Response surface methodology for optimization of biodiesel production by the Penicillium commune NRC 2016 and its mutants

Background: With increasing energy requirements and limited fuel resources, �nding cheap renewable sources is essential. The objective of our current economy was to enhance biodiesel production from the Penicillium commune NRC 2016. Methods: Four mutagenic agents were used to improve the lipid production from P. commune NRC 2016; a physical agent was gamma radiation and the chemical mutagenic agents including ethyl methanesulfonate (EMS), ethidium bromide (EB), and sodium azide (NaN 3 ). Inter-Simple Sequence Repeats molecular (ISSR) marker was used to compare the wild type of P. commune NRC 2016 and the resulting mutants and the result showed a major difference between the wild type and its mutants. Response surface methodology was used to optimize the culture conditions for lipid production by P. commune NRC 2016 and the induced mutants using hydrolysate that was produced from bagasse using B. cereus 3SME. Results: The maximum lipid content (g/l) for P. commune NRC 2016 wild type, gamma mutant, EB mutant, and EMS mutant were 2.01, 2.55, 1.71, and, 2.27, respectively. Gas chromatography analysis for biodiesel compositions produced from P. commune NRC 2016 wild type and its mutants were mainly C16-C18 that suitable to produce biodiesel. The physical properties such as density, viscosity, cloud point, pour point, and cetane number for biodiesels from P. commune NRC 2016 and its mutants were similar to standard biodiesel and could be applied on large scale.


Background
Biofuels are becoming more attractive and important due to the decreasing reserves of fossil fuels and environmental concerns.
Among them, biodiesel produced from vegetable oils and animal fats is rather attractive for being biodegradable, clean, nontoxic, and renewable as well as having similar properties to conventional diesel fuels [1].The lipids produced by oleaginous microorganisms have several bene ts including their high lipid content and rapid growth rate resulting in great production [2][3][4].
Mutations are heritable alterations in the genetic material that occurred suddenly.At the molecular level, mutation referred to any heritable alterations in a genome's nucleotide sequence [5].The mutagenic agents included a variety of physical and chemical factors that had been demonstrated to be mutagenic in microorganisms for increasing lipid production [6,7].The physical mutagenic agent such as -ray is mostly produced by the isotopes Cobalt-60 and Caesium-137 [8].Ethyl methanesulphonate (EMS), propanamide, ethyl nitrosourea, and sodium azide (NaN 3) are the most widely used chemical mutagens that do not require expensive equipment [9].Genetic markers are lengthy or short DNA sequences that are linked to the target gene and served as indicators or ags such as Inter-Simple Sequence Repeats (ISSR) [10].ISSR marker approach is quick and inexpensive doesn't require the sequence information, has good repeatability, and only required one step of PCR [11,12].Response surface methodology (RSM), is an e cient experimental strategy to seek optimal conditions for multivariable systems in fermentation processes with acceptable results, replacing the media optimization using the traditional methods which were time-consuming, expensive, and often lead to misinterpretation of results [13].Alternative low-cost carbon sources for microbial cultivation could be lignocellulosic biomass [14].P. commune NRC 2016 is chosen for biodiesel production in our previous study [15,16].The objective of our current economy was to enhance biodiesel production from P. commune NRC 2016 using the following steps: Firstly, the bagasse hydrolysis and the fermentable sugars were produced using Bacillus cereus 3SME to reduce the cost of biodiesel production.Secondly, the P. commune NRC 2016 was treated with different chemical and physical mutagens for improving biodiesel production.Thirdly, RSM was applied to optimize fungal lipid production to increase biodiesel production by the P. commune NRC 2016 wild type and its mutants.

Microorganisms
P. commune NRC 2016 and B. cereus 3SME strain which were previously isolated, identi ed, and kept in the Gene Bank under accession numbers KU752217 and MW522550, respectively [15,17] 2.2 Treatment of the P. commune NRC 2016 with various mutagens Different physical and chemical mutagens were used to induction the mutant from P. commune NRC 2016.

Physical mutagen
The ray was used as a physical mutagen.The source of radiation was Cobalt 60 gamma cell 220 located at National Center for Radiation Research and Technology, Nasr city, Cairo, Egypt the Gamma chamber (MC20, Russia) [18].P. commune NRC 2016 was grown in the medium of minimal salt according to [19] which contained (g/l): NaCl (0.30), MgSO 4 .7H 2 O (0.30), KH 2 PO 4 (0.30), NH 4 NO 4 (0.30) and Agar (20) then exposed to radiation with dose levels of a low and quick rate.The low rate 0.638 rad/sec for 10.0, 20.0, 30.0, 40.0, and 50.0 Gray and quick rate 1.014 kilo-gray/h.for 1.0, 2.0, 3.0, 4.0, 5.0 and 8.0 kilo Gray.Three replicates were used for each dose.After irradiation, the fungi were screened for lipid production.

Chemical mutagen
The chemical mutagens including NaN 3 , Et Br, and EMS were used to induction of mutants for P. commune NRC 2016 after growing in the medium of minimal salt [19] the mutagens were treated for intervals time 15, 30, 45, and 60 min with different concentrations of the NaN 3 0.001, 0.75, 1.5, 2.25, 3, and 3.75 M, the Et Br 1, 6, 15, 30, and 60 mg/ml, and the EMS 0.001, 0.4 and 0.8 M. Three replicates were used for each concentration.

Stability test for the fungal mutant
Examination of the stability for all selected fungal mutants resulting from the physical and chemical mutagens that produced lipids for three generations.

DNA Extraction
For the DNA extraction from P. commune NRC 2016 wild type, and its mutant.Regardless of the homogenization method, a modi ed CTAB extraction method was used [20,21].

Inter simple sequence repeat (ISSR) technique
ISSR-PCR reactions were conducted according to [22].Five anchored primers were synthesized by Euro ns, Germany.The primer names and primer sequences were shown in Table (1).The reaction conditions were optimized and the following reagents were mixed in a nal volume of 25 µl: 1 x of green GoTaq Flexi buffer; 1.5mM of MgCl 2 ; 200 µM of dNTPs (Promega); 25pucM of primer; 1 U of GoTaq Flexi DNA Polymerase (Promega); 25ng of template DNA and up to 25 µl double distilled water.Ampli cation was carried out in a T100-Bio-Rad Gradient Thermal cycler.The following programmer was used to amplify the DNA: 94°C/5min (1 cycle); [94°C/45 secs 45°C/50 sec, 72°C/1.5 min] (40 cycles); 72°C/7 min (1 cycle), and 4°C (in nitive).A volume of 10µl of the ISSR-PCR product was resolved using 1.5% agarose gel electrophoresis containing ethidium bromide.A 100 bp DNA marker was used as a DNA molecular weight standard.Results were visualized on a UV transilluminator and photographed by Molecular Imager® Gel Doc™ System with Image Lab™ Software, Bio-Rad.

Optimization of the culture conditions for the wild and mutant fungal lipid production
The physicochemical and nutritional conditions have been optimized based on the response surface methodology (RSM), for the lipid production from P. commune NRC 2016 wild type and its mutants as follows:

Experimental design and statistical analysis
The prepared inoculum size of P. commune NRC 2016 was 6.4 ×10 5 spore/ml for all the following experiments.After autoclaving, the inoculum size for all strains was adjusted at 1% by using the lipid production medium modi ed [23] and using the hydrolysate of fermentable sugar that was produced from bagasse by using B. cereus 3SME [17] instead the carbon source as recycling of agro-industrial by-products as a mean of cost-effective production medium was applied.P. commune NRC 2016 wild type and its mutants by using EMS, Et Br, and ray mutagens 47 quadratic Box-Behnken runs were carried out using ve independent variables as follows; incubation period (A), initial pH (B), incubation temperature (C), xylose concentration (D), and peptone concentration (E).Variables including their ranges and their summary were presented in Tables (2).A statistical model, describing the relationships between the different variables and the produced lipid was developed.The signi cance and accuracy of this model were evaluated by statistical analysis of variance (ANOVA) and coe cient of determination R2, respectively.Design-Expert software (version 7, Stat-Ease Inc., Minneapolis, USA) has been used for experimental design, statistical analysis of the data, and numerical optimization of productivity.Table (2) Summary of variables and ranges of study for P. commune NRC 2016 wild type and its mutants by using EMS, Et Br, and ray mutagens

Numerical optimization and validation
The model had been numerically optimized for maximum lipid production.The different conditions had been predicted for maximum lipid productivity and nally validated by experimental application of the predicted values and results were compared.

Biodiesel production
Biodiesels were produced from P. commune NRC 2016 wild type and its mutants [24].Experiments were planned to use HCl as an acid catalyst and the reaction temperature was 25ºC.Reactions of extracted fungal lipids were performed with magnetically stirring (900 rpm) using methanol to oil ratio of 60:1 molar and a catalyst concentration of 8 wt% relative to microbial oil.The reactor was then immersed in a thermostatic bath at the reaction temperature for 8 h.The fatty acid methyl esters (FAMEs) layer was collected and the crude glycerol was washed ve times with petroleum ether: diethyl ether (80:20%) as well as the same volume of water.The upper organic layers were put together with the rst FAME layer and the solvent was removed on a rotary evaporator leaving the residue containing the FAMEs.

Gas chromatography analysis
Gas chromatography analysis of fatty acid methyl esters (FAMEs) for P. commune NRC 2016 wild type and its mutants was performed.The GC model 7890B from Agilent Technologies was equipped with a ame ionization detector at Central Laboratories Network, National Research Centre, and Cairo, Egypt.Separation was achieved using a Zebron ZB-FAME column (60 m x 0.25 mm internal diameter x 0.25 µm lm thickness).Analyses were carried out using hydrogen as the carrier gas at a ow rate of 1.8 ml/min at a split-1:50 mode, injection volume of 1 µl, and the following temperature program: 100°C for 3 min; rising at 2.5°C /min to 240°C and held for 10 min.The injector and detector (FID) were held at 250°C and 285°C, respectively.

Physical properties of biodiesel
Fatty acid methyl esters from P. commune NRC 2016 wild type and its mutants.The fungal FAME was blended with diesel at a concentration of 5% with a low stirring rate.The mixture was stirred for 20 min and left to reach equilibrium before analysis.To measure the physical properties of fatty acid methyl esters (FAMEs) were blended with petroleum diesel at a concentration of 5% and determining density was (ASTM D1298), the viscosity was (ASTM D445), cloud point was (ASTM D2500), pour point was (ASTM D97), and cetane number was (ASTM D613).

Induction of mutants
The mutants of P. commune NRC 2016 were developed by using various mutagenesis strategies and selected initially based on growing on speci c lipid media.Several potent mutants thus selected were evaluated for their lipid production ability.Table (3) indicated the lipid production from the various potent mutants obtained through physical ( ray) and chemical mutagenesis (NaN 3, Et Br, and EMS).It was observed that lipid productivity of 0.81, 1.47, 0.41, 0.93, and 0.90 g/l were obtained from wild-type, ray, NaN 3 , Et Br, and EMS, respectively.The stability of three generations for lipid production from P. commune NRC 2016 mutants indicated that were given stable characters in the case of ray mutants, Et Br mutants, and EMS mutants while the NaN 3 mutants resulted in an unstable charter.

Comparative analysis of P. commune NRC 2016 wild type and its mutants using ISSR analysis
In this study, ISSR molecular marker technique was used to differentiate between the wild type of P. commune NRC 2016and its mutants.A total of 5 primers were used and 53 and 39 bands were recorded from P. commune NRC 2016 shown in Figure (1).The most polymorphic primers were ISSR-1 which produced 6 bands, followed by primer ISSR-4, primer ISSR-5, primer ISSR-6, and primer ISSR-10 which produced 7, 14, 12, and 14 bands, respectively (Table 4).Some primers produced several bands, while others produced a few bands.ISSR-primers produced polymorphism ranging from 16.667 to 100%.Primer ISSR-10 produced the highest percentage of polymorphism (100%) and did not produce any monomorphic band, while primer ISSR-4 produced the lowest percentage of polymorphism (0) with 7 monomorphic bands.The mean band frequency of each primer ranged from 0.458 to 1.

Optimization of the culture conditions for the lipid production from P. commune NRC 2016 wild-type and its mutants
To reduce the cost of lipid production by P. commune NRC 2016 wild type and mutants, the agriculture waste of bagasse with B. cereus 3SME which produced fermentable sugars was used.The RSM had employed for a statistical optimization of lipid production from P. commune NRC 2016 wild type and its mutants.A total sum of 47 runs with different combinations of ve parameters was carried out and the results were shown in Table (5).The lipid content by wild type ranged from 0.005 to 2.01 g/l.The lipid content from the mutant by ray ranged from 0.005 to 2.55 g/l.The lipid content from mutant by Et Br ranged from 0.005 to 1.705 g/l.The lipid content from mutant by EMS ranged from 0.005 to 2.27 g/l.
The interactive effects of the ve parameters for P. commune NRC 2016 wild type and its mutants were deduced by analysis of variance (ANOVA) of the results, regression coefficient, F values, P values of variables as shown in Tables (6-9).
The wild-type ANOVA data was shown in Tables (6).The model was a significant as F-value was 5.91 and A, C, E, C 2 , and A 2 E were all significant model terms.The model R 2 was 0.721, adequate precision of 9.538, stander deviation was 0.367, and the mean was 0.426.The mutant by ray ANOVA was shown in Tables (7).The model was significant as F-value was 13.89 and A, AB, AC, BC, A 2 , ABC, ABD, ACD, A 2 B, and A 2 C were all significant model terms.The model R 2 was 0.927, the adequate precision of 15.761, the stander deviation was 0.266, and the mean was 0.404.The mutant by EMS ANOVA was shown in Table (9).The model was significant as F-value was 6.99 and C, AB, AC, AD, AE, C 2 , ABC, ABE, ACD, ACE, and BDE were all significant model terms.The model R 2 was 0.884, the adjusted of 0.757, the adequate precision of 10.199, the stander deviation was 0.272, and the mean was 0.343.
The final equation in terms of actual factors for mutant by EMS was as follows: . Lipid g/l from P. The production of lipid from P. commune NRC 2016 wild type and its mutants had been numerically optimized and applied practically to validate the Box-Behnken model.The results in Figure ( 4) revealed the validation of the model at different conditions for predicted maximizing lipid production compared with actual value, chosen value using 1 g/l carbon and nitrogen source, and synthesis medium.For the wild type, the lipid-increasing rate was 59.70%.The predicted lipid value was 1.46, the maximum lipid 2.01 g/l produced at which incubation time was 7 days, pH 6, at a temperature of 25°C, xylose 3 g/l, and peptone 3 g/l compared to results from the synthetic media before using surface response optimization 0.965 g/l.Because the main purpose of this study was to reduce cost so the run was chosen at 1.895 g/l.since it was very close to the optimum, while 1 g xylose and 1 g peptone were used.For the ray mutant lipid, the increasing rate was 42.75%.The predicted lipid was 2.34, the maximum lipid 2.55 g/l was produced at which the incubation time was 7 days, pH 6, at a temperature of 25°C, xylose 3 g/l, and peptone 3 g/l compared to results on synthetic media before using surface response optimization 1.46 g/l.Because the main purpose of this study was to reduce cost so the chosen 2.17 g/l.For the Et Br mutant lipid, the increasing rate was 36.73%.The predicted maximizing lipid was1.47, the maximum lipid 1.705 g/l produced at which incubation time was 5 days, pH 7, at a temperature of 20°C, xylose 2 g/l, and peptone 2 g/l compared to results on synthetic media before using surface response optimization 0.705 g/l.Because the main purpose of this study was to reduce cost so the run was chosen at 1.465 g/l.For the EMS mutant lipid, the increasing rate was 60.35%.The predicted maximizing lipid was 1.62, the maximum lipid 2.27 g/l produced at which incubation time was 7 days, pH 8, at a temperature of 25°C, xylose 1 g/l, and peptone 3 g/l compared to results on synthetic media before using surface response optimization 0.90 g/l.Because the main purpose of this study was to reduce cost so the run was chosen at 1.48 g/l.

Physical properties of the biodiesels
The blending biodiesel (B5) from P. commune NRC 2016 and F. oxysporum NRC 2017 wild types and mutants.Table (11) indicated their physical properties are accepted with ASTM D975 (standard biodiesel for B5).

Discussion
Because of the limited storage capacity of fossil fuels and environmental concerns, biofuels are becoming more desirable and essential.Biodiesel generation from biomass was formerly thought to be a potential alternative to fossil fuels [14].The physical and chemical mutagenic agents were utilized to induce signi cant improvements with a high recurrence rate [25].In this study, four different mutagenic agents were utilized to enhance lipid production by P. commune NRC 2016 and the maximum lipid productivity was 1.47, 0.90, 0.93, and 0.41 g/l was obtained using ray, EMS, EtBr, and NaN 3, respectively.The current nding was matched with [26], who stated that Epicoccum nigrum was used to manufacture digoxin and that ray was employed to raise output to ve-fold after a 1 kilo gray ray dose.[27] studied after that mutagenization with NaN 3 , the lipid concentration of Chlorella desiccate was 275 mg/l.[28] also studied the Et Br that caused point mutations in the absence of an exogenous metabolic activation system.Another study by [29] who was utilized another mutagenic EMS to improve the lipid production of a Chlorella vulgaris mutant strain (UV715), resulting in a mutant with a lipid content and biomass that were 67% and 35% than the wild-type, respectively.The ISSR marker, in the study, could be used as a molecular marker to distinguish between the wild type of P. commune NRC 2016 and its mutants and the results indicated the differences among them.This nding matched the ndings of previous research on Aspergillus avus strains using the ISSR markers as an ideal genetic marker [30].The optimization of medium growth conditions and nutritional parameters was required to achieve higher lipid accumulation by oleaginous microorganisms [31].In our previous work, the optimum conditions for P. commune NRC 2016 lipid content were with the inoculum size adjusted to 1%, the medium pH adjusted to 7.0 incubation for ve days at a temperature of 20ºC using xylose as a carbon source, and peptone as nitrogen source [16].In the current study, the response surface methodology (RSM) was used to optimize the condition for lipid production using P. commune NRC 2016 wild type and its mutants grown on the fermentable sugars produced by growing B. cereus 3SME on bagasse as cheap, carbon source [17].For statistical optimization of different factors affecting production lipid production from P. commune NRC 2016 wild type and its mutants were employed.The increasing rate of lipid production using Box-Behnken 59.70, 42.75, 36.73, and 60.35% were obtained from wild type, ray, NaN3, Et Br, and EMS, respectively.Similar to [14] used the response surface methodology (RSM) approach to optimize the Yarrowia lipolytica TISTR 5151 lipid production in simultaneous sacchari cation and fermentation, with lipid content ranging from 1.45 to 6.63 g/l.The main fatty acids for P. commune NRC 2016 and its mutants in the current study by using Gas chromatography analysis were mainly C16-C18 and it should be noted that these fatty acids were similar to those of plant oils and animal fats.The current study for the analysis of fatty acids was similar to the study [32].The physical properties in this study occurred on blended biodiesel (B5) produced by P. commune NRC 2016 wild type and its mutants the resulting biodiesel properties indicated that biodiesel would have had excellent fuel properties similar to [33].

Conclusion
The fermentable sugars hydrolysate that was produced from bagasse using B. cereus 3SME was reused in the biodiesel production from P. commune NRC 2016 and its mutants to reduce the biodiesel cost.The different chemical and physical mutagens were used for improving the produced biodiesel by P. commune NRC 2016.Response surface methodology (RSM) was applied to optimize P. commune NRC 2016 lipid production to increase biodiesel production from the wild-type and mutant isolates and caused signi cant productivity increases.Gas chromatography analysis of the four tested fungal isolates revealed the fatty acids suitable for biodiesel production.The physical properties of biodiesel produced from the four fungal isolates were similar to standard biodiesel (ASTM D975).Tables Table (1    3D plotting of the interactions and effect of different factors on lipid production from P. commune NRC 2016 wild type (A), mutant by using ray (B), mutant by using Et Br (C), and mutant by using EMS (D)

Declarations
Actual and predicted lipid production values using P. commune NRC 2016 EB mutant based on Box-Behnken design by P.

)
Primer name and primer sequence used in the ISSR analysis

Figures Figure 1 PCR
Figures

Table ( 2
(11)mmary of variables and ranges of study for P. commune NRC 2016 wild type and its mutants by using EMS, Et Br, and ray mutagens Table(11)Physical properties obtained from blending biodiesel P. commune NRC 2016 wild type and its mutants as compared with standard diesel ASTM D975 (standard biodiesel for B5) 3 sodium azide, Et Br: ethidium bromide, EMS: ethyl methanesulfonate, M: mole Table (4) Statistical analysis of ISSR primers used in P. commune NRC 2016 wild type and its mutants and the ampli cation results A: Incubation period, B: pH, C: Temperature, E: Peptone Table (10) acid percentage of P. commune NRC 2016 and mutants