Submerged cultivation of Nigrospora sp. in batch and fed-batch modes for microbial oil production

Abstract Microbial lipids are a valuable source of potential biofuels and essential polyunsaturated fatty acids. The optimization of the fermentation conditions is a strategy that affects the total lipid concentration. The genus Nigrospora sp. has been the target of investigations based on its potential bioherbicidal action. Therefore, this study developed a strategy to maximize the biomass concentration and lipid accumulation by Nigrospora sp. in submerged fermentation. Different media compositions and process variables were investigated in shaken flasks and bioreactor in batch and fed-batch modes. Maximum biomass concentration and lipid accumulations were 40.17 g/L and 21.32 wt% in the bioreactor, which was 2.1 and 5.4 times higher than the same condition in shaken flasks, respectively. This study presents relevant information to the production of fungal lipids since few investigations are exploring the fed-batch strategy to increase the yield of fungi lipids, as well as few studies investigating Nigrospora sp. to produce lipids. Graphical Abstract


Introduction
oleaginous microorganisms are microbial species that can accumulate significant amounts of triacylglycerols.Filamentous fungi such as Aspergillus oryzae, Claviceps purpurea, Humicola lanuginose, Mortierella isabellina, Mortierella vinacea, and Mucor circinelloides have been reported as microorganisms capable of accumulating lipids (Ma et al. 2018;Ivančić et al. 2021).The oils produced by fungi present a high potential for the production of biofuels, while they are also an alternative for the production of essential polyunsaturated fatty acids (PuFAs), such as arachidonic, γ-linolenic, eicosapentaenoic and docosahexaenoic acids.
The genus Nigrospora sp. is studied to produce bioactive secondary metabolites.Nonetheless, lipid production is not addressed.A study isolated five fungi with potential in the production of microbial oil, and Nigrospora sp.produced approximately 20 wt% of microbial oil by solid-state fermentation (Peng and Chen 2007).Another study evaluated the lipid production capacity of 150 fungal strains isolated from the Brazilian Pampa Biome, while Nigrospora sp.presented promising results with maximum lipid accumulation of 11.28 wt% (51% PuFAs) (Tonato et al. 2018).Nevertheless, the lipid accumulation should be higher to make an industrial process feasible for microbial oil production.one possibility to increase this accumulation is to modify the fermentation media and process conditions and investigate the possibility to operate the process in the fed-batch (FB) mode.Furthermore, the choice of the carbon source is a significant factor because it can significantly benefit the yield and lipid composition of the microbial oil due to differences in the metabolism of microorganisms.Additionally, the cost of raw materials is a decisive aspect to be considered in the definition of total production costs.Substrates such as glucose, lactose, starch, oils, xylose, glycerol, acetic acid, and ethanol are carbon sources applied to intensify microbial oil production (Wang et al. 2022).
Therefore, the nature and cultivation specifications of the microorganism can influence the lipid accumulation of oleaginous species.The fermentation strategy (batch, fed-batch, and repeated fed-batch) has been analyzed in the microbial fermentation processes because it can influence the final yield of the bioproduct.Studies are reported using batch fermentation to produce lipids by species of fungi (Tonato et al. 2018(Tonato et al. , 2019)).The fed-batch cultivation is efficient for biotechnological processes that involve the production of bioproducts, such as biopolymers, fatty acids, and single-cell proteins (Scheel et al. 2021).
The purpose of this study was to maximize the biomass concentration and lipid accumulation by the fungus Nigrospora sp. in submerged fermentation.Media composition and process variables were investigated in an orbital shaker in batch and fed-batch modes.For the bioreactor, the process investigated the influence of stirring rate and aeration in batch and fed-batch modes.Also, the composition of microbial oil was presented for all fermentations.

Microbial growth and lipid accumulation in shaken flasks
Figure S1 presents the sugar, biomass, and ph profiles obtained at 25, 28, and 33 °C during 7 days of fermentation.The ph of the medium, regardless of the temperature, showed two distinct behaviors.In the first two days of fermentation, the ph decreased slightly (5.8 to 5.3).In the same period, approximately 80% of the initial sugar was consumed by the fungus.From the second to the seventh day of fermentation, the ph raised to close to 8.0.These characteristics show a relationship between ph variation in the fermentation process and the consumption of glucose.These changes in the ph during the microbial growth are dependent on the carbon and nitrogen sources of the fermentation medium.The reduction of ph in the first two days was associated with the production of organic acids during the metabolism of the carbon sources.After the depletion of sugar, the fungus metabolizes the acids produced in the first stage, increasing the ph of the medium (Galhaup et al. 2002).
For the biomass, at 25 and 28 °C, the stationary phase was reached on the fourth day of fermentation, whereas for 33 °C the biomass was slightly increased until the seventh day.Temperature affected the initial rate of sugar consumption, where the highest initial rate was obtained at 28 °C.The growth of Nigrospora sp. was associated with the consumption of sugar in the media because the biomass was accumulated until all sugar was metabolized.Afterward, the growth was stabilized, showing that the final concentration of biomass is dependent on sugar concentration in the media (Subhash and Mohan 2014).
Table 1 presents the biomass concentration, lipid accumulation, and biochemical parameters for the temperatures evaluated after 7 days of fermentation.The application of the Tukey test for the conversion factors and yields of biomass and lipids, for each temperature, showed statistical differences (p < 0.05) (Table 1).The temperature influenced most of the results obtained.Some exceptions were observed, in which the values do not differ statistically from the others, as occurred with the Y X/S (substrate conversion factor in biomass) at 25 and 33 °C and the values obtained in the biomass concentration (g/L).This concentration was not influenced by temperature (p < 0.05), which the highest value was obtained at 33 °C (11.88 g/L).This result did not differ statistically from the conditions of 28 °C (11.76 g/L) and 25 °C (10.67 g/L).Temperatures near 30 °C are frequently most appropriate for fungi cultivation aiming to obtain a high biomass concentration because around this temperature the enzymes show increased metabolic activity (Subhash and Mohan2014).These data are also in agreement with another study (Mironov et al. 2018), in which Mortierella alpina was cultivated for the production of lipids 20 to 28 °C and the highest concentration of biomass (17.7 g/L) was obtained at 28 °C.
At 28 and 33 °C, the lipid yield was higher (1.24 and 1.02 wt%) than 0.84% obtained at 25 °C.These values differed from each other, showing a higher lipid yield at 28 °C.Maximum production of lipids for temperatures close to 30 °C for different fungi is reported (Mironov et al. 2018).The highest values of biochemical parameters such as µ max , Y X/S , and Y P/X , were observed at 28 °C.For Y P/S , the highest value was achieved at 25 °C.The highest µ max was observed at 28 °C (1.81/h).The conditions of 33 °C (1.76/h) and 25 °C (1.40/h) showed lower values.Considering Y X/S , the highest value was observed for 28 °C (1.24 g/g), significantly different from 33 °C (1.18 g/g) and 25 °C (1.13 g/g).A similar scenario was observed for Y P/X and Y P/S , where 28 °C showed the highest values (0.0124 g/g and 0.0145 g/g, respectively).These results differed from 25 °C (0.0084 g/g and 0.0896 g/g, respectively) and 33 °C (0.0102 g/g and 0.014 g/g, respectively).
From the analysis of data presented in Figure S1 and Table 1, two main responses can be achieved: 28 °C is the most appropriate temperature and biomass concentration is dependent on sugar concentration.The biomass concentration and lipid accumulation obtained in this first set of experiments are not enough to make the bioprocess feasible for the production of microbial oil.Therefore, other strategies should be proposed.Table S1 presents data referring to the cultivation of Nigrospora sp. in shaken flasks using different media compositions in batch and fed-batch fermentations.
Most of the responses obtained with the different formulations of fermentation media, for biomass concentration and lipid yield observed in Table S1, showed significant differences in the Tukey's test at 95% confidence level (p < 0.05).It demonstrates that the optimization of the fermentation medium influenced most of the responses obtained for biomass concentration (g/L), lipid yield (% by weight), and conversion factors (g/g) (Y X/S , Y P/X , Y P/S ).
In this case, all fermentations were conducted at 28 °C.Increasing initial glucose concentration from 10 to 100 g/L in batch fermentations increased the final biomass concentration and lipid accumulation.The highest value of biomass was observed para FB G100_CSL10 (29.93 g/L).This condition differed from all other conditions.The B G100_CSL10 reported up to 22.19 g/L, which did not differ from B G100 (18.80 g/L).The lowest results were verified for FB G20 (6.20 g/L) and FB S20 (9.40 g/L).A different scenario was observed for oil production, in which B G100 presented the most promising results (3.94%), while it differed from all other experimental conditions.The lowest results were obtained for B G10 (1.24%), FB S10 (1.30%), and FB G20 (1.40%), which differed from all other conditions.
Considering Y X/S , Y P/X , and Y P/S , the highest results were concentrated in the conditions involving B G10 and B G100 .For Y X/S , B G10 had the highest result (1.24 g/g) and differed from all other conditions.This behavior was verified for B G100_CSL10 and B G100 , which reported significant values (0.21 g/g and 0.20 g/g).The same scenario was observed for Y P/S , with 0.015 g/g for B G10 and 0.008 g/g for B G100 .The exception was observed for Y P/X , where the best condition was B G100 (0.039 g/g), but did not differ from FB G60 (0.033 g/g), FB G100 (0.030 g/g), FB S60 (0.028 g/g), and FB S100 (0.026 g/g).The least promising results were observed for FB S20 for all conditions (0.09, 0.013, and 0.001 g/g).The lowest value of lipid accumulation in the fermentation B G100_CSL10 may be attributed to the reduction of the C/N ratio because the CSL is a nitrogen-rich source (Shaigani et al. 2021).The fed-batch fermentations were not effective to increase the biomass concentration and lipid accumulation.FB G100_CSL10 was effective to increase biomass concentration but failed in lipid accumulation.Both carbon sources evaluated in the fed-batch fermentations (glucose and sucrose) presented similar biomass concentrations (approximately 13 g/L) at concentrations higher than 60 g/L.
Nevertheless, glucose showed to be more effective in lipid accumulation (approximately 15% more lipid than using sucrose).These results are corroborated by a study that used Cunninghamella echinulata with carbon sources (glucose, xylose, and lignocellulosic residues) in shaken flasks.The ideal condition for lipid accumulation and high biomass concentration was obtained when using glucose as a carbon source at 100 g/L (Zikou et al. 2013).

Microbial growth and lipid accumulation in STR
In the third set of experiments, aiming to increase the biomass concentration in the media and the lipid accumulation, fermentations were carried out in an STR bioreactor in batch and fed-batch modes (Table S2).The use of a bioreactor was effective to increase biomass concentration and lipid accumulation.In batch mode, the maximum biomass concentration was 32.50 g/L with 16.7 wt% of lipid accumulation (assay 3).It was statistically different (p < 0.05) from other assays of the CCD.Comparing with fermentation in shaken flasks (B G100 ), the biomass concentration and lipid accumulation increased by 72.9% and 324%.one reason for this result is the aeration, which was considerably improved in the STR bioreactor in relation to shaken flasks.The importance of aeration in biomass production and lipid accumulation can be seen by comparing assays 1 and 3 (stirring rate of 100 rpm) and 2 and 4 (stirring rate of 200 rpm).It was confirmed by ANoVA presented in Table S3, where aeration was the most significant variable, followed by the interaction between aeration and stirring rate.In both situations, the increase of the aeration from 0.5 to 2 vvm improved biomass production and lipid accumulation.This increase was more accentuated in assays with the lowest stirring rate (100 rpm).
The increase in stirring rate increases the shear forces, which may influence the morphology of the microorganism, decrease viscosity, increase the rate of oxygen transfer, and damage the mycelium, thus causing a reduction in productivity.otherwise, low stirring rates cause a reduction of dissolved oxygen concentration in the system, which may be detrimental to the microorganism in the biosynthesis of secondary metabolites.A study with the filamentous fungus Cunninghamella bainieri 2A1 in a 5 L bioreactor for lipid production reported that the control of stirring rate and aeration was important to increase lipid production in the bioreactor (Saad et al. 2014).Furthermore, Mortierella isabellina cultivated in a 3 L batch bioreactor with glucose as substrate indicated higher values of biomass and lipid content when compared to the values observed in the assays in shaken flasks.These findings were obtained with a lower stirring rate and a higher concentration of dissolved oxygen (Chatzifragkou et al. 2010).
Fed-batch fermentation was performed in the best condition (assay 8) at 100 rpm and 2 vvm for biomass production and lipid accumulation in batch fermentations in the STR bioreactor.According to the results, in fed-batch fermentations in the STR, biomass and lipid increased by 23.6 and 27.6%.At this point, a process condition with satisfactory biomass and lipid accumulation was reached, which is comparable to other studies focusing on the production of microbial oil with conventional oleaginous microorganisms (Shaigani et al. 2021).
The use of fed-batch fermentation to increase the yield of biomass and lipids of oil-producing microorganisms is more efficient than batch mode.In batch cultivation, the nutrients are added in the beginning while in the fed-batch the nutrients are added during the fermentation, preventing the inhibition by carbon source.Studies have been performed using this fed-batch to increase the accumulation of lipids in various oil-producing microorganisms.A study reported up to 37.2 g/L of biomass and 64.5 wt% of lipid in fed-batch in a bioreactor with Rhodosporidium toruloides (Tchakouteu et al. 2017).These values were higher than those obtained in batch fermentation.In other studies, using oleaginous microorganisms grown in fed-batch modes, such as the yeasts Rhodotorula glutinis (Karamerou et al. 2017) and Rhodosporidium toruloides Y4 (Fei et al. 2016), the efficacy of this mode of cultivation to increase the biomass and lipid concentration was confirmed.

Fatty acids composition
The fatty acid composition of the lipids obtained from the biomass of Nigrospora sp. is presented in Table S4 and Figure S2.The fatty acids with the highest concentration identified in the lipids extracted from this fungus, regardless of fermentation mode, were oleic acid (C18:1n9c), linoleic acid (C18:2n6c), and palmitic acid (C16:0).Generally, the concentration of fatty acids is different from cultivation in shaken flasks or bioreactor.Following this trend, the mode of fermentation also affected the fatty acid composition.In shaken flasks, a higher concentration of monounsaturated fatty acids (MuFA) was obtained, ranging from 130.785 to 370.927 mg/g.In the sequence, saturated fatty acids (SFA) and polyunsaturated fatty acids (PuFA) ranged from 98.329 to 207.833 and 49.442 to 169.052 mg/g.
In the bioreactor, the fatty acid profile was similar to the shaken flasks (MuFA > SFA > PuFA), but the concentrations of fatty acids were lower.otherwise, the synthesis of essential PuFA such as arachidonic, docosahexaenoic and eicosapentaenoic acids was observed only in fermentations carried out in the STR.The improvement in the mixing and oxygen transfer rate inside the bioreactor had a positive influence on the synthesis of long-chain PuFA (Sun et al. 2018), mainly arachidonic, docosahexaenoic and eicosapentaenoic acids, which are highly desirable due to their positive effect on human health.

Conclusions
This study presented a strategy to maximize the biomass concentration and lipid accumulation by Nigrospora sp. in submerged fermentation.Maximum biomass concentration and lipid accumulations were 40.17 g/L and 21.32 wt%.These values were obtained with a bioreactor operating at 100 rpm and aeration of 2 vvm in the fed-batch mode, with a glucose concentration of 100 g/L and an intermittent feeding strategy from the second to the sixth day of fermentation.The results provided relevant information to the production of fungal lipids on a pilot scale, especially contributing to outcomes about exploring the fed-batch strategy to increase microbial oil yields.

Table 1 .
Influence of temperature on biomass concentration, oil accumulation, and biochemical parameters.
a-cdifferent letters in the same column indicate a significant difference (p < 0.05) by tukey's test.µ max : maximum specific growth rate; Y X/s : biomass yield on substrate; Y P/X : oil yield on biomass; Y P/s : oil yield on substrate.