Transcriptomic profiling of an evolved Yarrowia lipolytica strain: tackling hexanoic acid fermentation to increase lipid production from short-chain fatty acids.

BACKGROUND: Short-chain fatty acids (SCFAs) are cost-effective carbon sources for an affordable production of lipids. Hexanoic acid, the acid with the longest carbon chain in the SCFAs pool, is produced in anaerobic fermentation of organic residues and its use is very challenging, even inhibiting oleaginous yeasts growth. RESULTS: In this investigation, an adaptive laboratory evolution (ALE) was performed to improve Yarrowia lipolytica ACA DC 50109 tolerance to high hexanoic acid concentrations. Following ALE, the transcriptomic analysis revealed several genetic adaptations that improved the assimilation of this carbon source in the evolved strain compared to the wild type (WT). Indeed, the evolved strain presented a high expression of the up-regulated gene YALI0 E16016g, which codes for FAT1 and is related to lipid droplets formation and responsible for mobilizing long-chain acids within the cell. Strikingly, acetic acid and other carbohydrate transporters were over-expressed in the WT strain. CONCLUSIONS: A more tolerant yeast strain able to attain higher lipid content under the presence of high concentrations of hexanoic acid has been obtained. Results provided novel information regarding the assimilation of hexanoic acid in yeasts.

Cutaneotrichosporon curvatum and Yarrowia lipolytica as key players for green chemistry: efficient oil producers from food waste via the carboxylate platform.

Cutaneotrichosporon curvatum and Yarrowia lipolytica can accumulate microbial oils using short-chain fatty acids (SCFA) as carbon sources. SCFAs-rich media often contain significant amounts of nitrogen that prevent high carbon:nitrogen (C:N) ratios necessary to boost lipid production. This work assessed the intrinsic ability of C. curvatum and Y. lipolytica to produce high amounts of microbial oils from these unusual carbon sources. Results demonstrated that minor differences in SCFA concentration (only 2 g/L) had a significant effect on yeast growth and lipid production. A C:N of 80 promoted yeast growth at all SCFA concentrations and favored SCFA consumption at 19 g/L SCFAs. The different SCFA uptake preferences in C. curvatum and Y. lipolytica highlighted the importance of considering the SCFA profile to select a suitable yeast strain for microbial oils production. At the most challenging SCFA concentration (19 g/L), 57.2% ±1.6% (w/w) and 78.4 ± 0.6% (w/w) lipid content were obtained in C. curvatum and Y. lipolytica, respectively. These values are among the highest reported for wild-type strains. To circumvent the challenges associated with media with high nitrogen content, this report also proved struvite precipitation as an effective method for increasing lipid production (from 17.9 ± 3.9% (w/w) to 41.9 ± 2.6% (w/w)) after nitrogen removal in food waste-derived media.

Slight variations in SCFA concentrations have a relevant effect on yeast lipid productionHigh nitrogen availability is crucial to promote cell growth at very high SCFA concentrationsC:N effect on cell growth and lipid production is specie-specific and may depend on yeast robustnessYeast strains have diverse SCFA preferences and differently metabolize these acidsStruvite precipitation effectively removes nitrogen from real digestates increasing C:N.

Microbial co-cultures for biochemicals production from lignocellulosic biomass: A review.

Global reliance on fossil oil should shift to cleaner alternatives to get a decarbonized society. One option to achieve this ambitious goal is the use of biochemicals produced from lignocellulosic biomass (LCB). The inherent low biodegradability of LCB and the inhibitory compounds that might be released during pretreatment are two main challenges for LCB valorization. At microbiological level, constraints are mostly linked to the need for axenic cultures and the preference for certain carbon sources (i.e., glucose). To cope with these issues, this review focuses on efficient LCB conversion via the sugar platform as well as an innovative carboxylate platform taking advantage of the co-cultivation of microorganisms. This review discusses novel trends in the use of microbial communities and co-cultures aiming at different bioproducts co-generation in single reactors as well as in sequential bioprocess combination. The outlook and further perspectives of these alternatives have been outlined for future successful development.

Production of short-chain fatty acids (SCFAs) as chemicals or substrates for microbes to obtain biochemicals.

Carboxylic acids have become interesting platform molecules in the last years due to their versatility to act as carbon sources for different microorganisms or as precursors for the chemical industry. Among carboxylic acids, short-chain fatty acids (SCFAs) such as acetic, propionic, butyric, valeric, and caproic acids can be biotechnologically produced in an anaerobic fermentation process from lignocellulose or other organic wastes of agricultural, industrial, or municipal origin. The biosynthesis of SCFAs is advantageous compared to chemical synthesis, since the latter relies on fossil-derived raw materials, expensive and toxic catalysts and harsh process conditions. This review article gives an overview on biosynthesis of SCFAs from complex waste products. Different applications of SCFAs are explored and how these acids can be considered as a source of bioproducts, aiming at the development of a circular economy. The use of SCFAs as platform molecules requires adequate concentration and separation processes that are also addressed in this review. Various microorganisms such as bacteria or oleaginous yeasts can efficiently use SCFA mixtures derived from anaerobic fermentation, an attribute that can be exploited in microbial electrolytic cells or to produce biopolymers such as microbial oils or polyhydroxyalkanoates. Promising technologies for the microbial conversion of SCFAs into bioproducts are outlined with recent examples, highlighting SCFAs as interesting platform molecules for the development of future bioeconomy.

Phosphate limitation as crucial factor to enhance yeast lipid production from short-chain fatty acids.

Microbial lipids for chemical synthesis are commonly obtained from sugar-based substrates which in most cases is not economically viable. As a low-cost carbon source, short-chain fatty acids (SCFAs) that can be obtained from food wastes offer an interesting alternative for achieving an affordable lipid production process. In this study, SCFAs were employed to accumulate lipids using Yarrowia lipolytica ACA DC 50109. For this purpose, different amounts of SCFAs, sulfate, phosphate and carbon: phosphate ratios were used in both synthetic and real SCFAs-rich media. Although sulfate limitation did not increase lipid accumulation, phosphate limitation was proved to be an optimal strategy for increasing lipid content and lipid yields in both synthetic and real media, reaching a lipid productivity up to 8.95 g/L h. Remarkably, the highest lipid yield (0.30 g/g) was achieved under phosphate absence condition (0 g/L). This fact demonstrated the suitability of using low-phosphate concentrations to boost lipid production from SCFAs.

Key role of fluorescence quantum yield in Nile Red staining method for determining intracellular lipids in yeast strains.

BACKGROUND: Microbial lipids are found to be an interesting green alternative to expand available oil sources for the chemical industry. Yeasts are considered a promising platform for sustainable lipid production. Remarkably, some oleaginous yeasts have even shown the ability to grow and accumulate lipids using unusual carbon sources derived from organic wastes, such as volatile fatty acids. Recent research efforts have been focused on developing rapid and accurate fluorometric methods for the quantification of intracellular yeast lipids. Nevertheless, the current methods are often tedious and/or exhibit low reproducibility. RESULTS: This work evaluated the reliability of different fluorescence measurements (fluorescence intensity, total area and fluorescence quantum yield) using Nile Red as lipid dye in two yeast strains (Yarrowia lipolytica ACA-DC 50109 and Cutaneotrichosporon curvatum NRRL-Y-1511). Different standard curves were obtained for each yeast specie. Fermentation tests were carried with 6-month difference to evaluate the effect of the fluorometer lamp lifetime on lipid quantification. CONCLUSIONS: Fluorescence quantum yield presented the most consistent measurements along time and the closer estimations when compared with lipids obtained by conventional methods (extraction and gravimetrical determination). The need of using fluorescence quantum yield to estimate intracellular lipids, which is not the common trend in studies focused on microbial lipid production, was stressed. The information here provided will surely enable more accurate results comparison.

Insights into cell robustness against lignocellulosic inhibitors and insoluble solids in bioethanol production processes.

Increasing yeast robustness against lignocellulosic-derived inhibitors and insoluble solids in bioethanol production is essential for the transition to a bio-based economy. This work evaluates the effect exerted by insoluble solids on yeast tolerance to inhibitory compounds, which is crucial in high gravity processes. Adaptive laboratory evolution (ALE) was applied on a xylose-fermenting Saccharomyces cerevisiae strain to simultaneously increase the tolerance to lignocellulosic inhibitors and insoluble solids. The evolved strain gave rise to a fivefold increase in bioethanol yield in fermentation experiments with high concentration of inhibitors and 10% (w/v) of water insoluble solids. This strain also produced 5% (P > 0.01) more ethanol than the parental in simultaneous saccharification and fermentation of steam-exploded wheat straw, mainly due to an increased xylose consumption. In response to the stress conditions (solids and inhibitors) imposed in ALE, cells induced the expression of genes related to cell wall integrity (SRL1, CWP2, WSC2 and WSC4) and general stress response (e.g., CDC5, DUN1, CTT1, GRE1), simultaneously repressing genes related to protein synthesis and iron transport and homeostasis (e.g., FTR1, ARN1, FRE1), ultimately leading to the improved phenotype. These results contribute towards understanding molecular mechanisms that cells might use to convert lignocellulosic substrates effectively.

Microalgae production for nitrogen recovery of high-strength dry anaerobic digestion effluent.

Dry anaerobic digestion (D-AD) generates nitrogen-rich effluents that are normally neglected in the circular bioeconomy. The high turbidity and ammonium content hamper nitrogen recovery from these effluents via biological processes, such as microalgae culture. The goal of this study was to demonstrate microalgae growth viability in high-strength D-AD effluents in order to recover nitrogen (N) as microalgae biomass. According to the experimental factorial design conducted in batch reactors, ammonium was identified as the critical inhibitory compound for microalgae growth while turbidity did not exhibit a significantly negative effect. Instead, turbidity resulted advantageous since it promoted high nitrogen uptake rates and biomass production. The presence of organic turbidity resulted in a positive effect that boosted Chlorella growth in a stream with higher ammonium (350 mg NH4+-N L-1) and turbidity (175 NTU) than the inhibition thresholds reported in the literature, reaching 98.7% of N recovery as microalgae biomass. When microalgae culture was scaled up in a photobioreactor operated in continuous mode, microalgae biomass was effectively produced while recovering 100% of N at a hydraulic retention time of 10 days. By imposing long exposure times and high turbidity, Chlorella adaptation to high-strength D-AD effluent resulted in high N uptake and biomass production. This study demonstrated not only the most influencing factor and the optimal NH4+-N and turbidity combination, but also the viability of using D-AD effluents as culture media for microalgae biomass production.

Carboxylic acids production via anaerobic fermentation: Microbial communities' responses to stepwise and direct hydraulic retention time decrease.

Carboxylic acids, traditionally produced from fossil fuels, might be generated from renewable biomass resources via anaerobic fermentation. Considering that the microbial activity of this bioprocess is ruled by the imposed hydraulic retention time (HRT), this investigation explored the relationship between process stability and microbial community. Stepwise and direct HRT reduction strategies were assessed in terms of waste bioconversion into volatile fatty acids (VFAs). Microbial community dynamics revealed a microbial specialization along the HRT decrease. The direct implementation of low HRT resulted in drastic microbial fluctuations, leading to process failure at HRT below 6 days. Stepwise strategy for HRT reduction favored microbial adaptation, supporting maximum bioconversions efficiencies (32 % VFACOD/tCODin) at low HRT values (HRT 4 days). Microbial similarity analysis revealed Clostridiales, Lactobacillales and Bacteroidales orders as keystone microorganisms involved in VFAs production, being responsible for protein degradation and propionic acid accumulation.

Statistical correlation between waste macromolecular composition and anaerobic fermentation temperature for specific short-chain fatty acid production.

To properly exploit short-chain fatty acids (SCFAs) in the chemical industry, it is of foremost importance to ensure stable SCFA profile production via anaerobic fermentation (AF). The different macromolecular distribution of food wastes (FWs) used as feedstock might be crucial for process outcome. Targeting at a specific SCFAs profile and yield, this study explored the statistical correlation between the macromolecular composition of FWs and the produced SCFAs in batch-AFs at 25 °C and 55 °C. Principal Component Analysis (PCA) revealed that the carbohydrates fraction was directly related with butyric acid accumulation, regardless of process temperature. Nevertheless, operational temperature resulted in a pH change, which ultimately affected the process fate. PCA of 25 °C-batch-AF showed a positive correlation between high carbohydrate content and longer-chain acids accumulation. By contrast, 55 °C-AF resulted in higher product specificity than at 25 °C, mainly due to butyrate-type fermentation of carbohydrates. Batch results were further validated in a semicontinuous reactor. Prevailing SCFAs and high bioconversion efficiencies relied on 3 main FWs characteristics: high carbohydrate content (>77% w/w), high carbohydrate/protein ratio (≥10) and high soluble organic matter content. Results obtained herein allowed predicting a specific SCFAs profile based on FWs composition, which is relevant for setting proper downstream technologies.

Bioproducts generation from carboxylate platforms by the non-conventional yeast Yarrowia lipolytica.

In recent years, there has been a growing interest in the use of renewable sources for bio-based production aiming at developing sustainable and feasible approaches towards a circular economy. Among these renewable sources, organic wastes (OWs) can be anaerobically digested to generate carboxylates like volatile fatty acids (VFAs), lactic acid, and longer-chain fatty acids that are regarded as novel building blocks for the synthesis of value-added compounds by yeasts. This review discusses on the processes that can be used to create valuable molecules from OW-derived VFAs; the pathways employed by the oleaginous yeast Yarrowia lipolytica to directly metabolize such molecules; and the relationship between OW composition, anaerobic digestion, and VFA profiles. The review also summarizes the current knowledge about VFA toxicity, the pathways by which VFAs are metabolized and the metabolic engineering strategies that can be employed in Y. lipolytica to produce value-added biobased compounds from VFAs.

Tuning microbial community in non-conventional two-stage anaerobic bioprocess for microalgae biomass valorization into targeted bioproducts.

Unspecific microorganisms consortia are normally used in anaerobic biodegradation of solid wastes. However, these consortia can be tuned to optimally obtain determined bioproducts. In this study, high value-added products and biogas were obtained via an innovative two-stage anaerobic bioprocess from microalgae biomass. The anaerobic fermentation (AF) entailed the production of short-chain fatty acids (SCFAs) and subsequently, only the solid spent of AF effluent was valorized for methane production via conventional anaerobic digestion (AD). Applied conditions in AF (25 °C, HRT 8 days) favored Firmicutes predominance (64%) enabling a conversion efficiency of 32.1% g SCFAs-COD/g CODin. Opposite, a wider microbial biodiversity was determined in the AD reactor (35 °C, HRT 20 days), being mainly composed by Firmicutes (28.6%), Euryarchaeota (17.7%) and Proteobacteria (15.3%). AD of the AF-solid spent reached 168.9 mL CH4 /g CODin. Strikingly, operational conditions imposed mediated a microbial specialization that maximized product output.

Insights on the microbial communities developed during the anaerobic fermentation of raw and pretreated microalgae biomass.

Short-chain fatty acids (SCFAs) are considered building blocks for bioproducts in the so-called carboxylate platform. These compounds can be sustainably produced via anaerobic fermentation (AF) of organic substrates, such as microalgae. However, SCFAs bioconversion efficiency is hampered by the hard cell wall of some microalgae. In this study, one thermal and two enzymatic pretreatments (carbohydrases and proteases) were employed to enhance Chlorella vulgaris biomass solubilization prior to AF. Pretreated and non-pretreated microalgae were assessed in continuous stirred tank reactors (CSTRs) for SCFAs production. Aiming to understand microorganisms' roles in AF depending on the employed substrate, not only bioconversion yields into SCFAs were evaluated but microbial communities were thoroughly characterized. Proteins were responsible for the inherent limitation of raw biomass conversion into SCFAs. Indeed, the proteolytic pretreatment resulted in the highest bioconversion (33.4% SCFAs-COD/CODin), displaying a 4-fold enhancement compared with raw biomass. Population dynamics revealed a microbial biodiversity loss along the AF regardless of the applied pretreatment, evidencing that the imposed operational conditions specialized the microbial community. In fact, a reduced abundance in Euryarchaeota phylum explained the low methanogenic activity, implying SCFAs accumulation. The bacterial community developed in the reactors fed with pretreated microalgae exhibited high acidogenic activities, being dominated by Firmicutes and Bacteroidetes. Firmicutes was by far the dominant phylum when using protease (65% relative abundance) while Bacteroidetes was prevailing in the reactor fed with carbohydrase-pretreated microalgae biomass (40% relative abundance). This fact indicated that the applied pretreatment and macromolecule solubilization have a strong effect on microbial distribution and therefore in SCFAs bioconversion yields.

Life cycle assessment of volatile fatty acids production from protein- and carbohydrate-rich organic wastes.

Volatile fatty acids (VFAs) are platform molecules with numerous applications. They can be obtained by adjusting the operational conditions of anaerobic digestion to avoid methanogenesis while focusing on fermentative stages. There are gaps in the knowledge of how, from a life-cycle perspective, the fermentative process performs in VFAs production from waste, including environmental consequences of substituting common commodities in the current market. Mass and energy balances of VFAs production from protein-rich microalgal and carbohydrate-rich agro-industrial wastes were used herein as a key source of inventory data for life cycle assessment. Two waste treatment options were considered: (i) VFAs production (anaerobic fermentation) plus anaerobic digestion of the resulting waste after VFAs separation, and (ii) anaerobic digestion of the original waste for bioenergy. Several scenarios were formulated to evaluate their life-cycle performance. VFAs production generally shows a better environmental behaviour than conventional anaerobic digestion, principally due to the substitution of conventional chemicals.

Assessment of different Bacillus coagulans strains for l-lactic acid production from defined media and gardening hydrolysates: Effect of lignocellulosic inhibitors.

Cellulose valorisation has been successfully addressed for years. However, the use of hemicellulosic hydrolysates is limited due to the presence of C5-sugars and inhibitors formed during pretreatment. Bacillus coagulans is one of the few bacteria able to utilize both C6- and C5-sugars to produce l-lactic acid, but its susceptibility to the lignocellulosic inhibitors needs further investigation. For such a purpose, the tolerance of different B. coagulans strains to increasing concentrations of inhibitors is studied. The isolated A162 strain reached the highest l-lactic acid productivity in all cases (up to 2.4 g L-1  h-1), even in presence of 5 g L-1 of furans and phenols. Remarkably, most of furans and phenolic aldehydes were removed from defined media and hemicellulosic gardening hydrolysate after fermentation with A162. Considering the high productivities and the biodetoxifying effect attained, A162 could be pointed out as a great candidate for valorisation of mixed sugars from hemicellulosic hydrolysates with high inhibitors concentration, promoting the implementation of lignocellulosic biorefineries.

Efficient utilization of hydrolysates from steam-exploded gardening residues for lactic acid production by optimization of enzyme addition and pH control.

The expansion of urban green areas has boosted the accumulation of gardening lignocellulosic residues that could be potentially used to produce platform chemicals like lactic acid. However, when using lignocelluloses, pretreatment step, such as steam explosion, is often needed to favour sugar release. Considering that the conversion of glucose from cellulose has been widely addressed, this work is focused on the valorisation of the steam-exploded gardening liquid fraction rich in hemicellulosic sugars. Since oligomeric sugars are usually solubilized during steam explosion, an enzymatic hydrolysis step was required in some cases to increase the monosaccharides content. Although the presence of inhibitors released during pretreatment (e.g. formic acid) hindered hydrolysis yields, the addition of hemicellulases and the enzyme dosage optimization resulted in 85%, 89% and 95% of glucose, xylose and arabinose release from soluble oligomers, respectively. Lactobacillus pentosus CECT4023T was used for lactic acid fermentation of C6 and C5 sugars from the hydrolysate with the highest sugars concentration, that did not require enzymatic hydrolysis. Xylose consumption was hampered due to the inhibitory effect of acids that produced pH drop. Different pH control systems were applied and automatic NaOH addition in bioreactor resulted in 21 g L-1 of lactic acid (95% of the maximum theoretical yield) that implied 44% increase in lactic acid production when compared with flask fermentation. These results provide new insights for the valorisation of emerging lignocellulosic materials like gardening residues into high added-value products.

VOLATILE FATTY ACIDS FROM ORGANIC WASTES AS NOVEL LOW-COST CARBON SOURCE FOR Yarrowia lipolytica.

Microbial oil biosynthesis is envisaged as a promising technology for sustainable production of chemicals and fuels. Sugar-based substrates are the most typical carbon sources used for this purpose where metabolic pathways and stoichiometry are well known. However, the use of low-cost substrates is crucial for the economic viability of the process. Volatile fatty acids (VFAs) are considered to be a novel low-cost carbon source for microbial lipid production. They can be utilized by oleaginous yeasts to produce and store fatty acids in form of intracellular lipid bodies. In this work, Yarrowia lipolytica growth and substrate consumption were evaluated using the major VFAs present in anaerobic effluents. Individual VFAs as well as synthetic mixtures were tested at different concentrations to determine uptake rates and potential toxicity. Increasing VFA chain length resulted in greater biomass yield although, when added individually, 4 g Carbon/L VFA (e.g. 6.45 g/L of caproic and 10 g/L of acetic acid) caused inhibitory effects. Remarkably, biomass growth increased by 2.5-fold on real anaerobic fermentation effluent compared with synthetic mixtures. When real digestate was supplemented with synthetic VFAs up to 26.5 g/L, the inhibitory effect of the acids was counterbalanced. The results provided evidence of robustness of Y. lipolytica towards low-cost fermentation effluents and present this yeast as a promising candidate for the sustainable production of microbial oil using real digestates.

Volatile fatty acids as novel building blocks for oil-based chemistry via oleaginous yeast fermentation.

Microbial oils are proposed as a suitable alternative to petroleum-based chemistry in terms of environmental preservation. These oils have traditionally been studied using sugar-based feedstock, which implies high costs, substrate limitation, and high contamination risks. In this sense, low-cost carbon sources such as volatile fatty acids (VFAs) are envisaged as promising building blocks for lipid biosynthesis to produce oil-based bioproducts. VFAs can be generated from a wide variety of organic wastes through anaerobic digestion and further converted into lipids by oleaginous yeasts (OYs) in a fermentation process. These microorganisms can accumulate in the form of lipid bodies, lipids of up to 60% wt/wt of their biomass. In this context, OY is a promising biotechnological tool for biofuel and bioproduct generation using low-cost VFA media as substrates. This review covers recent advances in microbial oil production from VFAs. Production of VFAs via anaerobic digestion processes and the involved metabolic pathways are reviewed. The main challenges as well as recent approaches for lipid overproduction are also discussed.

Agroindustrial waste as a resource for volatile fatty acids production via anaerobic fermentation.

This study evaluated the feasibility of the anaerobic digestion as a sustainable valorisation strategy for volatile fatty acids production from agroindustrial waste (cucumber, tomato and lettuce). High bioconversion efficiencies were reached by operating the reactors at 25 °C, 3 g VS·d-1·L-1 with pH adjustment. Cucumber fermentation achieved the highest bioconversion (52.6%), whereas tomato degradation was the least efficient bioprocess (40.1%) due to the low pH (5.6) that partially inhibited the hydrolytic and acidogenic activities. In all cases, carboxylic acid profiles were mainly composed of volatile fatty acids with even carbon number. The developed microbial community exhibited high hydrolytic and acidogenic activities associated to carbohydrates degradation. This microbial population was dominated by Firmicutes phylum and showed a lack of acetogenic bacteria related with CH4 production, resulting in a remarkably high VFAs accumulation.

Volatile Fatty Acids Production from Microalgae Biomass: Anaerobic Digester Performance and Population Dynamics during Stable Conditions, Starvation, and Process Recovery.

Disturbances in anaerobic digestion (AD) negatively impact the overall reactor performance. These adverse effects have been widely investigated for methane generation. However, AD recently appeared as a potential technology to obtain volatile fatty acids (VFAs) and thus, the impact of process disturbances must be evaluated. In this sense, microbial response towards a starvation period of two weeks was investigated resulting in a conversion of organic matter into VFAs of 0.39 ± 0.03 COD-VFAs/CODin. However, the lack of feeding reduced the yield to 0.30 ± 0.02 COD-VFAs/CODin. Microbial analysis revealed that the starvation period favored the syntrophic acetate-oxidizing bacteria coupled with hydrogenotrophic methanogens. Finally, the system was fed at 9 g COD/Ld resulting in process recovery (0.39 ± 0.04 COD-VFAs/CODin). The different microbiome obtained at the end of the process was proved to be functionally redundant, highlighting the AD robustness for VFAs production.