A semi-continuous algal-bacterial wastewater treatment process coupled with bioethanol production.

Harnessing the biomass energy potential through biofuel production offers new outlets for a circular economy. In this study an integrated system which combine brewery wastewater treatment using algal-bacterial aggregates instead of activated sludge was developed. The use of algal-bacterial aggregates can eliminate the aeration requirements and significantly reduce the high biomass harvesting costs associated with algal monocultures. A sequencing batch reactor (SBR) setup operating with and without biomass recirculation was used to investigate pollutant removal rates, aggregation capacity and microbial community characteristics under a range of hydraulic retention times (HRTs) and solid retention times (SRTs). It was observed that biomass recirculation strategy significantly enhanced aggregation and pollutant removal (i.e., 78.7%, 94.2% and 75.2% for d-COD, TKN, and PO43--P, respectively). The microbial community established was highly diverse consisting of 161 Bacterial Operational Taxonomic Units (B-OTUs) and 16 unicellular Eukaryotic OTUs (E-OTUs). Escalation the optimal conditions (i.e., HRT = 4 d, SRT = 10 d) at pilot-scale resulted in nutrient starvation leading to 38-44% w/w carbohydrate accumulation. The harvested biomass was converted to bioethanol after acid hydrolysis followed by fermentation with Saccharomyces cerevisiae achieving a bioethanol production yield of 0.076 g bioethanol/g biomass. These data suggest that bioethanol production coupled with high-performance wastewater treatment using algal-bacterial aggregates is feasible, albeit less productive concerning bioethanol yields than systems exclusively designed for third and fourth-generation biofuel production.

Laboratory evolution strategies for improving lipid accumulation in Yarrowia lipolytica.

Oleaginous microorganisms are of high biotechnological interest being considered as alternative sources of oil (single cell oil-SCO). Current research for increasing productivity of oleaginous microorganisms is focused on the overexpression of genes implicated in lipid synthesis, the inactivation of genes implicated in storage lipid turnover, and on the suppression of competitive to lipid biosynthesis pathways. An alternative strategy, described here, relies on evolution of Yarrowia lipolytica under alternating environments that promote growth, encourage storage lipid synthesis, and reward high energy-containing cells. Derived populations were characterized biochemically, especially on their ability to accumulate lipids, and compared with the starting strain. Interestingly, lipid-accumulating ability early in the evolution was decreased compared with the starting strain. Subsequently, oleaginous lineages dominated, leading to populations able to accumulate lipids in high amounts. A population obtained after 77 generations was able to accumulate 44% w/w of lipid, which was 30% higher than that of the starting strain. We conclude that evolution-based strategies can be utilized as a robust tool for improving lipid accumulation capacity in oleaginous microorganisms.

Sources of microbial oils with emphasis to Mortierella (Umbelopsis) isabellina fungus.

The last years a constantly rising number of publications have appeared in the literature in relation to the production of oils and fats deriving from microbial sources (the "single cell oils"-SCOs). SCOs can be used as precursors for the synthesis of lipid-based biofuels or employed as substitutes of expensive oils rarely found in the plant or animal kingdom. In the present review-article, aspects concerning SCOs (economics, biochemistry, substrates, technology, scale-up), with emphasis on the potential of Mortierella isabellina were presented. Fats and hydrophilic substrates have been used as carbon sources for cultivating Zygomycetes. Among them, wild-type M. isabellina strains have been reported as excellent SCO-producers, with conversion yields on sugar consumed and lipid in DCW values reported comparable to the maximum ones achieved for genetically engineered SCO-producing strains. Lipids produced on glucose contain γ-linolenic acid (GLA), a polyunsaturated fatty acid (PUFA) of high dietary and pharmaceutical importance, though in low concentrations. Nevertheless, due to their abundance in oleic acid, these lipids are perfect precursors for the synthesis of 2nd generation biodiesel, while GLA can be recovered and directed to other usages. Genetic engineering focusing on over-expression of Δ6 and Δ12 desaturases and of C16 elongase may improve the fatty acid composition (viz. increasing the concentration of GLA or other nutritionally important PUFAs) of these lipids.

Critical steps in carbon metabolism affecting lipid accumulation and their regulation in oleaginous microorganisms.

Oleaginous microorganisms are able to convert numerous agro-industrial and municipal wastes into storage lipids (single cell oil (SCO)) and are therefore considered as potential biofuel producers. While from an environmental and technological point of view the idea to convert waste materials into fuels is very attractive, the production cost of SCO is not currently competitive to that of conventional oils due to the low productivity of oleaginous microorganisms in combination with the high fermentation cost. Current strategies used to optimize the lipid-accumulating capacity of oleaginous microorganisms include the overexpression of genes encoding for key enzymes implicated in fatty acid and triacylglycerol synthesis, such as ATP-dependent citrate lyase, acetyl-CoA carboxylase, malic enzyme, proteins of the fatty acid synthase complex, glycerol 3-phosphate dehydrogenase and various acyltransferases, and/or the inactivation of genes encoding for enzymes implicated in storage lipid catabolism, such as lipases and acyl-CoA oxidases. Furthermore, blocking, even partially, pathways competitive to lipid biosynthesis (e.g., those involved in the accumulation of storage polysaccharide or organic acid and polyol excretion) can also increase lipid-accumulating ability in oleaginous microorganisms. Methodologies, such as adaptive laboratory evolution, can be included in existing workflows for the generation of strains with improved lipid accumulation capacity. In our opinion, efforts should be focused in the construction of strains with high carbon uptake rates and a reprogrammed coordination of the individual parts of the oleaginous machinery that maximizes carbon flux towards lipogenesis.

Storage lipid and polysaccharide metabolism in Yarrowia lipolytica and Umbelopsis isabellina.

Complex biochemical mechanisms are being involved in oleaginous microorganisms during storage lipid and polysaccharide metabolism. Detailed biochemical analyses and monitoring of key enzymes involved in carbon metabolism were performed in Yarrowia lipolytica and Umbelopsis isabellina, which are often used as model oleaginous microorganisms. It was found that during the early oleaginous phase, the carbon source (glucose) was channeled to lipid accumulation, but also to polysaccharide biosynthesis. However, during transition from the early to the late oleaginous phase, glucose was exclusively converted to lipids, while in U. isabellina, but not in Y. lipolytica, an additional conversion of cellular polysaccharides into lipids was observed. After glucose depletion in the growth medium, cellular storage material was degraded either for generating maintenance energy or for supporting further microbial growth, depending on the availability of essential nutrients in the growth medium. We demonstrated that in both microorganisms, reserve lipids were exclusively used as an intra-cellular carbon source in order to generate energy for maintenance purpose. When cellular storage material degradation was related to new cell mass production, a bioconversion of lipids into new lipid-free material, consisting of polysaccharides and proteins, was observed in Y. lipolytica, while new lipid-free material in U. isabellina was richer in proteins. Lipid and polysaccharide remodeling may occur in some cases in both microorganisms. This study revealed some new biochemical features of oleaginous microorganisms that may be crucial for the design of new biotechnological processes, such as the production of bio-molecules of industrial, technological, and medical interest.

Fatty acid biosynthesis during the life cycle of Debaryomyces etchellsii.

Fatty acid biosynthesis during the life cycle of the ascomycetous yeast Debaryomyces etchellsii cultivated on a non-fermentable substrate, i.e. glycerol, in nitrogen rich media (NRM) and nitrogen limited media (NLM) has been studied. Although considerable activities of key lipogenic enzymes, such as ATP citrate lyase (ACL) and malic enzyme (ME), were detected in vegetative cells during asexual proliferation (which occurred in the first growth stages in both NRM and NLM), lipid accumulation was restricted due to the high activities of NAD+-isocitrate dehydrogenase (NAD+-ICDH). A similar enzymatic profile has been found in ascii and free ascospores produced in NRM; thus lipid accumulation was low. On the contrary, very high activities of both ACL and ME and low activities of NAD+-ICDH were detected in ascii and free ascospores produced in NLM resulting in lipid accumulation. Neutral lipids (NL) were the predominant fraction of cellular lipids produced in vegetative cells and ascospores in both NRM and NLM. On the other hand, phospholipids (P) were the major polar lipids while glycolipids (G) were synthesized in low proportions. During transition from asexual to sexual phase, the percentage of NL increased with a significant decrease of P and, to a lesser extent, of G. High quantities of linoleic acid were found esterified in polar lipids, especially in P, during the vegetative stage of growth, while, with a few exceptions, during transition from asexual to sexual stage, linoleic acid concentration decreased markedly, mainly in P, while oleic acid concentration increased.

Feasibility of raw glycerol conversion into single cell oil by zygomycetes under non-aseptic conditions.

The use of plant oils as feedstock for the biodiesel manufacture has many drawbacks, thus, the interest has turned to single cell oil (SCO) as an alternative. However, the production of SCO is still too expensive, mainly due to the low oil productivity and the high cost of medium sterilization required. In this work raw glycerol was converted into SCO by oleaginous Zygomycetes under non-aseptic conditions on selective (i.e., containing essential oils and/or antibiotics) nitrogen limited media. The obtained data showed that although bacterial populations inhibited the fungal growth, lipid accumulation remained unaffected by the presence of bacteria in the growth medium compared to control experiments (conducted under aseptic conditions). Therefore, a two-stage process was developed in which growth was performed under aseptic conditions (1st stage) followed by lipid accumulation performed under non-aseptic conditions (2nd stage) in the presence of thyme essential oil as an antibacterial agent. Large amounts of lipids were accumulated inside the mycelia, yielding around 13% wt/wt of oil per glycerol consumed.

Morphological and metabolic shifts of Yarrowia lipolytica induced by alteration of the dissolved oxygen concentration in the growth environment.

Yarrowia lipolytica, an ascomycete with biotechnological potential, is able to form either yeast cells or hyphae and pseudohyphae in response to environmental conditions. This study shows that the morphology of Y. lipolytica, cultivated in batch cultures on hydrophilic (glucose and glycerol) and hydrophobic (olive oil) media, was not affected by the nature of the carbon source, nor by the nature or the concentration of the nitrogen source. By contrast, dissolved oxygen concentration (DOC) should be considered as the major factor affecting yeast morphology. Specifically, when growth occurred at low or zero DOC the mycelial and/or pseudomycelial forms predominated over the yeast form independently of the carbon and nitrogen sources used. Experimental data obtained from a continuous culture of Y. lipolytica on glycerol, being used as carbon and energy source, demonstrated that the mycelium-to-yeast form transition occurs when DOC increases from 0.1 to 1.5 mg l(-1). DOC also affected the yeast physiology, as the activity of enzymes implicated in lipid biosynthesis (i.e. ATP-citrate lyase, malic enzyme) was upregulated at high DOC whereas the activity of enzymes implicated in glycerol assimilation (such as glycerol dehydrogenase and kinase) remained fundamentally unaffected in the cell-free extract.

Patterns of major metabolites biosynthesis by different mushroom fungi grown on glucose-based submerged cultures.

The biosynthetic potential of four basidiomycetes (Agrocybe aegerita, Flammulina velutipes, Ganoderma applanatum and Pleurotus pulmonarius) and one ascomycete (Morchella esculenta) was examined in regard to biomass, intracellular (endopolysaccharides and lipids) and extracellular (exopolysaccharides) compounds' production in liquid media with glucose as substrate, in static and agitated cultures. Exopolysaccharides' production presented significant negative correlation with biomass, endopolysaccharides and lipids, while biomass was positively related to the production of endopolysaccharides and lipids. Maximum values of biomass, endo- and exo-polysaccharides obtained were quite impressive: P. pulmonarius produced 22.5 g/L of biomass, A. aegerita 60.4 % (w/w) of endopolysaccharides and F. velutipes 1.2 g/L of exopolysaccharides. Polysaccharides and lipids synthesized at the early growth stages were subjected to degradation as the fermentation proceeded. Mycelial lipids of all strains were highly unsaturated, dominated by linoleic acid, whereas glucose was the main building block of endopolysaccharides. The ability of the examined mushroom fungi to synthesize in high quantities biomass and polysaccharides, products with biotechnological and medicinal interest, renders these fungi as potential candidates in sugar-based bio-refineries.

Two cases of typical HUS in adults treated with an anti-C5 monoclonal antibody: a new perspective?

Hemolytic uremic syndrome (HUS) is a rare clinical entity, especially in adults. In its typical form the causative factor that triggers the cascade of immunologic and inflammatory events is a Shiga toxin-producing pathogen, found in the patient's stool. Renal and neurologic involvement usually prevails and requires immediate care. Regarding this potentially life-threatening condition, little is known and the mainstay is supportive care. However, some interesting evidence has come up about the use of eculizumab, an anti-C5 monoclonal antibody, mainly in pediatric patients with typical HUS. Herein, we present two cases with typical HUS caused by two different strains of Escherichia coli (Shiga toxin-producing enterohemorrhagic and enteropathogenic) who were both treated effectively with anti-C5 monoclonal antibodies (eculizumab and ravulizumab).

Poly-Unsaturated Fatty Acids (PUFAs) from Cunninghamella elegans Grown on Glycerol Induce Cell Death and Increase Intracellular Reactive Oxygen Species.

Cunninghamella elegans NRRL-1393 is an oleaginous fungus able to synthesize and accumulate unsaturated fatty acids, amongst which the bioactive gamma-linolenic acid (GLA) has potential anti-cancer activities. C. elegans was cultured in shake-flask nitrogen-limited media with either glycerol or glucose (both at ≈60 g/L) employed as the sole substrate. The assimilation rate of both substrates was similar, as the total biomass production reached 13.0-13.5 g/L, c. 350 h after inoculation (for both instances, c. 27-29 g/L of substrate were consumed). Lipid production was slightly higher on glycerol-based media, compared to the growth on glucose (≈8.4 g/L vs. ≈7.0 g/L). Lipids from C. elegans grown on glycerol, containing c. 9.5% w/w of GLA, were transformed into fatty acid lithium salts (FALS), and their effects were assessed on both human normal and cancerous cell lines. The FALS exhibited cytotoxic effects within a 48 h interval with an IC50 of about 60 µg/mL. Additionally, a suppression of migration was shown, as a significant elevation of oxidative stress levels, and the induction of cell death. Elementary differences between normal and cancer cells were not shown, indicating a generic mode of action; however, oxidative stress level augmentation may increase susceptibility to anticancer drugs, improving chemotherapy effectiveness.

Laboratory- and Pilot-Scale Cultivation of Tetraselmis striata to Produce Valuable Metabolic Compounds.

Marine microalgae are considered an important feedstock of multiple valuable metabolic compounds of high biotechnological potential. In this work, the marine microalga Tetraselmis striata was cultivated in different scaled photobioreactors (PBRs). Initially, experiments were performed using two different growth substrates (a modified F/2 and the commercial fertilizer Nutri-Leaf (30% TN-10% P-10% K)) to identify the most efficient and low-cost growth medium. These experiments took place in 4 L glass aquariums at the laboratory scale and in a 9 L vertical tubular pilot column. Enhanced biomass productivities (up to 83.2 mg L-1 d-1) and improved biomass composition (up to 41.8% d.w. proteins, 18.7% d.w. carbohydrates, 25.7% d.w. lipids and 4.2% d.w. total chlorophylls) were found when the fertilizer was used. Pilot-scale experiments were then performed using Nutri-Leaf as a growth medium in different PBRs: (a) a paddle wheel, open, raceway pond of 40 L, and (b) a disposable polyethylene (plastic) bag of 280 L working volume. Biomass growth and composition were also monitored at the pilot scale, showing that high-quality biomass can be produced, with important lipids (up to 27.6% d.w.), protein (up to 45.3% d.w.), carbohydrate (up to 15.5% d.w.) and pigment contents (up to 4.2% d.w. total chlorophylls), and high percentages of eicosapentaenoic acid (EPA). The research revealed that the strain successfully escalated in larger volumes and the biochemical composition of its biomass presents high commercial interest and could potentially be used as a feed ingredient.

Adaptation dynamics of Clostridium butyricum in high 1,3-propanediol content media.

Aim of the present study was to evaluate the effect of exogenous additions of 1,3-propanediol (1,3-PDO) on microbial growth and metabolites production of Clostridium butyricum VPI 1718 strain, during crude glycerol fermentation. Preliminary batch cultures in anaerobic Duran bottles revealed that early addition of 1,3-PDO caused growth cessation in rather low quantities (15 g/L), while 1,3-PDO additions during the middle exponential growth phase up to 70 g/L resulted in an almost linear decrease of the specific growth rate (µ), accompanied by reduced glycerol assimilation, with substrate consumption being used mainly for energy of maintenance requirements. During batch trials in a 3-L bioreactor, the strain proved able to withstand more than 70 g/L of both biologically produced and externally added 1,3-PDO, whereas glycerol assimilation and metabolite production were carried on at a lower rate. Adaptation of the strain in high 1,3-PDO concentration environments was validated during its continuous cultivation with pulses of 1,3-PDO in concentrations of 31 and 46 g/L, where no washout phenomena were noticed. As far as C. butyricum cellular lipids were concerned, during batch bioreactor cultivations, 1,3-PDO addition was found to favor the biosynthesis of unsaturated fatty acids. Also, fatty acid composition was studied during continuous cultures, in which additions of 1,3-PDO were performed at steady states. Lipids were globally more saturated compared to batch cultures, while by monitoring of the transitory phases, it was noticed that the gradual diol washout had an evident impact in the alteration of the fatty acid composition, by rendering them more unsaturated.

Adaptive laboratory evolution principles and applications in industrial biotechnology.

Adaptive laboratory evolution (ALE) is an innovative approach for the generation of evolved microbial strains with desired characteristics, by implementing the rules of natural selection as presented in the Darwinian Theory, on the laboratory bench. New as it might be, it has already been used by several researchers for the amelioration of a variety of characteristics of widely used microorganisms in biotechnology. ALE is used as a tool for the deeper understanding of the genetic and/or metabolic pathways of evolution. Another important field targeted by ALE is the manufacturing of products of (high) added value, such as ethanol, butanol and lipids. In the current review, we discuss the basic principles and techniques of ALE, and then we focus on studies where it has been applied to bacteria, fungi and microalgae, aiming to improve their performance to biotechnological procedures and/or inspect the genetic background of evolution. We conclude that ALE is a promising and efficacious method that has already led to the acquisition of useful new microbiological strains in biotechnology and could possibly offer even more interesting results in the future.

Single Cell Oil (SCO)-Based Bioactive Compounds: I-Enzymatic Synthesis of Fatty Acid Amides Using SCOs as Acyl Group Donors and Their Biological Activities.

Fatty acid amides (FAAs) are of great interest due to their broad industrial applications. They can be synthesized enzymatically with many advantages over chemical synthesis. In this study, the fatty acid moieties of lipids of Cunninghamella echinulata ATHUM 4411, Umbelopsis isabellina ATHUM 2935, Nannochloropsis gaditana CCAP 849/5, olive oil, and an eicosapentaenoic acid (EPA) concentrate were converted into their fatty acid methyl esters and used in the FAA (i.e., ethylene diamine amides) enzymatic synthesis, using lipases as biocatalysts. The FAA synthesis, monitored using in situ NMR, FT-IR, and thin-layer chromatography, was catalyzed efficiently by the immobilized Candida rugosa lipase. The synthesized FAAs exhibited a significant antimicrobial activity, especially those containing oleic acid in high proportions (i.e., derived from olive oil and U. isabellina oil), against several human pathogenic microorganisms, insecticidal activity against yellow fever mosquito, especially those of C. echinulata containing gamma-linolenic acid, and anticancer properties against SKOV-3 ovarian cancer cell line, especially those containing EPA in their structures (i.e., EPA concentrate and N. gaditana oil). We conclude that FAAs can be efficiently synthesized using microbial oils of different fatty acid composition and used in specific biological applications.

Patterns of Lignocellulosic Sugar Assimilation and Lipid Production by Newly Isolated Yeast Strains From Chilean Valdivian Forest.

Three yeast strains were isolated from decaying wood of Chilean Valdivian forest and identified as Meyerozyma guilliermondii, Scheffersomyces coipomensis, and Sugiyamaella paludigena. These strains were able to efficiently grow on the major monomers contained in Pinus spp. and Eucalyptus spp. wood that includes glucose (Glc), xylose (Xyl), and mannose (Man), showing at 28 °C higher uptake rates for Man, and in some cases for Glc, than for Xyl, used as single carbon sources. Nevertheless, in cultures performed on sugar mixtures, the strains displayed a notable preference for Glc. Additionally, in sugar mixtures, the absence of regulatory mechanisms in sugar assimilation (e.g., catabolic repression) was observed and documented when the activities of several enzymes involved in sugar assimilation (i.e., phosphoglucose isomerase, phosphomannose isomerase, and xylulokinase) were determined. The activity of the key enzymes involved in the onset of lipid accumulation (i.e., NAD+-ICDH) and in fatty acid (FA) biosynthesis (i.e., ATP:CL) indicated a significant accumulation of storage lipids (i.e., up to 24%, w/w) containing oleic and palmitic acids as the major components. The present paper is the first report on the potential of M. guilliermondii, S. coipomensis, and S. paludigena as oleaginous yeasts. We conclude that the new isolates, being able to simultaneously assimilate the major lignocellulosic sugars and efficiently convert them into oily biomass, present a biotechnological potential which deserve further investigation.

High-added value products from microalgae and prospects of aquaculture wastewaters as microalgae growth media.

Aquaculture plays an important role in human nutrition and economic development but is often expanded to the detriment of the natural environment. Several research projects, aimed at cultivating microalgae in aquaculture wastewaters (AWWs) to reduce organic loads and minerals, along with the production of microalgal cell mass and metabolic products, are underway. Microalgal cell mass is of high nutritional value and is regarded as a candidate to replace, partially at least, the fish meal in the fish feed. Also, microalgal cell mass is considered as a feedstock in the bio-fuel manufacture, as well as a source of high-added value metabolic products. The production of these valuable products can be combined with the reuse of AWWs in the light of environmental concerns related with the aquaculture sector. Many research papers published in the last decade demonstrate that plenty of microalgae species are able to efficiently grow in AWWs, mainly derived from fish and shrimp farms, and produce valuable metabolites reducing the AWW pollutant load. We conclude that bio-remediation of AWWs combining with the production of microalgae cell mass and specific metabolites is probably the most convenient and economical solution for AWWs management and can contribute to the sustainable growth of the aquaculture.

Microbial sources of polyunsaturated fatty acids (PUFAs) and the prospect of organic residues and wastes as growth media for PUFA-producing microorganisms.

The discovery of non-fish sources of polyunsaturated fatty acids (PUFAs) is of great biotechnological importance. Although various oleaginous microalgae and fungi are able of accumulating storage lipids (single cell oils - SCOs) containing PUFAs, the industrial applications utilizing these organisms are rather limited due to the high-fermentation cost. However, combining SCO production with other biotechnological applications, including waste and by-product valorization, can overcome this difficulty. In the current review, we present the major sources of fungi (i.e. members of Mucoromycota, fungoid-like Thraustochytrids and genetically modified strains of Yarrowia lipolytica) and microalgae (e.g. Isochrysis, NannochloropsisandTetraselmis) that have come recently to the forefront due to their ability to produce PUFAs. Approaches adopted in order to increase PUFA productivity and the potential of using various residues, such as agro-industrial, food and aquaculture wastes as fermentation substrates for SCO production have been considered and discussed. We concluded that several organic residues can be utilized as feedstock in the SCO production increasing the competitiveness of oleaginous organisms against conventional PUFA producers.

Gamma-linolenic acid production by Cunninghamella echinulata growing on complex organic nitrogen sources.

Growth of two strains of Cunninghamella echinulata on various nitrogen containing raw materials (corn gluten, corn steep, whey concentrate,yeast extract and tomato waste hydrolysate) yielded important amounts of biomass containing various quantities of gamma-linolenic acid (GLA) rich cellular lipids. Especially, growth on tomato waste hydrolysate (TWH) yielded 17.6 g/l of biomass containing 39.6% oil and significant quantities of GLA corresponding to 800 mg/l GLA. Mycelium-bounded proteolytic activity was detected during early growth stages on TWH and declined thereafter, increasing the concentration of assimilable nitrogen in the medium. However, addition of glucose in the medium during the stationary phase triggered the biosynthesis of reserve lipid, since an increase of the proportion of neutral lipids from 45% to 79% in total lipids was observed, while polar lipids decreased from 35% to 12% and from 20% to 9% for glycolipids plus sphingolipids and phospholipids, respectively.

Citric acid production by Yarrowia lipolytica cultivated on olive-mill wastewater-based media.

Yarrowia lipolytica ACA-DC 50109 cultivated on olive-mill wastewater (O.M.W.)-based media, enriched with commercial-industrial glucose, presented an efficient cell growth. Parameters of growth were unaffected by the presence of O.M.Ws in the growth medium. In diluted O.M.Ws enriched with high glucose amounts (initial sugar concentration, 65 g l(-1)), a notable quantity of total citric acid was produced (28.9 g l(-1)). O.M.W.-based media had a noteworthy stimulating effect on the production of citric acid, since both final citric acid concentration and conversion yield of citric acid produced per unit of sugar consumed were higher when compared with the respective parameters obtained from trials without added O.M.W. Adaptation of the strain in O.M.W.-based media favoured the biosynthesis of cellular unsaturated fatty acids (principally of oleic and palmitoleic acids). Additionally, a non-negligible decrease of the phenolic compounds in the growth medium [up to 15% (wt/wt)], a slight decrease of the phyto-toxicity, and a remarkable decolourisation of the O.M.W. were observed. All these results suggest the potentiality of O.M.Ws utilisation in the fermentation process of citric acid production.