Enhancement of Biodiesel Production from Fusarium oxysporum NRC 2017 with Various Mutagenesis and Optimization of the Cultural Conditions Using Response Surface Methodology

doi:10.21203/rs.3.rs-2865792/v1

Background: Climate change is still a problem we face that has an impact on us every day even as our world develops technologically. Energy has been and will continue to be a key engine of economic growth. The greenhouse gas emissions (GHG) from biodiesel are less than those of fossil fuels. The objective was to improve biodiesel production from a fungus called Fusarium oxysporum NRC 2017 using the bagasse hydrolysate of Bacillus cereus 3SME as a cheap carbon source.

Results: To enhance lipid accumulation in F. oxysporum NRC 2017, four mutagenic agents were used: gamma radiation (Ɣ ray); ethidium bromide (Et Br); ethyl methane sulfonate (EMS); and sodium azide (NaN₃). Using the Inter-Simple Sequence Repeats (ISSR) as a molecular biomarker, the findings demonstrated a significant difference between the F. oxysporum NRC 2017 wild type and induced mutants examined. The culture conditions were optimized for F. oxysporum NRC 2017 and F. oxysporum NRC 2017-1, F. oxysporum NRC 2017-2, and F. oxysporum NRC 2017-3 using the response surface methodology (RSM) and the maximum lipid content (g/l) was 2.40, 3.47, 3.81, and 2.63, respectively. According to gas chromatography (GC) analysis, the biodiesel compositions produced from the F. oxysporum NRC 2017 wild type and its mutants exhibited generally C16-C18 and were suitable for biodiesel synthesis. F. oxysporum NRC 2017 and mutant biodiesels indicated physical features that were close to those of standard biodiesel.

Conclusion: The biodiesels produced from the F. oxysporum NRC 2017 and its mutants could be used for large scale as eco-friendly products from a cheap carbon source.

Biological sciences/Cancer

Biological sciences/Microbiology

Earth and environmental sciences/Environmental sciences

F. oxysporum NRC 2017

mutation

ISSR

RSM

GC

Physical properties

Climate change issues and their impacts on our environment have prompted efforts worldwide to decrease the consumption of fossil fuels [1]. It was predicted that growing markets and developing economies would account for over 70% of the projected world energy demand, which would result in a growth in GHG emissions [2]. Therefore, it is essential to evaluate the ability of these growth economies to create bioenergy that can sustainably replace fossil fuels as well as the effects of these activities on the environment [3]. A substitute for fossil fuel, users might use biodiesel in place of or combined with fossil diesel but has improved cetane number, lower sulfur, non-toxicity, biodegradability, low aromatic compounds, and other characteristics that reduce GHG emissions [4, 5]. Around the world, research is taking place with the objective of increasing bioenergy and keeping costs down by utilizing a variety of substrates and original processing methods [6, 7]. Agricultural wastes are excellent sources of renewable energy [8]. Mutation and random strain screening have usually been the methodologies used in the classical strain development [9]. A diversity of agents, chemical and physical, have been demonstrated to cause mutations in organisms [10]. When an atom's unstable nucleus decays, gamma radiations are emitted, which have a shorter wavelength and a high energy per photon [11]. Effective mutations are induced by chemical mutagens [12]. The most common chemical mutagens that don't require expensive equipment include NaN₃, EMS, ethyl nitrosourea, and H₂O₂ [13]. A biomarker sequence in the genome is called ISSR [14]. RSM is a recent and advanced technique that yields acceptable findings for the optimization of ideal conditions for multivariable systems in fermentation processes, replacing the traditional time- and money-consuming methodologies [6]. F. oxysporum NRC 2017 was chosen for producing biodiesel in the previous finding [15]. Using bagasse hydrolyzed by B. cereus 3SM, F. oxysporum NRC 2017 biodiesel production costs will be reduced [16]. The F. oxysporum NRC 2017 was exposed to several mutagens to improve biodiesel production. RSM was used to enhance the biodiesel produced by the F. oxysporum NRC 2017 wild type and mutants.

Microorganisms

F. oxysporum NRC 2017 and B. cereus 3SME strains both have been isolated, identified, and deposited with the accession numbers MF62208 and MW522550, respectively, in the Gene Bank [15, 16]. F. oxysporum NRC 2017 was produced lipid production and B. cereus 3SME was produced fermentable sugar hydrolysate from bagasse.

Treatment with mutagens

Physical mutagen

F. oxysporum NRC 2017 was grown in a minimal salt medium Bidochka and Khachatourians, [17] using the Ɣ ray as a physical mutagen at the National Center for Radiation Research and Technology, Egypt. The radiation source was a Cobalt 60 Ɣ cell 220. From 10.0 Gray to 8.0 kilo Gray. For each dose, three replicates were examined [18]. Following irradiation, the fungi were investigated for lipid production [19].

Chemical mutagens

To induce a mutant in F. oxysporum NRC 2017, chemical mutagens such as NaN3, EB, and EMS were utilized. According to Bidochka and Khachatourians, [17], F. oxysporum NRC 2017 was cultivated on a minimal salt medium before being subjected to mutagenic treatments. Several concentrations of NaN₃ (0.75, 1.5, and 2.25 M), Et Br (6 to 60 mg/ml), and EMS (0.4 and 0.8 M) were applied to the mutagens for intervals of time (15 to 60 min). For each concentration, three replicates were used. According to Li et al. [19], the fungi were investigated for lipid production.

Fungal mutant stability test

The stability of each of the fungal mutants that were picked and induced by the physical and chemical mutagens by using the selective media according to Li et al. [19] was investigated and screened over three generations.

Molecular biomarker

Microbial strains

F. oxysporum NRC 2017 strain and its mutant names are indicated in Table (1).

Table (1) The name of F. oxysporum NRC 2017 with various induced mutagenesis

Extraction of DNA

Total DNA was extracted from the wild-type F. oxysporum NRC 2017, F. oxysporum NRC 2017-1, F. oxysporum NRC 2017-2, and F. oxysporum NRC 2017-3 using the modified CTAB extraction of DNA method according to Doyle and Doyle, [20].

Inter simple sequence repeat method

Total DNA was extracted from the wild-type and mutants of F. oxysporum NRC 2017, the Inter simple sequence repeat (ISSR) primers were performed by Attallah et al. [21]. Table (2) displayed the primer names and sequences.

Table (2) The ISSR analysis's primer names and sequences

Optimizing the growth conditions for lipids production of fungal

The culture conditions for the lipid production from the wild-type and mutants of F. oxysporum NRC 2017 were optimized using the response surface methodology (RSM). The inoculum size was 6.1 x 10⁵ spores/ml for all tested isolates at 1%. A more production was achievable for the bagasse hydrolysate of fermentable sugar produced using B. cereus 3SME as the sole carbon source [16] and utilizing medium using the modified lipid production medium [22]. Based on previous on Sayeda et al., [15], the four critical parameters for lipid production by F. oxysporum NRC 2017were applied. Table (3) presents the results of 29 quadratic Box-Behnken runs utilizing the (A) incubation period, (B) initial pH, (C) incubation temperature, and (D) yeast extract concentration for the F. oxysporum NRC 2017 wild type and its mutants. A statistical model was created to explain the relationships between the various variables and the synthesized lipid. The model had been numerically enhanced to produce the most lipids.

Table (3) Summary of study variables for the wild type and mutants of F. oxysporum NRC 2017

Physico-chemical analysis for the produced biodiesels

F. oxysporum NRC 2017 wild type and its mutants have been utilized to produce biodiesels, according to Vicente et al. [23]. At the Central Laboratories Network, National Research Centre, biodiesel gas chromatography (GC) analysis was carried out using an Agilent Technologies (GC) model 7890B equipped with a flame ionization detector. The different physical features of biodiesels were evaluated, which were blended with petroleum diesel at a concentration of 5%.

Formation of mutants

The F. oxysporum NRC 2017 mutants were obtained using a range of mutagenesis procedures, and they were selected based on their ability to grow on specific lipid media. The lipid accumulation from the several effective mutants has appeared in Table (4). The results showed that wild-type, Ɣ ray, NaN₃, Et Br, and EMS yielded lipids at values of 1.74, 1.14, 0.45, 1.74, and 1.62 g/l, respectively. The stability test for lipid synthesis from the Ɣ ray, EMS, and Et Br mutants had stable characteristics, whereas the NaN₃ mutant had an unstable charter.

Table (4) Lipid accumulation from different mutants and the wild-type strain of F. oxysporum NRC 2017

ISSR analysis as comparative analysis of F. oxysporum NRC 2017 wild type and mutants

Using a total of 5 ISSR primers. The 39 bands were obtained, and the PCR results are shown in Table (5). Polymorphism exhibited using ISSR primers ranged from 0 to 80%. Primer ISSR-5 yielded the highest percentage of polymorphism (80%) and two monomorphic bands, whereas primers ISSR-6 and ISSR-10 yielded the lowest percentage of polymorphism 0 with 5 and 3 monomorphic bands. Each primer's mean band frequency varied from 0.725 to 1.

Table (5) An analysis using statistics of the ISSR primers amplification results used in the F. oxysporum NRC 2017 and mutants

According to the ISSR dendrogram in Figure (1) for F. oxysporum NRC 2017 and induced mutants, the F. oxysporum NRC 2017-1 was further from the wild type and is therefore considered to be more variable.

Figure (1) Using ISSR data, a dendrogram was constructed to show the genetic fingerprint and relationship between the wild type and mutants of F. oxysporum NRC 2017.

Optimizing the conditions for the production of lipids from the wild-type and mutant of F. oxyporium NRC 2017

Data in Table (6) show that maximum lipid content by F. oxysporum NRC 2017 was produced 2.405 g/l on (run 25). The maximum lipid content from F. oxysporum NRC 2017-1 was produced 3.81 g/l on (run 5). The maximum lipid content from F. oxysporum NRC 2017-2 was produced 2.625 g/l on (run 23). The maximum lipid content from F. oxysporum NRC 2017-3 was produced 3.47 g/l on (run 12).

Table (6) Based on the Box-Behnken design, actual and predicted lipid production values for the wild type and mutants of F. oxysporum NRC 2017

Analysis of variance (ANOVA) of the results, regression coefficient, F values, and P values of variables as presented in Tables (7 a-d) was used to evaluate the interaction effect of the four factors for F. oxysporum NRC 2017 wild type and its mutants. The relationships between varying factors and their effects on lipid accumulation by F. oxysporum NRC 2017 wild-type and mutants were presented by the three-dimensional (3D) response surface plots created by Design-Expert software as illustrated in Figure (2 a-d).

The Table (7 a) presented the F. oxysporum NRC 2017 ANOVA data. The model was significant since the F-value was 8.27 and the Prob > F values for A, AB, AD, BD, A², ABC, ACD, BCD, A²C, and AB² were all signiﬁcant model were < 0.05. The model R² was 0.927398. Moreover, adequate precision of 11.32457.

The ﬁnal model term equation for lipid production (g/l) by F. oxysporum NRC 2017 = 50.25599 - 7.56723 x A - 13.2272*B -0.29281*C - 3.67972*D + 1.157656*A*B + 0.078125*A* C -1.21854*A*D - 0.04287*B* C +2.21375* B*C + 0.096417* C*D + 0.582161* A²+1.210518* B² + 0.022656* A* B* C + 0.033792* A* C*D - 0.06092*B * C*D - 0.01789* A²* C - 0.16172* A* B²

Table (7 a) F. oxysporum NRC 2017 lipid production analyzed data using an ANOVA

P-values < 0.05 are classified as significant

Figure (2 a) The interactions and effects of various variables on the lipid production of F. oxysporum NRC 2017 is plotted in 3D

The Table (7 b) presented the mutant by F. oxysporum NRC 2017-1 ANOVA data. The model was significant since the F-value was 33.32 and the Prob > F values for C, AC, BC, BD, A², B², C², D², A²B, and A²C were all signiﬁcant model were < 0.05. The model R² was 0.980953. Moreover, adequate precision of 18.82789.

The ﬁnal model term equation for lipid production (g/l) by F. oxysporum NRC 2017-1 = 108.012 -28.3945*A -14.4326*B -1.33339*C + 9.370306*D + 3.548281 A*B + 0.433219*A*C + 0.036375*B*C -1.12875 *B *C -0.21025 C*D + 1.50243 A² + 0.402219* B² - 0.01166* C² - 0.43272*D² + 0.03175 *B*C *D - 0.14211 A² * B -0.02595* A²* C - 0.10703*A*B²

Table (7 b) F. oxysporum NRC 2017-1 lipid production analyzed data using an ANOVA

P-values < 0.05 are classified as significant

Figure (2 b) The interactions and effects of various variables on the lipid production of F. oxysporum NRC 2017-1 is plotted in 3D

The Table (7 c) presented the mutant by Et Br ANOVA data. The model was significant since the F-value was 15.11 and the Prob > F values for A, B, C, D, BC, A², B², C², A²D, and AB² were all signiﬁcant model were < 0.05. The model R² was 0.91893. Moreover, adequate precision of 13.06721.

The ﬁnal model term equation for lipid production (g/l) by F. oxysporum NRC 2017-2 = 87.71814 - 12.7659*A + 24.8959*B + 0.178741* C - 6.94333*D + 3.181094*A*B + 1.821875*A*D + 0.101688*B*C + 0.204654*A² + 1.757052* B² - 0.01452*C² - 0.11115* A² *D - 0.26141* A* B²

Table (7 c) F. oxysporum NRC 2017-2 mutant lipid production analyzed data using an ANOVA

Figure (2 c) The interactions and effects of various variables on the lipid production of F. oxysporum NRC 2017-2 is plotted in 3D

The Table (7 d) presented the mutant by F. oxysporum NRC 2017-3 ANOVA data. The model was significant since the F-value was 9.58 and the Prob > F values for A, AB, AC, BC, BD, A², C², D², BCD, A²B, and A²C were all signiﬁcant model were < 0.05. The model R² was 0.988476. Moreover, adequate precision of 25.75927.

The ﬁnal model term equation for lipid production (g/l) by F. oxysporum NRC 2017-3 = 114.2056 - 32.4888*A- 7.32375*B -2.07354* A + 9.368348*D+1.906875*A*B + 0.690563*A*C - 0.04917*A*D - 0.02862*B* C - 1.07333* B*D - 0.1815*A*D + 2.186579* A² - 0.01105* C² - 0.60877*C² + 0.00875*A*B *C + 0.031333*B *C *D - 0.13891* A² * B - 0.04731* A²* C

Table (7 d) ANOVAoflipid production values for lipid production by F. oxysporum NRC 2017-3

Figure (2 d) The interactions and effects of various variables on the lipid production of F. oxysporum NRC 2017-3 is plotted in 3D

Depending on the practically validate Box-Behnken model, the synthesis of lipids from the F. oxysporum NRC 2017 wild type and mutants have been optimized. The model was validated under different variables for predicted lipid production maximum in comparison to the actual value, chosen value with a lower nitrogen source, and synthetic medium, as shown in the data in Figure (3). For F. oxysporum NRC 2017-1, the lipid-increasing rate was 28.09%. The chosen value was 2.35 g/l which was very close to the optimal, even though it was only for 6 days. The maximal lipid content was 2.41 g/l, the predicted lipid content was 2.42 g/l, and the results achieved from the synthetic media without the use of surface response optimization were 1.74 g/l.

For F. oxysporum NRC 2017-1, the lipid-increasing rate of increase was 70.07%. The produced lipid was compared to the findings on synthetic media without applying surface response optimization at 1.14 g/l; the predicted lipid was 3.42, the maximum lipid, and the chosen value was 3.81 g/l.

For F. oxysporum NRC 2017-2, the lipid-increasing rate of increase was 33.84%. The produced lipid was compared to the findings on synthetic media without applying surface response optimization at 1.74 g/l; the predicted lipid and the maximum lipid were 2.64 g/l, and the chosen value was 2.22 g/l.

For F. oxysporum NRC 2017-3, the lipid-increasing rate of increase was 53.31%. The produced lipid was compared to the findings on synthetic media without applying surface response optimization at 1.62 g/l; the predicted lipid and the maximum lipid were 3.47 g/l, and the chosen value was 2.62 g/l.

Figure (3) Actual and predicted lipid production values for the wild type and mutants of F. oxysporum NRC 2017 based on the Box-Behnken design

Physico-chemical analysis for the produced biodiesels

The induced mutants and F. oxysporum NRC 2017 wild-type both exhibited fatty acid compositions that are principally in the C16–C18 range (Table 8). The physical characteristics of the blending biodiesel (B5) produced from F. oxysporum NRC 2017 wild types and mutants are presented in Table (9) and are comparable with ASTM D975.

Table (8) F. oxysporum NRC 2017 wild type and mutant fatty acid percent

SFA: Saturated fatty acids, USFA: Unsaturated fatty acids

Table (9)F. oxysporum NRC 2017 wild type and mutant physical properties for blending biodiesel comparing to ASTM D975 standard

One of the most critical problems in the current period is climate change. People suffer from the consumption of greenhouse gases, which can cause things including contributing to respiratory problems, harsh weather, and shortages in the food supply [24]. Finding cheap renewable sources to produce eco-friendly biofuels had expressed concerns due to rising energy needs and a restriction of fuel resources [25]. The purpose of this economic study was to increase the generation of biodiesel as a clean biofuel to reduce the greenhouse gases emissions from the F. oxysporum NRC 2017 when it was cultured on bagasse digested with B. cereus 3SME [16]. Many chemical and physical agents have been shown to cause in microorganisms mutations [26]. The total fatty acid of many microalgae rises as a consequence of mutagenesis [27]. The present study reported most productivity quantity of lipid was produced using F. oxysporum NRC 2017 utilizing Ɣ ray, NaN₃, Et Br, and EMS at maximum efficiency of 1.14, 0.45, 1.74, and 1.62 g/l, respectively. The work matched with the current finding by Asker et al., [28], who reported that Ɣ ray improved the trehalose biosynthesis enzyme in Corynobacterium nitrilophilus NRC. Furthermore, it was observed that NaN3 was an extremely effective mutagen in barley and certain other crop species and a major mutagen for microbiology [29]. Moreover, Dwivedi et al. [30] established a mutant from P. oxalicum SA-8 ITCC 6024 using a combination mutagenesis approach UV-irradiation and Et Br, which caused a 1.87-fold increase the activity of the alkali-tolerant xylanase. According to Adsul et al. [31], the P. janthinellum NCIM 1171 strain developed a mutation that includes exposure to EMS which raised the synthesis of cellulase enzyme. The ideal biomarkers for a broad range of studies are ISSR markers [14]. This study target is using the ISSR that could be applied to differentiate between the F. oxysporum NRC 2017 wild type and the resulting mutants. This work is similar to the examined the genetic variety of bacterial mutants using the same technique [32]. Depended on the microorganisms type and the cultivation conditions [33]. In the previous findings, they performed the lipid accumulation of F. oxysporum NRC 2017 and the optimal conditions for biomass and lipid content were reached to 49.42%. By utilizing an inoculum size of 1%, adjusting the pH to 5.0, and incubating for 9 days at a 30^oC with using a carbon source (xylose) and a nitrogen source (yeast extract) [15]. In the current work, F. oxysporum NRC 2017 wild type, and the induced mutants were cultivated on the hydrolysate obtained by growing B. cereus 3SME on bagasse as a cheap carbon source. The RSM was applied to optimize lipid production conditions. Using the Box-Behnken approach, lipid production has risen. The findings from of F. oxysporum NRC 2017, of F. oxysporum NRC 2017-1, of F. oxysporum NRC 2017-2, and of F. oxysporum NRC 2017-3 were 28.09%, 70.07%, 33.84%, and 53.31%, respectively. Similar to Saccharomyces cerevisiae bioethanol yield was improved by Ghazanfar et al., [34] using the RSM, which resulted in the greatest ethanol output of 72.0 g/l. The constituents of the extracted lipid were identified using gas-liquid chromatography (GC). Many microorganisms produced lipids with fatty acid profiles identical to those found in the plant [35]. In line with the current study's GC analysis of the wild types and mutants of the F. oxysporum NRC 2017 strain, the major fatty acids were mainly C16-C18. The physical properties of B5 biodiesel produced by F. oxysporum NRC 2017 wild types and induced mutants were similar to the study with Pimentel et al. [36]. The physical characteristics of the blended biodiesel results showed that the study's biodiesel production similar the standards recommended by ASTM D975.

To lower the cost of producing biodiesel, which could reduce greenhouse gas emissions and improve sustainability in society and the environment, F. oxysporum NRC 2017 and induced mutants were utilized to ferment the bagasse hydrolysate that was produced by B. cereus 3SME. F. oxysporum NRC 2017 applied multiple physical and chemical genetic mutations to improve the biodiesel it produced. RSM was applied to optimize biodiesel production condtions and achieved remarkable productivity increases. The fatty acids acceptable for the production of biodiesel were detected by GC analysis of all tested isolates. The four fungal isolate-produced biodiesels' physical characteristics were similar to those of standard biodiesel.

Acknowledgments

Not applicable

Authors’ contributions

All authors certify that they have participated sufficiently in contributing to the intellectual content, concept, writing the manuscript and all authors have read and approved the manuscript”, and ensure that this is the case. All the participant researchers contribute to this work and this research was from the thesis of Abdelhamid, S.A., Enhancement of Biodiesel Production Using Agricultural Residues, Ph.D., Ain Shams University, Egypt. S.A.A. (corresponding author) did the particle part and wrote the paper and confirmed that all listed authors have approved the manuscript before submission, including the names and order of authors, and that all authors receive the submission and all substantive correspondence with editors, as well as the full paper. E.H.E. the main supervisor from Ain Shams University revised the written part, M.S.A., S.K.A., and S.S.M. the supervisor from National Research Centre, A.G.A. helped me in ISSR, and MMA helped me in the RSM program. And all were revised the particular part.

Funding

Not applicable

Availability of data and materials

The datasets generated during and/or analyzed during the current study are available from the corresponding author upon reasonable request.

Competing interests

The authors declare that they have no competing interests.

Consent for publication

Not applicable

Abbreviations

Not applicable

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Table (1) The name of F. oxysporum NRC 2017 with various induced mutagenesis

Name	Mutagen
*F. oxysporum* NRC 2017	Wild type
*F. oxysporum* NRC 2017-1	Ɣ ray
*F. oxysporum* NRC 2017-2	Et Br
*F. oxysporum* NRC 2017-3	EMS

Table (2) The ISSR analysis's primer names and sequences

Primer	Sequence
ISSR- 1	5'-AGAGAGAGAGAGAGAGYC-3'
ISSR- 4	5'-ACACACACACACACACYG-3'
ISSR- 5	5'-GTGTGTGTGTGTGTGTYG-3'
ISSR- 6	5'-CGCGATAGATAGATAGATA-3'
ISSR- 10	5'-GACAGACAGACAGACAAT-3'

Table (3) Summary of study variables for the wild type and mutants of F. oxysporum NRC 2017

Factor	Name	Minimum	Minimum
A	Incubation period (day)	6	10
B	pH	5	7
C	Temperature (°C)	25	35
D	Yeast extract (g/l)	1.5	3

Table (4) Lipid accumulation from different mutants and the wild-type strain of F. oxysporum NRC 2017

Mutagenic material	Lethal dosage	Preferred dosage	Fungal biomass (g/l)	Fungal lipid (g/l)	Productivity (%)
None	--		4.95	1.74	35.11
Ɣ ray	5 K-Gr	2 K-Gr	1.91	1.14	59.40
NaN₃	1.5 M	0.75 M	0.78	0.45	57.88
Et Br	60 mg/ml	30 mg/ml	5.29	1.74	32.89
EMS	0.8 M	0.4 M	3.59	1.62	44.18

Ɣ ray: Gamma radiation; NaN₃ Sodium azide; Et Br: Ethidium bromide; EMS: Ethyl methane sulfonate; K-Gr: Kilo-Gray, M: mole

Table (5) An analysis using statistics of the ISSR primers amplification results used in the F. oxysporum NRC 2017 and mutants

Primer name	ISSR Primers
Primer name	scored bands (NSB)	polymorphic bands (NPB)	Polymorphism (PPB)%	Monomorphic bands	Unique bands	Mean of band frequency
ISSR-1	10	5	50	5	2	0.725
ISSR-4	11	5	45.455	6	1	0.818
ISSR-5	10	8	80	2	1	0.725
ISSR-6	5	0	0	5	0	1
ISSR-10	3	0	0	3	0	1
Total	39	18	--	21	4	4.268
Average	7.8	3.6	35.11	4.2	0.8	0.8536

Table (6) Based on the Box-Behnken design, actual and predicted lipid production values for the wild type and mutants of F. oxysporum NRC 2017

Run no.	Lipid (g/l)
	*F. oxysporum* NRC 2017		*F. oxysporum* NRC 2017-1		*F. oxysporum* NRC 2017-2		*F. oxysporum* NRC 2017-3
	Actual value	Predicated Value	Actual Value	Predicated Value	Actual value	Predicated value	Actual value	Predicated value
1	0.615	0.46	2.42	2.33	1.865	1.74	1.64	1.73
2	0.05	0.14	0.04	0.04	0.25	0.41	-0.04	-0.06
3	0.355	0.43	0.235	0.025	0.505	0.60	0.35	0.32
4	0.795	0.62	-0.025	-0.08	-0.23	-0.18	0.26	0.14
5	0.035	0.166	3.81	3.41	2.22	2.22	2.62	2.60
6	2.35	2.16	0.92	0.94	0.155	0.22	0.33	0.28
7	0.03	0.02	0.16	0.31	1.22	1.22	1.72	1.77
8	0.33	0.11	0.39	0.25	0.06	0.43	-0.025	-0.088
9	0.71	0.52	-0.025	-0.029	0.945	0.80	-0.005	-0.039
10	0.05	0.24	0.58	0.72	0.08	0.41	1.65	1.70
11	0.14	0.18	1.26	1.21	0.19	0.23	0.73	0.63
12	0.3	0.16	2.36	2.57	1.77	2.26	3.47	3.41
13	2.08	2.17	0.035	0.18	2.39	2.14	0.82	0.97
14	0.12	0.21	0.215	0.36	-0.13	-0.12	0.235	0.38
15	0.21	0.62	1.205	1.27	1.08	0.96	0.085	0.38
16	0.62	0.66	0.03	0.05	0.02	0.56	0.15	0.038
17	0.42	0.66	0.475	0.62	-0.005	-0.53	0.71	0.86
18	0.645	0.46	2.265	2.33	1.96	1.74	1.70	1.73
19	0.625	0.46	2.425	2.33	1.82	1.74	1.67	1.73
20	0.105	0.23	-0.045	-0.12	0.39	0.42	-0.025	-0.009
21	0.63	0.46	2.3	2.33	1.55	1.74	1.665	1.73
22	0.425	0.25	2.54	2.67	2.56	2.24	2.20	2.07
23	0.045	0.30	1.22	1.45	2.63	2.52	0.345	0.35
24	0.915	0.69	0.35	0.29	0.14	0.36	1.095	0.97
25	2.405	2.49	2.075	1.83	2.255	2.02	3.285	3.18
26	0.1	0.182	0.3	0.168	0.16	0.41	0.165	0.12
27	0.61	0.46	2.25	2.33	1.76	1.74	1.815	1.73
28	0.015	0.26	1.565	1.71	1.065	1.11	0.25	0.40
29	-0.11	-0.02	0.03	0.004	0.22	0.38	0.13	0.08

Table (7 a) F. oxysporum NRC 2017 lipid production analyzed data using an ANOVA

Source	Sum of squares	df	Mean square	F value	p-value Prob> F
Model	11.21704	17	0.659826	8.265305	0.0005
A-Incubation period	1.9208	1	1.9208	24.06088	0.0005
B-pH	0.074259	1	0.074259	0.930209	0.3555
C-Temperature	0.082013	1	0.082013	1.027329	0.3326
D-Yeast extract	0.157626	1	0.157626	1.974501	0.1876
AB	0.682689	1	0.682689	8.551697	0.0138
AC	0.023639	1	0.023639	0.296114	0.5972
AD	1.509827	1	1.509827	18.91283	0.0012
BC	0.000689	1	0.000689	0.008632	0.9276
BD	1.342702	1	1.342702	16.81934	0.0018
CD	0.000352	1	0.000352	0.004404	0.9483
A²	0.912157	1	0.912157	11.42612	0.0061
B²	0.191253	1	0.191253	2.395729	0.1499
ABC	0.821289	1	0.821289	10.28787	0.0083
ACD	1.027689	1	1.027689	12.87334	0.0043
BCD	0.834939	1	0.834939	10.45886	0.0080
A²C	0.682826	1	0.682826	8.553407	0.0138
AB²	0.55793	1	0.55793	6.9889	0.0228
Residual	0.878139	11	0.079831
Pure Error	0.00075	4	0.000188
Cor Total	12.09518	28

P-values < 0.05 are classified as significant

Table (7 b) F. oxysporum NRC 2017-1 lipid production analyzed data using an ANOVA

Source	Sum of squares	df	Mean square	F value	p-value Prob> F
Model	32.01538	17	1.883258	33.32432	< 0.0001
A-Incubation period	0.0162	1	0.0162	0.28666	0.6030
B-pH	0.0882	1	0.0882	1.560703	0.2375
C-Temperature	0.59405	1	0.59405	10.51174	0.0078
D-Yeast extract	0.045501	1	0.045501	0.805143	0.3888
AB	0.006202	1	0.006202	0.109737	0.7467
AC	0.516602	1	0.516602	9.141287	0.0116
BC	4.649414	1	4.649414	82.27158	< 0.0001
BD	0.279577	1	0.279577	4.947119	0.0480
CD	0.087764	1	0.087764	1.552989	0.2386
A²	6.889032	1	6.889032	121.9017	< 0.0001
B²	5.348611	1	5.348611	94.6439	< 0.0001
C²	2.205158	1	2.205158	39.02037	< 0.0001
D²	1.537209	1	1.537209	27.20098	0.0003
BCD	0.226814	1	0.226814	4.013485	0.0704
A²B	1.723313	1	1.723313	30.4941	0.0002
A²C	1.436938	1	1.436938	25.42668	0.0004
AB²	0.244388	1	0.244388	4.324457	0.0617
Residual	0.621643	11	0.056513
Pure error	0.02953	4	0.007383
Cor total	32.63702	28

P-values < 0.05 are classified as significant

Table (7 c) F. oxysporum NRC 2017-2 mutant lipid production analyzed data using an ANOVA

Source	Sum of squares	df	Mean square	F value	p-value Prob> F
Model	22.06076	12	1.838397	15.1133	< 0.0001
A-Incubation period	2.5538	1	2.5538	20.99456	0.0003
B-pH	3.973134	1	3.973134	32.66279	< 0.0001
C-Temperature	4.054926	1	4.054926	33.33519	< 0.0001
D-Yeast extract	1.209013	1	1.209013	9.939185	0.0062
AB	0.125139	1	0.125139	1.028757	0.3255
AD	0.068252	1	0.068252	0.56109	0.4647
BC	4.136139	1	4.136139	34.00283	< 0.0001
A²	0.888378	1	0.888378	7.303273	0.0157
B²	3.005424	1	3.005424	24.70733	0.0001
C²	3.544761	1	3.544761	29.14117	< 0.0001
A²D	0.592963	1	0.592963	4.874697	0.0422
AB²	1.457776	1	1.457776	11.98424	0.0032
Residual	1.946256	16	0.121641
Pure Error	0.09183	4	0.022958
Cor Total	24.00702	28

P-values < 0.05 are classified as significant

Table (7 d) ANOVA of lipid production values for lipid production by F. oxysporum NRC 2017-3

Source	Sum of squares	df	Mean square	F value	p-value Prob> F
Model	28.46779	17	1.674576	55.50297	< 0.0001
A-Incubation period	0.525104	1	0.525104	17.40431	0.0016
B-pH	0.00245	1	0.00245	0.081204	0.7810
C-Temperature	0.1058	1	0.1058	3.506687	0.0879
D-Yeast extract	0.001204	1	0.001204	0.039911	0.8453
AB	0.180625	1	0.180625	5.986724	0.0324
AC	0.310806	1	0.310806	10.30152	0.0083
AD	0.087025	1	0.087025	2.884399	0.1175
BC	5.006406	1	5.006406	165.9348	< 0.0001
BD	0.16	1	0.16	5.303119	0.0418
CD	0.009506	1	0.009506	0.31508	0.5858
A²	1.888727	1	1.888727	62.60091	< 0.0001
C²	2.052538	1	2.052538	68.03032	< 0.0001
D²	3.155359	1	3.155359	104.5828	< 0.0001
ABC	0.1225	1	0.1225	4.0602	0.0690
BCD	0.2209	1	0.2209	7.321618	0.0204
A²B	1.646502	1	1.646502	54.57248	< 0.0001
A²C	4.775408	1	4.775408	158.2785	< 0.0001
Residual	0.33188	11	0.030171
Pure Error	0.01868	4	0.00467
Cor Total	28

P-values < 0.05 are classified as significant

Table (8) F. oxysporum NRC 2017 wild type and mutant fatty acid percent

Fatty acid	*F. oxysporum* NRC 2017	*F. oxysporum* NRC 2017-1	*F. oxysporum* NRC 2017-2	*F. oxysporum* NRC 2017-3
Palmitic acid (C16:0)	10.9	16.33	16.2	13.93
Palmitoleic acid (C16:1)	4.23	0.95	1.71	2.08
Margaric acid (C17:0)	0.29	-	-	0.28
Stearic acid (C18:0)	8.16	11.61	9.17	9.26
Elaidic acid (C18:1)	9.17	-	-	0.1
Oleic acid (C18:1)	-	23.04	20.54	21.35
Linolelaidic acid (C18:2)	24.24	5.52	8.84	9.49
Linoleic acid (C18:2)	7.77	23.26	23.69	20.11
Linolenic acid (C18:3)	2.38	8.02	8.44	9.19
Arachidic acid (C20:0)	-	0.39	0.32	0.39
cis-11,14-Eicosadienoic acid (C20:2)	17.11	4.45	5.53	6.29
Heneicosanoic acid (C21:0)	0.43	0.33	0.42	0.22
Behenic acid (C22:0)	-	0.41	0.3	0.47
cis-13,16-Docosadienoic acid (C22:2)	1.51	0.38	-	0.48
Tricosanoic acid (C23:0)	10.98	2.8	2.96	3.99
SFA	33.07	34.39	31.26	30.70
USFA	66.71	65.62	68.74	69.29

SFA: Saturated fatty acids, USFA: Unsaturated fatty acids

Table (9) F. oxysporum NRC 2017 wild type and mutant physical properties for blending biodiesel comparing to ASTM D975 standard

Physical properties	Test method ASTM	*F. oxysporum* NRC 2017	*F. oxysporum* NRC 2017-1	*F. oxysporum* NRC 2017-2	*F. oxysporum* NRC 2017-3	ASTM D975 for B5
Fuel composition	--	Mix	Mix	Mix	Mix	Mix
Density (g/ml)	D1298	0.82	0.83	0.81	0.82	0.85
Kin. Viscosity (mm²/s)	D445	3.31	3.22	3.24	3.41	1.90 to 4.10
Cloud point (^ºC)	D2500	-2	-1.1	0.00	-1	-35.00 to 5.00
Pour point (^ºC)	D97	-4	-3	-2	-2	-35.00 to -15.00
Cetane no. (min)	D613	47.27	52.36	50.55	49.68	40.00 to 55.00

No competing interests reported.

Enhancement of Biodiesel Production from Fusarium oxysporum NRC 2017 with Various Mutagenesis and Optimization of the Cultural Conditions Using Response Surface Methodology

Status:

Version 1

Abstract

Figures

Introduction

Materials and methods

Results

Discussion

Conclusion

Declarations

References

Tables

Additional Declarations

Status:

Version 1