Scale-up and fed-batch cultivation strategy for the enhanced co-production of microbial lipids and carotenoids using renewable waste feedstock.

Agro-industrial by-product valorization as a feedstock for the bioproduction of high-value products has demonstrated a feasible alternative to handle the environmental impact of waste. Oleaginous yeasts are promising cell factories for the industrial production of lipids and carotenoids. Since oleaginous yeasts are aerobic microorganisms, studying the volumetric mass transfer (kLa) could facilitate the scale-up and operation of bioreactors to grant the industrial availability of biocompounds. Scale-up experiments were performed to assess the simultaneous production of lipids and carotenoids using the yeast Sporobolomyces roseus CFGU-S005 and comparing the yields in batch and fed-batch mode cultivation using agro-waste hydrolysate in a 7 L bench-top bioreactor. The results indicate that oxygen availability in the fermentation affected the simultaneous production of metabolites. The highest production of lipids (3.4 g/L) was attained using the kLa value of 22.44 h-1, while higher carotenoid accumulation of 2.58 mg/L resulted when agitation speed was increased to 350 rpm (kLa 32.16 h-1). The adapted fed-batch mode in the fermentation increased the production yields two times. The fatty acid profile was affected according to supplied aeration and after the fed-batch cultivation mode. This study showed the scale-up potential of the bioprocess using the strain S. roseus in the obtention of microbial oil and carotenoids by the valorization of agro-industrial byproducts as a carbon source.

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.

Extraction of Phenolic Compounds from Palm Oil Processing Residues and Their Application as Antioxidants.

The side streams derived from the palm oil production process, namely palm kernel cake, palm pressed fibre, palm kernel shells and empty fruit bunches, were evaluated as sources of phenolic compounds. Among these streams, kernel cake had the highest total phenolic content (in mg of gallic acid equivalents (GAE) per g of dry sample) with a value of 5.19, whereas the empty fruit bunches had the lowest value (1.79). The extraction time and liquid-to-solid ratio were investigated to optimize the phenolic extraction. Kernel cake exhibited the highest total phenolic content (5.35 mg/g) with a liquid-to-solid ratio of 40:1 during 20 min of extraction. The main phenolic compounds of the extracts deriving from all byproduct streams were also identified and quantified with HPLC-DAD. Pyrogallol, 4-hydroxybenzoic acid, gallic acid and ferulic acid were the main compounds found in kernel cake extracts. Empty fruit bunch and pressed fibre extracts were also rich in 4-hydroxybenzoic acid, while pyrogallol was the predominant compound in kernel shell extracts. All extracts showed antioxidant activity as it was indicated from the results of DPPH analysis and subsequently tested in sunflower oil aiming to prolong its shelf life. The addition of 0.8% kernel cake extract increased the induction time of sunflower oil more than 50%. According to the results obtained in this study, kernel cake extracts could be considered as a value-added co-product with a potential application as antioxidants in the food industry.

Valorization of industrial waste and by-product streams via fermentation for the production of chemicals and biopolymers.

The transition from a fossil fuel-based economy to a bio-based economy necessitates the exploitation of synergies, scientific innovations and breakthroughs, and step changes in the infrastructure of chemical industry. Sustainable production of chemicals and biopolymers should be dependent entirely on renewable carbon. White biotechnology could provide the necessary tools for the evolution of microbial bioconversion into a key unit operation in future biorefineries. Waste and by-product streams from existing industrial sectors (e.g., food industry, pulp and paper industry, biodiesel and bioethanol production) could be used as renewable resources for both biorefinery development and production of nutrient-complete fermentation feedstocks. This review focuses on the potential of utilizing waste and by-product streams from current industrial activities for the production of chemicals and biopolymers via microbial bioconversion. The first part of this review presents the current status and prospects on fermentative production of important platform chemicals (i.e., selected C2-C6 metabolic products and single cell oil) and biopolymers (i.e., polyhydroxyalkanoates and bacterial cellulose). In the second part, the qualitative and quantitative characteristics of waste and by-product streams from existing industrial sectors are presented. In the third part, the techno-economic aspects of bioconversion processes are critically reviewed. Four case studies showing the potential of case-specific waste and by-product streams for the production of succinic acid and polyhydroxyalkanoates are presented. It is evident that fermentative production of chemicals and biopolymers via refining of waste and by-product streams is a highly important research area with significant prospects for industrial applications.

Sustainable Production of Novel Oleogels Valorizing Microbial Oil Rich in Carotenoids Derived from Spent Coffee Grounds.

Sustainable food systems that employ renewable resources without competition with the food chain are drivers for the bioeconomy era. This study reports the valorization of microwave-pretreated spent coffee grounds (SCGs) to produce oleogels rich in bioactive compounds. Microbial oil rich in carotenoids (MOC) was produced under batch fermentation of Rhodosporidium toruloides using SCG enzymatic hydrolysates. Candelilla wax (CLW) could structure MOC and sunflower oil at a 3.3-fold lower concentration than that of carnauba wax (CBW). MOC-based oleogels with 10% CBW and 3% CLW showed an elastic-dominant and gel-like structure (tan δ ≪ 1), providing gelation and oil binding capacity (>95%). Dendritic structures of CBW-based oleogels and evenly distributed rod-like crystals of CLW-based ones were observed via polarized light microscopy. MOC-based oleogels exhibited similar Fourier-transform infrared spectroscopy spectra. X-ray diffractograms of oleogels were distinguished by the oil type that presented ß'-type polymorphism. MOC-based oleogels could be applied in confectionary products and spreads as substitutes for trans fatty acids, reformulating fat-containing food products.

Techno-economic evaluation and life-cycle assessment of poly(3-hydroxybutyrate) production within a biorefinery concept using sunflower-based biodiesel industry by-products.

This study presents techno-economic evaluation of a biorefinery concept using biodiesel industry by-products (sunflower meal and crude glycerol) to produce poly(3-hydroxybutyrate) (PHB), crude phenolic extracts (CPE) and protein isolate (PI). The PHB production cost at two annual production capacities ($12.5/kg for 2,500 t PHB/year and $7.8/kg for 25,000 t PHB/year) was not cost-competitive to current PHB production processes when the revenues derived from co-products were not considered. Sensitivity analysis projected the economic viability of a biorefinery concept that could achieve a minimum selling price of $1.1/kg PHB similar to polypropylene. The annual PHB production capacity and the identification of marketable end-uses with respective market prices for the co-products CPE and PI were crucial in attaining process profitability. Greenhouse gas emissions (ca. 0.64 kg CO2-eq/kg PHB) and abiotic depletion potential (61.7 MJ/kg PHB) were lower than polypropylene. Biorefining of sunflower meal and crude glycerol could lead to sustainable PHB production.

Olive Oil Oleogel Formulation Using Wax Esters Derived from Soybean Fatty Acid Distillate.

Oleogelation is an emerging technology to structure oils, which can be widely used to substitute saturated and trans fats. Extra virgin olive oil is widely recognized for its high nutritional value, but its utilization in oleogel production is currently limited. In this study, extra virgin olive oil was utilized for the production of a novel oleogel using wax esters derived from soybean fatty acid distillate (SFAD), a byproduct of industrial soybean oil refining. Different concentrations (7%, 10%, 20%, w/w) of SFAD-wax esters were used to evaluate the minimum concentration requirement to achieve oleogelation. Analyses of the mechanical properties of oleogel showed a firmness of 3.8 N, which was then reduced to around 2.1-2.5 N during a storage period of 30 days at 4 °C. Rheological analysis demonstrated that G' is higher than G″ at 20-27 °C, which confirms the solid properties of the oleogel at this temperature range. Results showed that SFAD was successfully utilized for the oleogelation of olive oil, resulting in a novel oleogel with desirable properties for food applications. This study showed that industrial fatty side streams could be reused for the production of value-added oleogels with novel food applications.

Enrichment of fermentation media and optimization of expression conditions for the production of EAK(16) peptide as fusions with SUMO.

EAK(16) (AEAEAKAKAEAKAEAK) belongs to a novel class of self-assembling peptides, which is being investigated in research and industry. SUMO belongs to the ubiquitin class of proteins and is a promising fusion partner currently in use. In this study, EAK(16) peptide fusions with hexa-histidine tagged SUMO have been constructed using Escherichia coli based pET expression vector. Intracellular expression of the SUMO-EAK(16) fusion using LB media has been optimized. Low-cost complex media (fungal autolysates, wheat and gluten hydrolysates) produced via a novel wheat-based biorefinery have been used as alternative fermentation media to LB. Shake flask cultures using either enriched LB or complex wheat-derived media containing 2 g/L of glucose resulted in intracellular SUMO-EAK(16) fusion protein production of approximately 250 mg/L fermentation volume which corresponded to 30-35% of the total bacterial protein expressed being the fusion protein. Fusion protein productivities up to five times higher were achieved when using a bioreactor.

Bioprocess development for the production of novel oleogels from soybean and microbial oils.

This study presents the production of novel oleogels via circular valorisation of food industry side streams. Sugarcane molasses and soybean processing side streams (i.e. soybean cake) were employed as fermentation feedstocks for the production of microbial oil. Fed-batch bioreactor fermentations carried out by the oleaginous yeast Rhodosporidium toruloides led to the production of 36.9 g/L total dry weight with an intracellular oil content of 49.8% (w/w) and 89.4 µg/g carotenoids. The carotenoid-rich microbial oil and soybean oil were evaluated as base oils for the production of wax-based oleogels. The wax esters, used as oleogelators, were produced via enzymatic catalysis, using microbial oil or soybean fatty acid distillate as raw materials. All oleogels presented a gel-like behaviour (G' > G″). However, the highest G' was determined for the oleogel produced from soybean oil and microbial oil-wax esters, which indicated a stronger network. Thermal analysis showed that this oleogel had a melting temperature profile up to 35 °C, which is favorable for applications in the confectionery industry. Also, texture analysis demonstrated that soybean oil-microbial oil wax oleogel was stable (1.9-2.2 N) within 30-days storage period. This study showed the potential of novel oleogels production through the development of bioprocesses based on the valorisation of various renewable resources.

A newly isolated Enterobacter sp. strain produces 2,3-butanediol during its cultivation on low-cost carbohydrate-based substrates.

2,3-Butanediol (BDO) is an important platform chemical with a wide range of applications in various industries. In the present study, a newly isolated wild Enterobacter sp. strain (FMCC-208) was evaluated towards its ability to produce BDO on media composed of sugars derived from sucrose refinery plant. Optimum values of temperature and pH as well as substrate inhibition were determined through batch experiments. The ability of the strain to convert various monosaccharides was also investigated. Maximum BDO concentrations of 90.3 and 10 g l-1 of acetoin were obtained during a fed-batch bioreactor experiment with cane molasses and sucrose employed as substrates. A high volumetric productivity was noted in a fed-batch experiment using molasses and sucrose as carbon sources at T = 37°C, in which 73.0 g l-1 of BDO together with 12.4 g l-1 of acetoin was produced where 1.15 g l-1 h-1 of diol/acetoin was produced. In previously pasteurized media, 70.0 g l-1 of BDO and 5.0 g l-1 of acetoin were produced (yield = 0.39 g g-1). Finally, besides BDO production, growth on molasses was accompanied by non-negligible decolorization (25-35%) of the residue. Therefore, the strain is a promising candidate for the conversion of sucrose-based materials into BDO.

Biodegradation and toxicity of emerging contaminants: Isolation of an exopolysaccharide-producing Sphingomonas sp. for ionic liquids bioremediation.

Ionic liquids (ILs) have been characterized as contaminants of emerging concern (CEC) that often resist biodegradation and impose toxicity upon environmental release. Sphingomonas sp. MKIV has been isolated as an extreme microorganism capable for biodegradation of major classes of ILs. Six imidazolium-, pyridinium- and ammonium-based ILs (pyridinium trifluoromethanesulfonate [Py][CF3SO3], 1-(4-pyridyl)pyridinium chloride [1-4PPy][Cl], 1-butyl-3-methylimidazolium bromide [BMIM][Br], 1-butyl-3-methylimidazolium methanesulfonate [BMIM][MeSO4], tetrabutylammonium iodide [n-Bu4N][I] and tetrabutylammonium hexafluorophosphate [n-Bu4N][PF6]) were used for microbial growth. The strain achieved 91% and 87% removal efficiency for cultures supplemented with 100 mg L-1 of [BMIM][MeSO4] and [n-Bu4N][I] respectively. The metabolic activity of MKIV was inhibited following preliminary stages of cultures conducted using [BMIM][MeSO4], [BMIM][Br], [Py][CF3SO3] and [n-Bu4N][PF6], indicating potential accumulation of inhibitory metabolites. Thus, a comprehensive toxicological study of the six ILs on Aliivibrio fischeri, Daphnia magna and Raphidocelis subcapitata was conducted demonstrating that the compounds impose moderate and low toxicity. The end-products from [BMIM][MeSO4] and [n-Bu4N][I] biodegradation were assessed using Aliivibrio fischeri, exhibiting increased environmental impact of the latter following biotreatment. MKIV produced 19.29 g L-1 of biopolymer, comprising mainly glucose and galacturonic acid, from 25 g L-1 of glucose indicating high industrial significance for bioremediation and exopolysaccharide production.

Synthesis and Characterization of Bacterial Cellulose from Citrus-Based Sustainable Resources.

Citrus juices from whole oranges and grapefruits (discarded from open market) and aqueous extracts from citrus processing waste (mainly peels) were used for bacterial cellulose production by Komagataeibacter sucrofermentans DSM 15973. Grapefruit and orange juices yielded higher bacterial cellulose concentration (6.7 and 6.1 g/L, respectively) than lemon, grapefruit, and orange peels aqueous extracts (5.2, 5.0, and 2.9 g/L, respectively). Compared to the cellulosic fraction isolated from depectinated orange peel, bacterial cellulose produced from orange peel aqueous extract presented improved water-holding capacity (26.5 g water/g, 3-fold higher), degree of polymerization (up to 6-fold higher), and crystallinity index (35-86% depending on the method used). The presence of absorption bands at 3240 and 3270 cm-1 in the IR spectrum of bacterial cellulose indicated that the bacterial strain K. sucrofermentans synthesizes both Iα and Iß cellulose types, whereas the signals in the 13C NMR spectrum demonstrated that Iα cellulose is the dominant type.

Fumaric acid production using renewable resources from biodiesel and cane sugar production processes.

The microbial production of fumaric acid by Rhizopus arrhizus NRRL 2582 has been evaluated using soybean cake from biodiesel production processes and very high polarity (VHP) sugar from sugarcane mills. Soybean cake was converted into a nutrient-rich hydrolysate via a two-stage bioprocess involving crude enzyme production via solid state fermentations (SSF) of either Aspergillus oryzae or R. arrhizus cultivated on soybean cake followed by enzymatic hydrolysis of soybean cake. The soybean cake hydrolysate produced using crude enzymes derived via SSF of R. arrhizus was supplemented with VHP sugar and evaluated using different initial free amino nitrogen (FAN) concentrations (100, 200, and 400 mg/L) in fed-batch cultures for fumaric acid production. The highest fumaric acid concentration (27.3 g/L) and yield (0.7 g/g of total consumed sugars) were achieved when the initial FAN concentration was 200 mg/L. The combination of VHP sugar with soybean cake hydrolysate derived from crude enzymes produced by SSF of A. oryzae at 200 mg/L initial FAN concentration led to the production of 40 g/L fumaric acid with a yield of 0.86 g/g of total consumed sugars. The utilization of sugarcane molasses led to low fumaric acid production by R. arrhizus, probably due to the presence of various minerals and phenolic compounds. The promising results achieved through the valorization of VHP sugar and soybean cake suggest that a focused study on molasses pretreatment could lead to enhanced fumaric acid production.

Optimization and cost estimation of novel wheat biorefining for continuous production of fermentation feedstock.

A wheat-based continuous process for the production of a nutrient-complete feedstock for bioethanol production by yeast fermentation has been cost-optimized. This process could substitute for the current wheat dry milling process employed in industry for bioethanol production. Each major wheat component (bran, gluten, starch) is extracted and processed for different end-uses. The separate stages, liquefaction and saccharification, used currently in industry for starch hydrolysis have been integrated into a simplified continuous process by exploiting the complex enzymatic consortium produced by on-site fungal bioconversions. A process producing 120 m3 h-1 nutrient-complete feedstock for bioethanol production containing 250 g L-1 glucose and 0.85 g L-1 free amino nitrogen would result in a production cost of $0.126/kg glucose.

Process design and optimization of novel wheat-based continuous bioethanol production system.

A novel design of a wheat-based biorefinery for bioethanol production, including wheat milling, gluten extraction as byproduct, fungal submerged fermentation for enzyme production, starch hydrolysis, fungal biomass autolysis for nutrient regeneration, yeast fermentation with recycling integrated with a pervaporation membrane for ethanol concentration, and fuel-grade ethanol purification by pressure swing distillation (PSD), was optimized in continuous mode using the equation-based software General Algebraic Modelling System (GAMS). The novel wheat biorefining strategy could result in a production cost within the range of dollars 0.96-0.50 gal(-1) ethanol (dollars 0.25-0.13 L(-1) ethanol) when the production capacity of the plant is within the range of 10-33.5 million gal y(-1) (37.85-126.8 million L y(-1)). The production of value-added byproducts (e.g., bran-rich pearlings, gluten, pure yeast cells) was identified as a crucial factor for improving the economics of fuel ethanol production from wheat. Integration of yeast fermentation with pervaporation membrane could result in the concentration of ethanol in the fermentation outlet stream (up to 40 mol %). The application of a PSD system that consisted of a low-pressure and a high-pressure column and employing heat integration between the high- and low-pressure columns resulted in reduced operating cost (up to 44%) for fuel-grade ethanol production.

Development of an oat-based biorefinery for the production of L(+)-lactic acid by Rhizopus oryzae and various value-added coproducts.

A novel oat-based biorefinery producing L(+)-lactic acid and various value-added coproducts (e.g., beta-glucan, anti-irritant solution) is proposed. Pearling is employed for sequential separation of bran-rich fractions for the extraction of value-added coproducts. Lactic acid production is achieved via fungal fermentation of Rhizopus oryzae on pearled oat flour. Maximum lactic acid concentration (51.7 g/L) and starch conversion yield (0.68 g/g) were achieved when an oat flour concentration of 116.5 g/L was used. Oxygen transfer played a significant role with respect to lactic acid production and starch conversion yield. Rhizopus oryzae produced a range of enzymes (glucoamylase, protease, phosphatase) for the hydrolysis of cereal flour macromolecules. Enzyme production during fungal fermentation has been reported. The proposed biorefining strategy could lead to significant operating cost reduction as compared to current industrial practices for lactic acid production from pure glucose achieved by bacterial fermentations.

Succinic acid production by immobilized cultures using spent sulphite liquor as fermentation medium.

Spent sulphite liquor (SSL) was used as carbon source for the production of succinic acid using immobilized cultures of Actinobacillus succinogenes and Basfia succiniciproducens on two different supports, delignified cellulosic material (DCM) and alginate beads. Fed-batch immobilized cultures with A. succinogenes in alginates resulted in higher sugar to succinic acid conversion yield (0.81g/g) than the respective yield achieved (0.65g/g) when DCM immobilized cultures were used. The final succinic acid concentration and yield achieved in fed-batch with immobilized cultures of B. succiniciproducens in alginates (45g/L and 0.66g/g) were higher than A. succinogenes immobilized cultures (35.4g/L and 0.61g/g) using nano-filtrated SSL as fermentation medium. Immobilized cultures of B. succiniciproducens in alginate beads were reused in four sequential fed-batch fermentations of nano-filtrated SSL leading to the production of 64.7g of succinic acid with a yield range of 0.42-0.67g/g and productivity range of 0.29-0.65g/L/h. The immobilized cultures improved the efficiency of succinic acid production as compared to free cell cultures.

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.

Optimal design of upstream processes in biotransformation technologies.

In this work a mathematical programming model for the optimal design of the bioreaction section of biotechnological processes is presented. Equations for the estimation of the equipment cost derived from a recent publication by the US National Renewable Energy Laboratory (NREL) are also summarized. The cost-optimal design of process units and the optimal scheduling of their operation can be obtained using the proposed formulation that has been implemented in software available from the journal web page or the corresponding author. The proposed optimization model can be used to quantify the effects of decisions taken at a lab scale on the industrial scale process economics. It is of paramount important to note that this can be achieved at the early stage of the development of a biotechnological project. Two case studies are presented that demonstrate the usefulness and potential of the proposed methodology.

Magnetically modified bacterial cellulose: A promising carrier for immobilization of affinity ligands, enzymes, and cells.

Bacterial cellulose (BC) produced by Komagataeibacter sucrofermentans was magnetically modified using perchloric acid stabilized magnetic fluid. Magnetic bacterial cellulose (MBC) was used as a carrier for the immobilization of affinity ligands, enzymes and cells. MBC with immobilized reactive copper phthalocyanine dye was an efficient adsorbent for crystal violet removal; the maximum adsorption capacity was 388mg/g. Kinetic and thermodynamic parameters were also determined. Model biocatalysts, namely bovine pancreas trypsin and Saccharomyces cerevisiae cells were immobilized on MBC using several strategies including adsorption with subsequent cross-linking with glutaraldehyde and covalent binding on previously activated MBC using sodium periodate or 1,4-butanediol diglycidyl ether. Immobilized yeast cells retained approximately 90% of their initial activity after 6 repeated cycles of sucrose solution hydrolysis. Trypsin covalently bound after MBC periodate activation was very stable during operational stability testing; it could be repeatedly used for ten cycles of low molecular weight substrate hydrolysis without loss of its initial activity.