitting its 16S rRNA gene partial sequence into the NCBI database.The phylogenetic relationship among the present isolates and the other close members of NCBI was constructed using neighborjoining analysis in MEGA X 10.1.7 software [18].

Determination of bacterial growth curves
The growth curves of the bacterial isolates grown in nutrient broth (NB), Luria-Bertani (LB) broth, and tryptic soy broth (TSB) were determined using the turbidimetric method [19].In 250 mL conical flasks, 50 mL of each medium was prepared and autoclaved.The flasks were inoculated with 100 µL of each bacterial inoculum (1.5 × 10 8 CFU/mL, OD 600 = 0.1).The flasks were incubated at 37 °C under shaking (150 rpm).The optical density was read at 600 nm at intervals of 10 h until the stationary phase was reached.The flasks without bacterial inoculums served as controls.The experiment was conducted three times.

Fermentation conditions
The freshly prepared inoculum (100 µL of 1 × 10 7 CFU/ mL) of each bacterial isolate was inoculated in 500 mL flasks containing 100 mL of three different broth culture media: NB, LB, and TSB.The flasks were incubated for 48 h at 37 °C and 150 rpm.Flasks containing media without bacterial inoculum were used as controls.Triplicates were made for all fermentations.

Extraction and GC/MS analysis of alkanes
Hexane was added to the bacterial cultures in a ratio of 1:1 (v/v) and homogenized well.The solvent layers were then separated and concentrated using a rotary evaporator at 40 °C and 130 rpm under pressure.Each extract (1 mg) was redissolved in 10 mL of hexane.The GC/MS system used in this study was the Agilent Technologies 7890A GC/5977A MSD supplied with a TR-5MS GC column (30 m, 0.25 mm ID, and 0.25 μm film).The sample (1 μL) was injected into the column, and the oven temperature was initially set at 30 °C for 1 min.The temperature was then increased at a rate of 10 °C/min until reaching 200 °C, where it was held for an additional 1 min.The carrier gas, helium, was used at a flow rate of 20 mL/min.The retention times of the sample peaks were compared with NIST11.L standard reference compounds.The alkane standard mixture (C 7 -C 40 , Millipore Sigma ™ Supelco ™ ) was used to quantify the alkanes in the samples.

Effect of carbon sources on alkane production
In conical flasks, the basal medium consisted of the following components per liter: KH 2 PO 4 (1.3 g), MgSO 4 .7H 2 O (0.2 g), NaCl (5 g), (NH 4 ) 2 SO 4 (1 g), and yeast extract (5 g) was supplemented with different carbon sources including glucose, sucrose, and sugar cane molasses, each at a concentration of 10 g/L.Flasks were then inoculated with 100 µL of a freshly prepared inoculum containing 1 × 10 7 CFU/mL.Flasks were incubated for 48 h at 37 °C and 150 rpm.Flasks containing media without bacterial inoculum were used as controls.Triplicates were prepared for all fermentations.Alkanes were extracted and analyzed using the method described above.

Results and discussion
The use of biofuels has become crucial in addressing the worldwide concerns of the energy crisis and climate change.Microbial alkanes provide a renewable, eco-friendly, and promising source for the sustainable production of biofuels [20].Unlike fossil fuels, biofuels derived from microbial alkanes not only decrease carbon emissions but also mitigate the effects of global warming [21].The low toxicity and biodegradability of bacterial alkanes make them eco-friendly alternatives to synthetic alkanes, serving various applications [22].

Determination of bacterial growth curves
From a commercial perspective, the growth of microorganisms is a significant challenge in the industrial production of valuable chemicals [26].Therefore, the growth curve for each bacterial isolate was determined in each culture medium.It was observed that the exponential phase of the three isolates began after 20 h of incubation and extended until 50 h.The stationary phase continued for 20 h, after which the growth rate declined (Fig. 2).Generally, the growth rate was higher in Fig. 1 The phylogenetic relationship among the isolates Camph.1,Camph.2,Camph.3, and the closely related species from the NCBI database using the neighbor-joining method in MEGA X10.1.7 software LB followed by TSB.The NB, on the other hand, had the lowest growth rate for all isolates (Fig. 2).
Interestingly, the profiles of alkanes released by the three bacterial isolates in the three tested culture media differed.For all isolates, the highest number of alkanes was detected in the LB medium (Fig. 3, 4  5).This finding aligns with previous studies that have reported a significant effect of medium composition on the profiles of volatile organic compounds released by microorganisms [27].

Effect of carbon sources on alkane production
Interestingly, various alkanes were produced by the three bacterial isolates using glucose, sucrose, and sugar cane molasses as carbon sources.This is consistent with previous studies that reported significant differences in hydrocarbon profiles produced by microorganisms based on carbon sources [28]   As stated above, the chain length of alkanes produced by the present isolates ranged from C 8 to C 31 (Tables 1, 2,3,4,5,6,7,8,9).Previous studies have reported that bacterial alkanes typically have chain lengths ranging from C 10 to C 36 , although this can vary depending on the bacterial strain and environmental conditions [29].The biosynthesis of n-alkanes by various bacteria including Desulfovibrio sp., Clostridium sp., Pseudomonas fluorescens, Vibrio furnissii M1, and Engineered Escherichia coli has been reported [30][31][32][33].Although endophytic bacteria were known within the biotechnology field for their ability to produce a great variety of sustainable safe, eco-friendly products, there are no reports about their ability to produce alkanes [34].Therefore, this study is the first documentation of alkane production by endophytic bacteria.
The alkanes are preferred as clean fuels, because they burn cleanly and easily, releasing a lot of heat and light energy [35].In the present study, the three Fig. 5 The heatmap displays the amounts of bio-alkanes (mg alkane/L culture) produced by B. aerophilus Camph.3 in NB, LB, and TSB media studied endophytic bacteria produced a variety of alkanes as mentioned above.Many of these alkanes are used in biofuel production.Octane and decane are the main constituents of gasoline.Octane is used in internal combustion engines.Nonane, decane, undecane, tetradecane, pentadecane, and hexadecane make up the majority of diesel, kerosene, and aviation fuel.Heptadecane, octadecane, ecosane, pentacosane, hexacosane, heptacosane, octacosane, and heneicosane are the main components of lubricating oil [36].media.Numerous of the produced alkanes, such as octane, nonane, decane, undecane, tetradecane, pentadecane, and hexadecane are used in biofuel production, such as gasoline, diesel, kerosene, and aviation fuel.Therefore, these endophytic bacteria may be promising and sustainable sources for alkane biofuel production.

Fig. 3
Fig. 3 The heatmap displays the amounts of bio-alkanes (mg alkane/L culture) produced by B. atrophaeus Camph.1 in NB, LB, and TSB media

Fig. 4
Fig.4 The heatmap displays the amounts of bio-alkanes (mg alkane/L culture) produced by B. spizizenii Camph.2 in NB, LB, and TSB media