Single-Cell Protein and Ethanol Production of a Newly Isolated Kluyveromyces marxianus Strain through Cheese Whey Valorization.

The present work examined the production of single-cell protein (SCP) by a newly isolated strain of Kluyveromyces marxianus EXF-5288 under increased lactose concentration of deproteinized cheese whey (DCW) and different temperatures (in °C: 20.0, 25.0, 30.0 and 35.0). To the best of the authors' knowledge, this is the first report examining the ability of Kluyveromyces marxianus species to produce SCP at T = 20.0 °C. Different culture temperatures led to significant differences in the strain's growth, while maximum biomass and SCP production (14.24 ± 0.70 and 6.14 ± 0.66 g/L, respectively) were observed in the cultivation of K. marxianus strain EXF-5288 in shake-flask cultures at T = 20.0 °C. Increased DCW lactose concentrations (35.0-100.0 g/L) led to increased ethanol production (Ethmax = 35.5 ± 0.2 g/L), suggesting that K. marxianus strain EXF-5288 is "Crabtree-positive". Batch-bioreactor trials shifted the strain's metabolism to alcoholic fermentation, favoring ethanol production. Surprisingly, K. marxianus strain EXF-5288 was able to catabolize the produced ethanol under limited carbon presence in the medium. The dominant amino acids in SCP were glutamate (15.5 mg/g), aspartic acid (12.0 mg/g) and valine (9.5 mg/g), representing a balanced nutritional profile.

A sustainable bioprocess to produce bacterial cellulose (BC) using waste streams from wine distilleries and the biodiesel industry: evaluation of BC for adsorption of phenolic compounds, dyes and metals.

BACKGROUND: The main challenge for large-scale production of bacterial cellulose (BC) includes high production costs interlinked with raw materials, and low production rates. The valorization of renewable nutrient sources could improve the economic effectiveness of BC fermentation while their direct bioconversion into sustainable biopolymers addresses environmental pollution and/or resource depletion challenges. Herein a green bioprocess was developed to produce BC in high amounts with the rather unexplored bacterial strain Komagataeibacter rhaeticus, using waste streams such as wine distillery effluents (WDE) and biodiesel-derived glycerol. Also, BC was evaluated as a bio-adsorbent for phenolics, dyes and metals removal to enlarge its market diversification. RESULTS: BC production was significantly affected by the WDE mixing ratio (0-100%), glycerol concentration (20-45 g/L), type of glycerol and media-sterilization method. A maximum BC concentration of 9.0 g/L, with a productivity of 0.90 g/L/day and a water holding capacity of 60.1 g water/g dry BC, was achieved at 100% WDE and ≈30 g/L crude glycerol. BC samples showed typical cellulose vibration bands and average fiber diameters between 37.2 and 89.6 nm. The BC capacity to dephenolize WDE and adsorb phenolics during fermentation reached respectively, up to 50.7% and 26.96 mg gallic acid equivalents/g dry BC (in-situ process). The produced BC was also investigated for dye and metal removal. The highest removal of dye acid yellow 17 (54.3%) was recorded when 5% of BC was applied as the bio-adsorbent. Experiments performed in a multi-metal synthetic wastewater showed that BC could remove up to 96% of Zn and 97% of Cd. CONCLUSIONS: This work demonstrated a low-carbon approach to produce low-cost, green and biodegradable BC-based bio-adsorbents, without any chemical modification. Their potential in wastewater-treatment-applications was highlighted, promoting closed-loop systems within the circular economy era. This study may serve as an orientation for future research towards competitive or targeted adsorption technologies for wastewater treatment or resources recovery.

Growth Response of Non-Conventional Yeasts on Sugar-Rich Media: Part 2: Citric Acid Production and Circular-Oriented Valorization of Glucose-Enriched Olive Mill Wastewaters Using Novel Yarrowia lipolytica Strains.

The global market for citric acid (CA) is one of the biggest and fastest expanding markets in the food industry. The CA production employing microbial bioprocessing with efficient GRAS strains and renewable waste streams is in line with the European Union binding targets for resource efficiency, sustainable consumption-production, and low-carbon technologies. In this work, the potential of three novel wild-type Yarrowia lipolytica strains (namely LMBF Y-46, LMBF Y-47 and ACA-YC 5033) regarding the production of CA and other valuable metabolites was tested on glucose-based media, and the most promising amongst the screened strains (viz. the strain ACA-YC 5033) was cultured on glucose-based media, in which part of the fermentation water had been replaced by olive-mill wastewaters (OMWs) in a novel approach of simultaneous OMW valorization and bioremediation. In the first part of this study, the mentioned strains were cultured under nitrogen-limited conditions with commercial (low-cost) glucose employed as a sole carbon source in shake-flask cultures at an initial concentration (S0) ≈ of 50 g/L. Variable quantities of secreted citric acid (CA) and intra-cellular compounds (viz. polysaccharides and lipids) were produced. All strains did not accumulate significantly high lipid quantities (i.e., maximum lipid in dry cell weight [DCW] values ≈30% w/w were noted) but produced variable CA quantities. The most promising strain, namely ACA-YC 5033, produced CA up to c. 24 g/L, with a yield of CA produced on glucose consumed (YCA/S) ≈ 0.45 g/g. This strain in stirred tank bioreactor experiments, at remarkably higher S0 concentrations (≈110 g/L) and the same initial nitrogen quantity added into the medium, produced notably higher CA quantities, up to 57 g/L (YCA/S ≈ 0.52 g/g). The potential of the same strain (ACA-YC 5033) to bioremediate OMWs and to produce value-added compounds, i.e., yeast cells, CA, and intra-cellular metabolites, was also assessed; under nitrogen-limited conditions in which OMWs had partially replaced tap water and significant glucose concentrations had been added (S0 ≈ 100 g/L, simultaneous molar ratio C/N ≈ 285 g/g, initial phenolic compounds [Phen0] adjusted to ≈1.0 g/L; these media were similar to the OMWs generated from the traditional press extraction systems) the notable CA quantity of 60.2 g/L with simultaneous YCA/S = 0.66 g/g, was obtained in shake flasks, together with satisfactory phenolic compounds removal (up to 19.5% w/w) and waste decolorization (up to 47.0%). Carbon-limited conditions with Phen0 ≈ 1.0 g/L favored the production of yeast DCW (up to 25.3 g/L), with equally simultaneous interesting phenolic compounds and color removal. The fatty acid profile showed that cellular lipids were highly unsaturated with oleic, linoleic and palmitoleic acids, accounting for more than 80% w/w. This study proposed an interesting approach that could efficiently address the biotreatment of toxic effluents and further convert them into circular-oriented bioproducts.

Bioconversion of underutilized brewing by-products into bacterial cellulose by a newly isolated Komagataeibacter rhaeticus strain: A preliminary evaluation of the bioprocess environmental impact.

A novel Komagataeibacter rhaeticus UNIWA AAK2 strain was used to produce bacterial cellulose (BC), valorizing brewers' spent grain (BSG) and brewer's spent yeast (BSY). Under optimal conditions (controlled pH = 6 and 30 g/L sugars), a maximum BC of 4.0 g/L was achieved when BSG aqueous extract (BSGE) was used. The substitution of yeast extract and peptone with BSY autolyzates did not show significant differences on BC concentration and productivity. The FTIR, SEM, and TGA analyses showed that the use of brewing by-products had no effect on the structure and thermal stability of the produced BC, compared to highly-pure and commercial substrates. The LCA of the developed bioprocess revealed that BSGE- and BSY-based media can reduce the carbon footprint of 1 kg dry BC by 76% compared to commercial-based-media. Beer by-products could serve as cost-effective resources to produce value-added and sustainable biopolymers such as BC, while minimizing waste and restructuring the brewing-industry.

Growth Response of Non-Conventional Yeasts on Sugar-Rich Media: Part 1: High Production of Lipid by Lipomyces starkeyi and Citric Acid by Yarrowia lipolytica.

Sugar-rich waste streams, generated in very high quantities worldwide, constitute an important source of environmental pollution. Their eco-friendly conversions into a plethora of added-value compounds through the use of microbial fermentations is currently a very "hot" scientific topic. The aim of this study, was to assess the potential of single cell oil (SCO), microbial mass and citric acid (CA) production by non-conventional yeast strains growing on expired ("waste") glucose. Six yeast strains (viz. Rhodosporidium toruloides DSM 4444, Rhodotorula glutinis NRRL YB-252, R. toruloides NRRL Y-27012, Yarrowia lipolytica LFMB Y-20, Y. lipolytica ACA-DC 50109 and Lipomyces starkeyi DSM 70296) were initially grown in shake flasks with expired glucose used as substrate under nitrogen limitation, in order to "boost" the cellular metabolism towards the synthesis of SCO and CA, and their growth response was quantitatively evaluated. Initial glucose concentration (Glc0) was adjusted at c. 50 g/L. Besides Y. lipolytica, all other yeast strains produced noticeable SCO quantities [lipid in dry cell weight (DCW) ranging from 25.3% w/w to 55.1% w/w]. Lipids of all yeasts contained significant quantities of oleic acid, being perfect candidates for the synthesis of 2nd generation biodiesel. The highest DCW production (=13.6 g/L) was obtained by L. starkeyi DSM 70296, while both Y. lipolytica strains did not accumulate noticeable lipid quantities, but produced non-negligible CA amounts. The most promising CA-producing strain, namely Y. lipolytica ACA-DC 50109 was further studied in stirred-tank bioreactor systems, while the very promising DCW- and SCO-producing L. starkeyi DSM 70296 was further studied in shake flasks. Both strains were grown on media presenting higher Glc0 concentrations and the same initial nitrogen quantity as previously. Indeed, L. starkeyi grown at Glc0 = 85 g/L, produced DCWmax = 34.0 g/L, that contained lipid =34.1% w/w (thus SCO was =11.6 g/L). The strain ACA-DC 50109 in stirred tank bioreactor with Glc0 ≈ 105 g/L produced CA up to 46 g/L (yield of CA produced on glucose consumed; YCA/Glc ≈ 0.45 g/g). Finally, in fed-batch bioreactor experiment, the significant CA quantity of 82.0 g/L (YCA/Glc = 0.50 g/g) was recorded. Concluding, "waste" glucose proved to be a suitable substrate for a number of non-conventional yeast strains. Y. lipolytica ACA-DC 50109 produced significant quantities of CA while L. starkeyi DSM 70296 was a very interesting DCW- and SCO-producing candidate. These strains can be used as potential cell factories amenable to convert glucose-based residues into the mentioned metabolic compounds, that present high importance for food, chemical and biofuel facilities.

Effect of Wheat Replacement by Pulse Flours on the Texture, Color, and Sensorial Characteristics of Crackers: Flash Profile Analysis.

Pulse flours are growing in popularity as alternatives to wheat in bakery products due to their high protein and nutritional value. However, the effect of different pulse species and substitution on sensory perception is unclear. The sensory perception of crackers made by partially replacing wheat with chickpea (40-80%) and lupin flour (10-30%) was evaluated using Flash profile analysis in association with instrumental analysis of texture and color. Flash profile analysis was conducted in Greece and Indonesia in order to allow culture comparison of the profiling of the samples and language by the subjects of the panel. Lightness (L∗) and hardness of crackers were decreased by the addition of pulses. Flash profile analysis indicated an association among color, texture, and sensory perception by judges. Derived attributes were associated with the physicochemical characteristics and raw materials of crackers for both panels. GPA analysis of Greek panel indicated that increasing the replacement of wheat led to the generation of more attributes regardless of pulse species, while the Indonesian panel was able to detect differences among pulse species.

Valorization of low-cost, carbon-rich substrates by edible ascomycetes and basidiomycetes grown on liquid cultures.

Three ascomycetes (Morchella vulgaris AMRL 36, M. elata AMRL 63, Tuber aestivum AMRL 364) and four basidiomycetes strains (Lentinula edodes AMRL 124 and 126, Agaricus bisporus AMRL 208 and 209) were screened for their ability to grow on liquid static flask cultures of glucose, glycerol, molasses and waste flour-rich hydrolysates with C/N ratio of 20 and produce biomass, exopolysaccharides and lipids. The profile of lipid fatty acids was also assessed. Selected strains were furthermore cultivated in C/N = 50. Results showed that substrate consumption, biomass formation and secondary metabolites production were strain, substrate and C/N ratio dependent. The maximum biomass (X), lipid (L) and exopolysaccharides (EPS) values noted were Xmax = 25.2 g/L (C/N = 20; molasses) and Lmax = 6.51 g/L (C/N = 50; rice cereal hydrolysates) by T. aestivum strain AMRL 364 and EPSmax = 2.41 g/L by M. elata strain AMRL 63 (C/N = 50; molasses), respectively. When C/N ratio of 50 was applied, biomass, lipid production and substrate consumption seem to be negatively affected in most of the trials. The adaptation and capability of the mushroom strains to be cultivated on substrates based on agro-industrial waste streams and infant food of expired shelf date offers the opportunity to set a circular oriented bioprocess.

Production of added-value microbial metabolites during growth of yeast strains on media composed of biodiesel-derived crude glycerol and glycerol/xylose blends.

A total of 11 yeast strains of Yarrowia lipolytica, Metschnikowia sp., Rhodotorula sp. and Rhodosporidium toruloides were grown under nitrogen-limited conditions with crude glycerol employed as substrate in shake flasks, presenting interesting dry cell weight (DCW) production. Three of these strains belonging to Metschnikowia sp. accumulated significant quantities of endopolysaccharides (i.e. the strain V.V.-D4 produced 11.0 g/L of endopolysaccharides, with polysaccharides in DCW ≈ 63% w/w). A total of six Y. lipolytica strains produced either citric acid or mannitol. Most of the screened yeasts presented somehow elevated lipid and polysaccharides in DCW values at the early steps of growth despite nitrogen appearance in the fermentation medium. Lipid in DCW values decreased as growth proceeded. R. toruloides DSM 4444 cultivated on media presenting higher glycerol concentrations presented interesting lipid-accumulating capacities (maximum lipid = 12.5 g/L, maximum lipid in DCW = 43.0-46.0% w/w, conversion yield on glycerol = 0.16 g/g). Replacement of crude glycerol by xylose resulted in somehow decreased lipid accumulation. In xylose/glycerol mixtures, xylose was more rapidly assimilated from glycerol. R. toruloides total lipids were mainly composed of triacylglycerols. Total cellular fatty acid composition on xylose presented some differences compared with that on glycerol. Cellular lipids contained mainly oleic and palmitic acid.

Valorization of Crude Glycerol, Residue Deriving from Biodiesel- Production Process, with the Use of Wild-type New Isolated Yarrowia lipolytica Strains: Production of Metabolites with Pharmaceutical and Biotechnological Interest.

BACKGROUND & OBJECTIVE: Crude glycerol (Glol), used as substrate for screening eleven natural Yarrowia lipolytica strains in shake-flask experiments. Aim of this study was to assess the ability of the screened strains to produce biomass (dry cell weight; X), lipid (L), citric acid (Cit), mannitol (Man), arabitol (Ara) and erythritol (Ery), compounds presenting pharmaceutical and biotechnological interest, in glycerol-based nitrogen-limited media, in which initial glycerol concentration had been adjusted to 40 g/L. METHODS: Citric acid may find use in biomedical engineering (i.e. drug delivery, tissue engineering, bioimaging, orthopedics, medical device coating, wound dressings). Polyols are considered as compounds with non-cariogenic and less calorigenic properties as also with low insulin-mediated response. Microbial lipids containing polyunsaturated fatty acids (PUFA) are medically and dietetically important (selective pharmaceutical and anticancer properties, aid fetal brain development, the sight function of the eye, hormonal balance and the cardio-vascular system, prevent reasons leading to type-2 diabetes, present healing and anti-inflammatory effects). RESULTS: All strains presented satisfactory microbial growth (Xmax=5.34-6.26 g/L) and almost complete substrate uptake. The principal metabolic product was citric acid (Citmax=8.5-31.7 g/L). Production of cellular lipid reached the values of 0.33-0.84 g/L. Polyols were also synthesized as strain dependent compounds (Manmax=2.8-6.1 g/L, Aramax ~2.0 g/L, Erymax= 0.5-3.8 g/L). The selected Y. lipolytica strain ACA-DC 5029 presented satisfactory growth along with synthesis of citric acid and polyols, thus, was further grown on media presenting an increased concentration of Glol~75 g/L. Biomass, lipid and citric acid production presented significant enhancement (Xmax=11.80 g/L, Lmax=1.26 g/L, Citmax=30.8 g/L), but conversion yield of citric acid produced per glycerol consumed was decreased compared to screening trials. Erythritol secretion (Erymax=15.6 g/L) was highly favored, suggesting a shift of yeast metabolism from citric acid accumulation towards erythritol production. Maximum endopolysaccharides (IPS) concentration was 4.04 g/L with yield in dry weight 34.2 % w/w. CONCLUSION: Y. lipolytica strain ACA-YC 5029 can be considered as a satisfactory candidate grown in high concentrations of crude glycerol to produce added-value compounds that interest pharmaceutical and biotechnology industries.

Production of Added-Value Chemical Compounds through Bioconversions of Olive-Mill Wastewaters Blended with Crude Glycerol by a Yarrowia lipolytica Strain.

Olive mill wastewaters (OMW) are the major effluent deriving from olive oil production and are considered as one of the most challenging agro-industrial wastes to treat. Crude glycerol is the main by-product of alcoholic beverage and oleochemical production activities including biodiesel production. The tremendous quantities of glycerol produced worldwide represent a serious environmental challenge. The aim of this study was to assess the ability of Yarrowia lipolytica strain ACA-DC 5029 to grow on nitrogen-limited submerged shake-flask cultures, in crude glycerol and OMW blends as well as in media with high initial glycerol concentration and produce biomass, cellular lipids, citric acid and polyols. The rationale of using such blends was the dilution of concentrated glycerol by OMW to (partially or fully) replace process tap water with a wastewater stream. The strain presented satisfactory growth in blends; citric acid production was not affected by OMW addition (Citmax~37.0 g/L, YCit/Glol~0.55 g/g) and microbial oil accumulation raised proportionally to OMW addition (Lmax~2.0 g/L, YL/X~20% w/w). Partial removal of color (~30%) and phenolic compounds (~10% w/w) of the blended media occurred. In media with high glycerol concentration, a shift towards erythritol production was noted (Erymax~66.0 g/L, YEry/Glol~0.39 g/g) simultaneously with high amounts of produced citric acid (Citmax~79.0 g/L, YCit/Glol~0.46 g/g). Fatty acid analysis of microbial lipids demonstrated that OMW addition in blended media and in excess carbon media with high glycerol concentration favored oleic acid production.

Production of added-value metabolites by Yarrowia lipolytica growing in olive mill wastewater-based media under aseptic and non-aseptic conditions.

Yarrowia lipolytica ACA-YC 5033 was grown on glucose-based media in which high amounts of olive mill wastewaters (OMWs) had been added. Besides shake-flask aseptic cultures, trials were also performed in previously pasteurized media while batch bioreactor experiments were also done. Significant decolorization (∼58%) and remarkable removal of phenolic compounds (∼51% w/w) occurred, with the latter being amongst the highest ones reported in the international literature, as far as yeasts were concerned during their growth on phenol-containing media. In nitrogen-limited flask fermentations the microorganism produced maximum citric acid quantity ≈19.0 g/L [simultaneous yield of citric acid produced per unit of glucose consumed (YCit/Glc)≈0.74 g/g]. Dry cell weight (DCW) values decreased at high phenol-containing media, but, on the other hand, the addition of OMWs induced reserve lipid accumulation. Maximum citric acid concentration achieved (≈52.0 g/L; YCit/Glc≈0.64 g/g) occurred in OMW-based high sugar content media (initial glucose added at ≈80.0 g/L). The bioprocess was successfully simulated by a modified logistic growth equation. A satisfactory fitting on the experimental data occurred while the optimized parameter values were found to be similar to those experimentally measured. Finally, a non-aseptic (previously pasteurized) trial was performed and its comparison with the equivalent aseptic experiment revealed no significant differences. Yarrowia lipolytica hence can be considered as a satisfactory candidate for simultaneous OMWs bioremediation and the production of added-value compounds useful for the food industry.