A Study Exploring the Effects of Cell Disruption Techniques on Lipid Recovery in Co-cultivated Microalgae and Oleaginous Yeast

Co-cultivation and effective downstream processing of microalgae and oleaginous yeast are potential strategies to enhance lipid yield. The findings of this work suggest that each cultivable partner should be screened and optimized for the best mutualistic co-cultures. In this case, T. pullulans was found to be the most effective yeast culture with microalgae S. obliquus, C. sorokiniana, and C. protothecoides along with additional cell disruption techniques. Co-cultures of C.protothecoides and T.pullulans had the highest relative lipid yield (RLY) of 68.71%, 59.32%, 55.33%, 53.19%, and 47.3% with sonication, microwave, osmotic shock, freeze-drying, and autoclave, respectively. Sonication was concluded as the best disruption technique with five out of nine co-cultures showing significant lipid enhancement. Even though T. pullulans was the best yeast partner with disruption techniques, co-culture of Y. lipolytica and S.obliquus (RLY 37.11%) produced lipids which was significantly higher than that of their respective monocultures (S. obliquus RLY 23.96%, Y. lipolytica RLY 4.96%) without any disruption process. R. glutinis and Y. lipolytica produced significantly enhanced lipid yield with C.sorokiniana and S. obliquus with sonication (RLY 51.44%) and osmotic shock (RLY 57.70%), respectively. FAME analysis reported that the co-cultures produced higher percentage of total FAME nearly 100% in the case of C. sorokiniana and R. glutinis with high content of saturated fatty acids (SFAs). The presence of biofuel precursors like palmitic acid, linoleic acid, oleic acid, and heptadecanoic acid, confirms their suitability for biofuel production.


Introduction
A country's insatiable appetite for economic growth, urbanization, and global industrialization have led to extensive coal, petroleum, and natural gas mining. Unfettered exploitation of fossil fuels has driven the world into a non-sustainable future with climatic changes, energy deficits, and global warming [1]. Constant demands for sustainable and economical fuel sources have ignited the research aspects for alternative energy sources. Biofuel production from agricultural waste as well as microbial biomass is considered a sustainable and renewable source of green energy that could compensate for the high energy demand [2]. Plantbased oils, animal fats, and microbial lipids are reservoirs of FAME, which can, in turn, be utilized to obtain biofuels. Raw materials for plant-based biofuel production require a vast arable region and good irrigation, making it non-sustainable and, therefore, non-dependable for energy production [3]. Although fats obtained from animal sources are cheap, the biodiesel generated from this source results in biodiesel with flashpoints, high viscosity, and high pour points [4].
Lipids obtained from microbial sources are beneficial for biofuel production since certain oleaginous microbes can accumulate up to 60% of lipids in their entire dry cell weight [5]. Even though bacteria and fungi can accumulate significant lipid contents under controlled conditions, their inherent lipid properties limit their use in biodiesel production [6]. Microalgae are aerobic photosynthetic organisms that can produce fatty acids in the form of triacylglycerides (TAGs), a significant feedstock for biofuel. Microalgae produce TAGs by utilizing either inorganic CO 2 or simple organic carbohydrates, making them the most sustainable source of biofuels [7]. On the other hand, oleaginous yeast is a heterotrophic unicellular organism widely present in nature that produces lipids primarily as TAGs. The symbiotic relationship between the two organisms occurs with the release of carbon dioxide by yeast via fermentation of sugar, which is then utilized by microalgae and in turn, provides nitrogen to the yeast by metabolism [8,9]. Arathi et al. have elaborated the qualitative and quantitative approaches for microalgae harvesting, lipid estimation, and molecular approaches that can be implemented to promote lipid accumulation and recovery [10][11][12]. Recent studies reveal that some nutrients like ferrous sulfate, sodium nitrate, and potassium phosphate affect the growth and lipid yield at specific concentrations in a co-culture medium [13]. Furthermore, certain trace elements released during cell lysis can be used up by yeast and algae for their growth. Thus, co-culturing of microalgae and yeast can enhance biomass and improve the accumulation of FAMEs in cells [14].
Microbial lipids possess numerous advantages; however, their production on a large scale has been severely constrained by their low productivity and high costs [15]. Microalgae have a rigid cell wall made up of several molecular components. The use of an efficient cell disruption technique can assist in high intracellular lipid yield from microalgal cells [16]. In this study, the three microalgal strains and three oleaginous yeast strains are examined for their potential to produce biomass and lipid yield when monoculture and cocultured in different combinations. To enhance the relative lipid yield, monocultures and cocultures are subjected to conventional cell disruption methods such as sonication, osmotic shock, microwave, freeze drying, and autoclaving. The most efficient cell disruption method for high lipid yield and high quality of the extracted product has been determined through direct comparison of the five different cell disruption methods. The lipid extracted from the monocultures of the microalgal and yeast strains and from the co-culture strains which showed higher lipid yield using cell disruption method have been subjected to FAME analysis using gas chromatography (GC-FID) and the FAME compounds essential for biofuel production have been evaluated.

Mono-and Co-cultivation Conditions
The microalgal strains were cultivated autotrophically in BG-11 medium at pH 7.5, light-to-dark photoperiod ratio of 16:8 h at a light intensity of 200 µmoles photons m −2 s −1 using white fluorescent light tubes. The cultures were incubated at room temperature for 14 days. The yeast cultures were grown in an MGYP medium, pH 6.5, at 28 °C in an orbital shaker at 110 rpm for 4 days.
The microalgae and oleaginous yeast strains were cocultivated in 500 mL Erlenmeyer flasks containing 100 mL BG-11 culture medium along with glucose (5 g L −1 ) as the organic carbon source and yeast extract (0.25 g L −1 ) as the nitrogen supplement. The pH of the media was initially set at 7.5. The microalgal cultures at the exponential phase were inoculated at a cell density of 2.68 × 10 6 cells/ml (Scenedesmus obliquus), 3.73 × 10 6 cells/ml (Chlorella protothecoides), and 2.37 × 10 6 cells/ml (Chlorella sorokiniana). The yeast culture strains were inoculated on the 10th day, as their growth rate is higher than the microalgal strains, with a cell density of 4.2 × 10 7 cells/ml (Rhodotorula glutinis), 1.64 × 10 7 cells/ml (Yarrowia lipolytica), and 4.4 × 10 6 cells/ ml (Trichosporon pullulans). The co-culture conditions were maintained the same as that of the microalgae monoculture experiment. The microalgae and yeast cells were co-cultured for 4 days [20].

Biomass Productivity
Following incubation, 100 ml of grown cultures were sampled to determine the biomass. The collected cultures were centrifuged at 6000 rpm for 8 min. The obtained pellet was rinsed twice with distilled water to remove the impurities. The pellet was dried in a hot air oven for 24 h at 70 °C, and then the quantity of biomass was measured by transferring the dried pellet into pre-weighed vials [21].

Cell Disruption
Cell disruption techniques assist in efficiently recovering lipids from microbial cells [22]. This study used five techniques for cell disruption, i.e., sonication, microwave treatment, osmotic shock (10% NaCl), autoclaving, and freeze-drying. For sonication, 100 ml of culture samples were centrifuged at 6000 rpm for 8 min, following which the pellet was collected by discarding the supernatant. Chloroform and methanol in a ratio of 1:2 was used to resuspend the pellet, which was then vortexed and subjected to probe sonication (Labman, Model PRO-250). The experiment was carried out at a frequency of 20 kHz at 40% amplitude for 15 min at 40 °C [23]. The sample was transferred into a 50 ml centrifuge tube for further analysis. Microwave pretreatment was performed in a commercially available microwave oven (Model MC2846BG, LG electronics Inc.) with an output power supply of 900 W. 100 ml of co-cultured microalgae-yeast samples were taken and treated at 90 °C for 30 s in a microwave oven and cooled for another 30 s. This process was repeated for 5 min [24]. Following the treatment, the samples were collected for further analysis. Hightemperature cell disruption by autoclave was carried out by treating 100 ml of the sample at 121 °C, 15 psi for 10 min [25]. The treated samples were then transferred to 50 ml centrifuge tubes for lipid extraction. Pre-treatment using 10% NaCl was carried out on 100 ml of the biomass sample overnight to generate osmotic shock. Osmotic pressure triggers cell wall rupture and aids in lipid release from the cell [26]. Cell disruption via freezing drying was obtained by placing plastic vials containing cultures at − 80 °C in an ultra-low temperature freezer and was frozen overnight. The destruction of the microalgal cell wall was aided by freezing and thawing the culture [27]. The samples collected from the cell disruption process of microwave, autoclave, osmotic shock, and freeze drying were centrifuged at 6000 rpm for 8 min and the pellets were resuspended in chloroform and methanol for the lipid extraction process, as explained in Sect. 1.4. All experiments were performed in triplicates.

Lipid Estimation
Lipids were extracted from the co-cultures of microalgae and yeast using Bligh and Dyer method with some modifications. This method added a 1:2 ratio of chloroform: methanol to the cultures and vortexed gently. The culture was left idle overnight, and then a 1:1 ratio of chloroform: water was added and vortexed gently. Following this, the treated samples were centrifuged for 5 min at 1000 rpm for layer separation, i.e., an aqueous top and an organic bottom. The bottom layer, which includes the lipid portion, was pipetted out, and the chloroform was evaporated at 60 °C in a water bath, after which the obtained lipid was analyzed using the gravimetric technique [24,28,29]. The lipid yield in percentage was mentioned as the relative lipid yield (RLY). The equation calculates the lipid content in percentage as mentioned below [30].

Transesterification and FAME Profiling
The transesterification of lipids was done by mixing dried lipids with toluene: methanol in a ratio of 1:2, followed by adding 1 M NaOCH 3 and incubating at room temperature for 1 h. 5 M NaCl and hexane were added after the incubation period and then centrifuged at 2500 rpm for 5 min to separate the phases. The upper layer containing hexane was pipetted out and evaporated at 65 °C [31]. The recovered FAME residues were analyzed for FAME profiling via GC-FID (Thermo Scientific Trace GC 1110) equipped with a 30 m × 0.25 mm capillary column, with Trace GOLD TG-5 MS as the stationary phase. The nitrogen was used as the carrier gas at a constant flow rate of 1 mL/min. The temperature of the injector and detector was maintained at 250 °C. Before injecting the samples, the oven temperature was ramped to 120 °C for 1 min and raised to 175 °C for 10 min at a rate of 10 °C min −1 , then raised to 210 °C for 10 min at the rate of 5 °C min −1 and raised to 230 °C for 9 min at the rate of 5 °C min −1 [32]. A 37-component FAME Mix (Sigma-Aldrich) was used as an external standard. A ratio of the partial area to the total area is used to calculate the percentage of individual fatty acids in samples.

Statistical Analysis
All experiments were conducted in triplicates and the mean values were considered for statistical analysis. Ordinary two-way ANNOVA was used with 95% confidence interval to determine the statistical significance of the results, which is represented as p-value. The results with p-value at least < 0.05 was considered statistically significant. All the analysis was carried out using GraphPad Prism version 8.0.

Biomass and Lipid Yield of Monocultures and Co-cultures
Microalgal and yeast cultures were chosen based on its reported ability to produce lipids. Chlorella and Scenedesmus are known to produce relatively high lipid content but have not been evaluated widely on its ability to produce Lipid content(%) = Lipid yield gL −1 Biomass production gL −1 × 100 lipids under co-cultivation conditions with yeasts, especially no studies report the optimization of cell disruption processes to enhance lipid recovery for co-cultures. Y. lipolytica, R. glutinis, and T. pullulans are chosen for co-cultivation because of its reported oleaginous properties. The biomass concentration and relative lipid yield obtained through the conventional method without employing additional cell disruption for three microalgal and yeast strains cultured separately and with various combinations of both are shown in Fig. 1. Even though yeast cultures show similar lipid yield in grams per liter the relative lipid yield of yeast cultures is low due to its high biomass content shown in Fig. 1. This type of low relative lipid yield due to higher biomass makes it difficult for the extraction of product with increased cost for downstream processing. Whereas algal cultures show lower biomass content with relatively higher lipid yield. Yeast cultures were incubated for a maximum of 4 days after which the cells attain the stationery and decline phase. The slow growth rate of microalgae results in a lower cell density even after 14 days of incubation. The inoculum for mono and cocultures was obtained by growing cells up to an optical density of 0.5, and 1 ml of these was used as seed inoculum for the experiment. The variation in the initial inoculum is negligible since each organism has varied cell size and different growth rate. For instance, in this study, among microalgal culture, C. protothecoides was added at a cell density of 3.73 × 10 6 cells/ml resulting in 0.95 ± 0.1 g L −1 biomass after 14 days of incubation, whereas C. sorokiniana produced 1.34 ± 0.02 g L −1 biomass with a lower cell inoculum of 2.37 × 10 6 cells/ml. The primary objective while inoculum preparation was to maintain the cells at logarithmic phase in order to ensure maximum viable cells and proper growth. C. sorokiniana produced the highest lipid yield among monocultures of microalgae with 40.99% relative lipid to that of biomass. The results are consistent with Li et al. (2014), who cultured the C.sorokiniana (UTEX 1602) under a mixotrophic condition and obtained a lipid yield of 45% [33]. Among yeast culture, Y. lipolytica showed the highest relative lipid yield of 4.96% along with the highest biomass content of 29.433 ± 1.99 g L −1 . Studies have shown that the lipid yield of Yarrowia lipolytica grown under higher acetic acid concentration and nitrogen-limiting condition was increased up to 35% [34].
Primary co-culture cultivation was performed using the optimized culture medium with a pH of 7.5 to examine the biomass and relative lipid yield produced by various microalgae and oleaginous yeast combinations. BG 11 media along with glucose and yeast extract was used for co-culturing to support the growth of microalgae as well as yeast cultures. Microalgal cultures were grown up to 10 days and yeast inoculums were added after analyzing the pH to the already-growing microalgal cultures. After 4 days of yeast inoculation, the biomass was obtained and lipids were extracted. The co-cultivation of microalgae and oleaginous yeast did not show desirable increase in the relative lipid yield for all co-cultures (shown in Fig. 2) but certainly has a Fig. 1 The biomass (g L −1 ) and relative lipid yield (%) obtained from monoculture and co-culture of microalgal and yeast strains. The floating bars represent the minimum to maximum biomass content plotted along the left Y-axis. The dot plot represents the relative lipid yield plotted along the right Y-axis mutualistic effect on certain combinations. S.obliquus and Y. lipolytica is one such co-culture where the relative lipid yield obtained from the co-culture (37.11% RLY) surpassed both the monoculture yield of 23.96% for S.obliquus and 4.96% for Y. lipolytica. Intense research on the co-cultures in the future could develop new insights in to the mutualistic effect in co-culturing conditions. There are several researches that provide multiple aspects to the enhancement in RLY in coculture. The pH of the culture medium is one such factor affected by co-culturing where the production of organic acids like formic acid, acetic acid, etc., by yeast cultures, is shown to reduce the pH of the media [35]. The formation of bicarbonate in microalgal cultures leads to a pH rise which can be compensated by the organic acid production by yeast. The organic acids produced by yeast are also utilized by the microalgae as carbon source [36]. Another study by Borowitzka, 2016 reported that microalgae release secondary metabolites that act as stimulators or inhibitors of other organisms [37] hence the need to optimize each organism in a co-culture condition. It is reported that the co-culture of microalgae and yeast with organic carbon in the medium leads to higher biomass and lipid recovery [38]. A study reported that the co-culture of Scenedesmus sp. and Candida pimensis with specific concentrations of inorganic micronutrients in a culture medium produced a significant increase in biomass with maximum lipid content [13]. Another study using Chlorella pyrenoidosa and Rhodotorula glutinis in a 3:1 ratio with an optimized carbon/nitrogen value achieved the maximum biomass yield of 6.12 ± 0.31 g L −1 and lipid yield of 2.48 ± 0.09 g L −1 [39]. Even though several studies have shown the mutualistic effect of co-culturing, the obtained results suggest that the co-cultivation may not be suitable for all cultures but can be highly efficient if screened for the best combinations along with additional processing steps to increase lipid yield.

Effective Cell Disruption Method
An effective cell disruption method can substantially aid in the downstream processing of any intracellular product and ensures high quality and enhanced product recovery with several economic prospects. In this study, we have analyzed five different in-vitro small-scale cell disruption techniques to analyze the potential of each on the relative lipid yield. The five different disruption methods used in this study belong to various categories including high moist heat (autoclave) and high dry heat (microwave) treatment, osmotic shock (10% NaCl), low-temperature treatment (freezing drying) as well as physical shear pressure (probe sonication) to determine an optimal cell disruption technique for co-cultured microalgae with yeast. The obtained Relative lipid yield results are depicted in Fig. 3. Results suggest that there is significant increase in the RLY while incorporating additional cell disruption techniques in downstream processing. The least effect was obtained for heat-based cell disruption techniques like autoclave and microwave. Even though there was increase in the RLY while using heat-based disruption, statistically significant increase in lipid yield was obtained only for the co-culture of C. protothecoides and T. pullulans. With autoclave, the obtained RLY was 47.35% (p-value < 0.0005) which was 23.63% without cell disruption. Similarly, while using the microwave the obtained RLY was 59.32% (p-value < 0.0001) which was increased from 23.63% for non-disrupted cells. Osmotic shock was another strategy that was used to disrupt the cells, in this work we have used Fig. 3 The relative lipid yield of microalgal monocultures with co-culture of microalgae and yeast using various cell disruption method a autoclave, b microwave, c osmotic shock, d freeze drying, and e sonication 10% NaCl to induce osmotic pressure for rupturing of cells. Co-cultures of S. obliquus and Y. lipolytica and C. protothecoides and T. pullulans produced significant increase in the RLY. All the other combinations also resulted in increase in lipid yield but not of statistical significance. The RLY of S. obliquus and Y. lipolytica and C. protothecoides and T. pullulans was increased from 37.11 to 23.63% for nondisrupted cells to 57.70% (p-value < 0.005) and 55.33% (p-value < 0.0001) respectively for 10% NaCl treated cells. Another approach for cell disruption was freeze-drying to disrupt the cells. Using freezing as the disruption strategy, the RLY of co-cultures, S. obliquus and T. pullulans and C. protothecoides and T.pullulans were increased from 14.72 to 23.63% for non-disrupted cells to 30.21% (p-value < 0.05) and 53.19% (p-value < 0.0001) respectively. Physical shear is one of the major strategies that could be employed for effective cell disruption. In this study, we have used probebased sonication for cell disruption and have obtained significant increase in RLY for co-cultures of S. obliquus and T. pullulans (non-disruption-14.72% and sonication-34.08%; p-value < 0.005), C. sorokiniana and R glutinis (non-disruption-35.00% and sonication-51.44%; p-value < 0.05), C. protothecoides and R. glutinis (non-disruption-20.20% and sonication-40.32%; p-value < 0.005), C. protothecoides and Y. lipolytica (non-disruption-20.26% and sonication-43.27%; p-value < 0.0005) and C. protothecoides and T. pullulans (non-disruption-23.63% and sonication-68.71%; p-value < 0.0001). Among the various co-cultures of microalgae with yeast, T. pullulans were demonstrated to be the most effective mutualistic partner with almost all combinations with microalgae showing significant increase in relative lipid yield. C. protothecoides and T. pullulans showed the highest RLY with all the cell disruption methods used.
Sonication was the most efficient method for disrupting cells with five out of nine microalgae and yeast co-cultures showing significant increase in RLY. Based on statistical analysis, sonication produced significantly more lipids than other methods and non-disrupted cells, resulting in the highest lipid yield of all co-cultures for C. protothecoides and T pullulans with RLY of 68.71%. Previous studies have reported the efficiency of sonication in order to obtain the highest lipid yield in microalgal cultures, viz., Nostoc sp, Chlorella sp., and Tolypothrix sp. Several studies have concluded that an appropriate cell disruption method for microalgal lipid extraction is one major factor for sustainable and economic biodiesel production [40]. Following sonication, osmotic shock and freeze drying are the best methods with two out of nine co-cultures showing significant increase in RLY. A study conducted by Rakesh et al. (2015) evaluated the highest lipid yield of 48.33% in Botryococcus sp (MCC31) using microwave and 36.18% in Chlorella sorokiniana (MIC-G5) with osmotic shock (15% NaCl) [41]. It has been reported that microwave and ultrasonication improve lipid extraction efficiency [42]. The cell disruption methods performed in microalgae were evaluated and reported that microwave pretreatment was the most effective for lipid recovery of 49% in N. oceanica [43]. Whereas, in respect to osmotic shock, the high salinity leads to osmotic stress in microalgae which leads to increased lipid accumulation in Chlorella vulgaris, Dunaliella sp., etc. [44,45]. Overall results and its analysis suggest that each co-culture has distinct kinds of interactions and mutualism cannot be hypothesized in all scenarios. Co-culturing organisms have to be optimized along with analyzing and determining appropriate downstream processing stages as well, which could ensure increase in the desirable product recovery.

Conclusion
The various co-culture combinations of yeast and microalgae were analyzed in terms of relative lipid yield. The coculture of yeast and microalgae are found to be the ideal partners compared to monocultures, which could lead to sustainable biodiesel. The lipid production was further enhanced by various cell disruption techniques and revealed that sonication produced significantly higher lipids than other techniques. Furthermore, fatty acid composition analysis confirmed the presence of major fatty acids required for biodiesel production. The mutualistic effect of coculturing has been shown in several studies, but evaluating the best combination and optimizing culture conditions with efficient downstream processing can enhance lipid yield.