Palm oil utilization for the simultaneous production of polyhydroxyalkanoates and rhamnolipids by <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis>

Direct utilization of palm oil for the simultaneous production of polyhydroxyalkanoates (PHAs) and rhamnolipids was demonstrated using Pseudomonas aeruginosa IFO3924. By secreted lipase, palm oil was hydrolyzed into glycerol and fatty acids. Fatty acids became favorable carbon sources for cell growth and PHA production via β-oxidation and glycerol for rhamnolipid production via de novo fatty acid synthesis. Both PHA and rhamnolipid syntheses started after the nitrogen source was exhausted and cell growth ceased. PHA synthesis continued until all fatty acids were exhausted, and at that time, PHA content in the cells reached a maximum, but stopped despite the remaining glycerol (<2g/l). In contrast, rhamnolipid synthesis continued until glycerol was exhausted.     

Polyhydroxyalkanoate production from whey by Pseudomonas hydrogenovora

Whey permeate from dairy industry was hydrolyzed enzymatically to cleave its main carbon source, lactose, to glucose and galactose. The hydrolysis products were chosen as carbon sources for the production of poly-3-hydroxybutyric acid (PHB) by Pseudomonas hydrogenovora. In shaking flask experiments, the utilization of whey permeate as a cheap substrate was compared to the utilization of pure glucose and galactose for bacterial growth under balanced conditions as well as for the production of PHB under nitrogen limitation. After determination of the inhibition constant Ki for sodium valerate on biomass production (Ki=1.84 g/l), the biosynthesis of PHA co-polyesters containing 3-hydroxybutyrate (3HB) and 3-hydroxyvalerate (3HV) units from hydrolyzed whey permeate and valerate was investigated. The application of hydrolyzed whey permeate turned out to be advantageous compared with the utilization of pure sugars. Therefore, fermentation under controlled conditions in a bioreactor was performed with hydrolyzed whey permeate to obtain detailed kinetic data (maximum specific growth rate, mu max=0.291/h, maximum polymer concentration, 1.27 g/l PHB), values for molecular mass distribution (weight average molecular weight Mw=353.5 kDa, polydispersity index PDI=3.8) and thermo analytical data. The fermentation was repeated with co-feeding of valerate (maximum specific growth rate, mu(max)=0.201/h, maximum polymer concentration, 1.44 g/l poly-(3HB-co-21%-3HV), weight average molecular weight M(w)=299.2 kDa, polydispersity index PDI=4.3).     

Production of poly(3-hydroxybutyrate) and poly(3-hydroxybutyrate-co-4-hydroxybutyrate) by Ralstonia eutropha from soybean oil

High-yield production of polyhydroxyalkanoates (PHAs) by Ralstonia eutropha KCTC 2662 was investigated using soybean oil and γ-butyrolactone as carbon sources. In flask culture, it was shown that R. eutropha KCTC 2662 accumulated PHAs during the growth phase. The optimum carbon to nitrogen ratio (C/N ratio) giving the highest cell and PHA yield was 20 g-soybean oil/g-(NH(4))(2)SO(4). The 4-hydroxybutyrate (4HB) fraction in the copolymer was not strongly affected by the C/N ratio. In a 2.5-L fermentor, a homopolymer of poly(3-hydroxybutyrate) [P(3HB)] was produced from soybean oil as the sole carbon source by batch and fed-batch cultures of R. eutropha with dry cell weights of 15-32 g/L, PHA contents of 78-83 wt% and yields of 0.80-0.82 g-PHA/g-soybean oil used. By co-feeding soybean oil and γ-butyrolactone as carbon sources, a copolymer of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)] could be produced with dry cell weights of 10-21 g/L, yields of 0.45-0.56 g-PHA/g-soybean oil used (0.39-0.50g-PHA/g-carbon sources used) and 4HB fractions of 6-10 mol%. Higher supplementation of γ-butyrolactone increased the 4HB fraction in the copolymer, but decreased cell and PHA yield.     

Simultaneous production of polyhydroxyalkanoates and rhamnolipids by Pseudomonas aeruginosa

The feasibility of the simultaneous production of polyhydroxyalkanoates (PHAs) and rhamnolipids, as a novel approach to reduce their production costs, was demonstrated by the cultivation of Pseudomonas aeruginosa IFO3924. Fairly large amounts of PHAs and rhamnolipids were obtained from the bacterial cells and the culture supernatant, respectively. Decanoate was a more suitable carbon source than ethanol and glucose for the simultaneous production, although glucose was suitable for cell growth without an induction period under pH control. The kind of carbon source affected PHA monomer composition markedly and PHA molecular weight slightly. Monorhamnolipids and dirhamnolipids were included in the rhamnolipids extracted from the culture supernatant using decanoate, glucose, or ethanol as the carbon source. Both PHAs and rhamnolipids were synthesized after the growth phase. PHA content in the cell reached a maximum when the carbon source was exhausted. After exhaustion of the carbon source, PHA content decreased rapidly, but rhamnolipid synthesis, which followed PHA synthesis, continued. This resulted in a time lag for the attainment of maximum levels of PHAs and rhamnolipids. The reusability of the cells used in rhamnolipid production was evaluated in the repeated batch culture of P. aeruginosa IFO3924 for the simultaneous production of PHAs and rhamnolipids. High concentrations of rhamnolipids in the culture supernatant were attained at the end of both the first and second batch cultures. High PHA content was achieved in the resting cells that were finally harvested after the second batch. Simultaneous production of PHAs and rhamnolipids will enhance the availability of valuable biocatalysts of bacterial cells, and dispel the common belief that the production cost of PHAs accumulated intracellularly is almost impossible to become lower than that of cells themselves.     

Characterization of medium-chain-length polyhydroxyalkanoate biosynthesis by Pseudomonas mosselii TO7 using crude glycerol

Polyhydroxyalkanoates (PHAs) are biopolyesters produced by microorganisms that are environmentally friendly. PHAs can be used to replace traditional plastic to reduce environmental pollution in various fields. PHA production costs are high because PHA must be produced from a carbon substrate. The purpose of this study was to find the strain that can used the BDF by-product as the sole carbon source to produce high amounts of medium-chain-length PHA. Three isolates were evaluated for potential PHA production by using biodiesel-derived crude glycerol as the sole carbon source. Among them, Pseudomonas mosselii TO7 yielded high PHA content. The PHA produced from P. mosselii TO7 were medium-chain-length-PHAs. The PHA content of 48% cell dry weight in 48 h with a maximum PHA productivity of 13.16 mg PHAs L^?1 h^?1. The narrow polydispersity index value of 1.3 reflected the homogeneity of the polymer chain, which was conducive to industrial applications.     

The penicillin G acylase production by B. megaterium is amino acid consumption dependent

Aiming at to enhance the production of penicillin G acylase (PGA) by Bacillus megaterium, we have performed flasks experiments using different medium composition. Using 51 g/L of casein hydrolyzed with Alcalase and 2.7 g/L of phenylacetic acid (PhAc), the following carbon substrates were tested, individually and combined: glucose, glycerol, and lactose (present in cheese whey). Glycerol and glucose showed to be effective nutrients for the microorganism growth but delayed the PGA production. Cheese whey always increased enzyme production and cell mass. However, lactose (present in cheese whey) was not a significant carbon source for B. megaterium. PhAc, amino acids, and small peptides present in the hydrolyzed casein were the actual carbon sources for enzyme production. Replacement of hydrolyzed casein by free amino acids, 10.0 g/L, led to a significant increase in enzyme production (app. 150%), with a preferential consumption of alanine, aspartic acid, glycine, serine, arginine, threonine, lysine, and glutamic acid. A decrease of the enzyme production was observed when 20.0 g/L of amino acids were used. Using the single omission technique, it was shown that none of the 18 tested amino acids was essential for enzyme production. The use of a medium containing eight of the preferentially consumed amino acids lead to similar enzyme production level obtained when using 18 amino acids. PhAc, up to 2.7 g/L, did not inhibit enzyme production, even if added at the beginning of the cultivation.     

真氧产碱杆菌在双营养（碳、氮）限制区内合成聚羟基烷酸酯

研究了真氧产碱杆菌以混合有机酸为碳源,硫酸铵为氮源,在双营养（碳、氮）限制区内聚羟基烷酸酯的生物合成。结果表明：双营养限制区的长度与聚羟基烷酸酯的产量呈正相关。同时,在对两种不同的双营养限制区实现方式进行比较后发现,首先限制碳源的双营养限制方式比首先限制氮源的双营养限制方式更有利于聚羟基烷酸酯的合成;在这两种不同营养限制方式下,PHAs的最高产量分别为3.72 g/L和2.55 g/L。     

Synthesis and production of polyhydroxyalkanoates by halophiles: current potential and future prospects

Biodegradable materials with plastic or elastomeric properties are in great demand for a variety of applications. Polyhydroxyalkanoates (PHAs), polyesters synthesized by microorganisms, possess such desired features. Industrial production of PHAs is currently achieved using recombinant Escherichia coli. Nevertheless, recent research on halophiles, salt requiring microorganisms, has shown a remarkable potential for biotechnological production of PHAs. The halophilic archaeon Haloferax mediterranei accumulates a co-polymer, i.e., poly(3-hydroxybutyrate-co-3-hydroxyvalerate) in large amounts using glucose, starch, and hydrolyzed whey as carbon sources. Chemical composition and molecular weight of PHAs produced by H. mediterranei can be modified depending on the substrate utilized as precursor. Phylogenetic studies on haloarchaeal enzymes able to polymerize the components of PHAs (i.e., PHA synthases) reveal a novel cluster, with a close relationship with PHA polymerases of bacteria and archaea found in marine-related niches. On the other hand, sequences of PHA synthases of two halophilic bacteria are more closely affiliated to synthases of Proteobacteria. Several bacterial species of the family Halomonadaceae accumulate PHAs. Halomonas boliviensis reached PHA yields and volumetric productivities close to the highest reported so far. Furthermore, H. boliviensis and other Halomonas species are able to co-produce PHA and osmolytes, i.e., ectoines and hydroxyectoine, in one process.     

Production of polyhydroxyalkanoates by mixed culture: recent trends and biotechnological importance

Polyhydroxyalkanoates (PHAs) are the polymers of hydroxyalkanoates that accumulate as carbon/energy or reducing-power storage material in various microorganisms. PHAs have been attracting considerable attention as biodegradable substitutes for conventional polymers. To reduce their production cost, a great deal of effort has been devoted to developing better bacterial strains and more efficient fermentation/recovery processes. The use of mixed cultures and cheap substrates can reduce the production cost of PHA. Accumulation of PHA by mixed cultures occurs under transient conditions mainly caused by intermittent feeding and variation in the electron donor/acceptor presence. The maximum capacity for PHA storage and the PHA production rate are dependent on the substrate and the operating conditions used. This work reviews the development of PHA research. Aspects discussed include metabolism and various mechanisms for PHA production by mixed cultures; kinetics of PHA accumulation and conversion; effects of carbon source and temperature on PHA production using mixed cultures; PHA production process design; and characteristics of PHA produced by mixed cultures.     

Production and characterization of polyhydroxyalkanoates in Pseudomonas aeruginosa ATCC 9027 from glucose, an unrelated carbon source

The production and characterization of polyhydroxyalkanoic acids (PHAs) from glucose in Pseudomonas aeruginosa ATCC 9027 is described. We determined that the synthesis of PHAs was not due to a complete lack of nitrogen source, as previously reported for other microorganisms. The synthesis of PHAs was observed during exponential growth and it depended on the carbon/nitrogen ratio in the culture. More significantly, the specific PHA accumulation rate in this phase was higher than that observed in the storage phase. This phenomenon was a consequence of higher extracellular production rates of gluconate and 2-ketogluconate detected during the storage phase. Therefore, the production of those acids decreased the synthesis of PHAs in P. aeruginosa. The maximum percentage of PHA accumulation obtained was 10.8% of the cell dry matter when all the glucose was consumed. The monomer composition of this PHA consisted only of saturated 3-hydroxy fatty acids (octanoic, decanoic, and dodecanoic acids) as shown by gas chromatography - mass spectroscopy and nuclear magnetic resonance analyses, where 3-hydroxydecanoic acid was the main component because of the high affinity of its PhaC synthase for this monomer. The physical properties of this PHA were determined by differential scanning calorimetry and gel permeation chromatography.     

Production of polyhydroxyalkanoates (PHAs) with canola oil as carbon source

Wautersia eutropha was able to synthesize medium chain length polyhydroxyalkanoates (PHAs) when canola oil was used as carbon source. W. eutropha was cultivated using fructose and ammonium sulphate as carbon and nitrogen sources, respectively, for growth and inoculum development. The experiments were done in a laboratory scale bioreactor in three stages. Initially, the biomass was adapted in a batch culture. Secondly, a fed-batch was used to increase the cell dry weight and PHA concentration to 4.36 g L(-1) and 0.36 g L(-1), respectively. Finally, after the addition of canola oil as carbon source a final concentration of 18.27 g L(-1) PHA was obtained after 40 h of fermentation. With canola oil as carbon source, the polymer content of the cell dry matter was 90%. The polymer was purified from dried cells and analyzed by FTIR, NMR and DSC using PHB as reference. The polymer produced by W. eutropha from canola oil had four carbon monomers in the structure of the PHA and identified by 1H and 13C NMR analysis as 3-hydroxybutyrate (3HB), 3-hydroxyvalerate (3HV), 3-hydroxyoctanoate (3HO), and 3-hydroxydodecanoate (3HDD).     

Polyhydroxyalkanoate biosynthesis in Bacillus cereus SPV under varied limiting conditions and an insight into the biosynthetic genes involved

Aims:  A new strain of Bacillus, Bacillus cereus SPV, was found to be capable of using a wide range of carbon sources for the production of polyhydroxyalkanoates (PHAs) ( Valappil et al. 2007b ). Limiting nutrient in the culture conditions is crucial for PHA production. In this study, B.   cereus SPV was grown in different culture conditions with limitation of potassium, nitrogen, sulphur and phosphorous to establish the impact of nutritional limitation on PHA production.
Methods and Results:  The PHA yields obtained were found to be 13·4, 38, 13·15 and 33·33% dcw for potassium, nitrogen, sulphur and phosphorus limitations, respectively. Gas chromatography–mass spectrometry analysis of the isolated polymers showed the presence of P(3HB) under nitrogen, sulphur and phosphate-limiting conditions and P(3HB-3HV) copolymer under potassium limiting conditions. This ability of B. cereus SPV to accumulate different PHA monomers from structurally unrelated carbon sources led to an interest in the molecular analysis of PHA biosynthesis in this organism. To achieve this, PCR was used to identify the polyhydroxyalkanoate biosynthetic genes in B. cereus SPV.
Conclusion:  Sequence analysis of the PCR products from B. cereus SPV revealed the sequence of the putative biosynthetic genes, and possible regions involved in substrate binding.
The nucleotide sequence reported in this paper is in the GenBank nucleotide sequence database under accession number DQ486135 .
Significance and Impact of the Study:  This is the first report comparing the capability of B. cereus SPV to produce PHAs under different culture conditions of potassium, nitrogen, sulfur and phosphate limitations. The results in this study suggest the unique ability of B. cereus SPV to supply both 3HB and 3HV monomers from a structurally unrelated carbon source, glucose.     

Production of targeted poly(3-hydroxyalkanoates) copolymers by glycogen accumulating organisms using acetate as sole carbon source

One of the main limitations in bacterial polyhydroxyalkanoate (PHA) production with mixed cultures is the fact that primarily polyhydroxybutyrate (PHB) homopolymers are generated from acetate as the main carbon source, which is brittle and quite fragile. The incorporation of different 3-hydroxyalkanoate (HA) components into the polymers requires the addition of additional carbon sources, leading to extra costs and complexity. In this study, the production of poly(3-hydroxybutyrate (3HB)-co-3-hydroxyvalerate (3HV)-co-3-hydroxy-2-methylvalerate (3HMV)), with 7-35C-mol% of 3HV fractions from acetate as the only carbon source was achieved with the use of glycogen accumulating organisms (GAOs). An enriched GAO culture was obtained in a lab-scale reactor operated under alternating anaerobic and aerobic conditions with acetate fed at the beginning of the anaerobic period. The production of PHAs utilizing the enriched GAO culture was investigated under both aerobic and anaerobic conditions. A polymer content of 14-41% of dry cell weight was obtained. The PHA product accumulated by GAOs under anaerobic conditions contained a relatively constant proportion of non-3HB monomers (30+/-5C-mol%), irrespective of the amount of acetate assimilated. In contrast, under aerobic conditions, GAOs only produced 3HB monomers from acetate causing a gradually decreasing 3HV fraction during this aerobic feeding period. The PHAs were characterized by gel permeation chromatography (GPC) and differential scanning calorimetry (DSC). The data demonstrated that the copolymers possessed similar characteristics to those of commercially available poly(3HB-co-3HV) (PHBV) products. The PHAs produced under solely anaerobic conditions possessed lower melting points and crystallinity, higher molecular weights, and narrower molecular-weight distributions, compared to the aerobically produced polymers. This paper hence demonstrates the significant potential of GAOs to produce high quality polymers from a simple and cheap carbon source, contributing considerably to the growing research body on bacterial PHA production by mixed cultures.     

Accumulation of polyhydroxyalkanoates by Microlunatus phosphovorus under various growth conditions

Microlunatus phosphovorus is an activated-sludge bacterium with high levels of phosphorus-accumulating activity and phosphate uptake and release activities. Thus, it is an interesting model organism to study biological phosphorus removal. However, there are no studies demonstrating the polyhydroxyalkanoate (PHA) storage capability of M. phosphovorus, which is surprising for a polyphosphate-accumulating organism. This study investigates in detail the PHA storage behavior of M. phosphovorus under different growth conditions and using different carbon sources. Pure culture studies in batch-growth systems were conducted in shake-flasks and in a bioreactor, using chemically defined growth media with glucose as the sole carbon source. A batch-growth system with anaerobic–aerobic cycles and varying concentrations of glucose or acetate as the sole carbon source, similar to enhanced biological phosphorus removal processes, was also employed. The results of this study demonstrate for the first time that M. phosphovorus produces significant amounts of PHAs under various growth conditions and with different carbon sources. When the PHA productions of all cultivations were compared, poly(3-hydroxybutyrate) (PHB), the major PHA polymer, was produced at about 20–30% of the cellular dry weight. The highest PHB production was observed as 1,421 mg/l in batch-growth systems with anaerobic–aerobic cycles and at 4 g/l initial glucose concentration. In light of these key results regarding the growth physiology and PHA-production capability of M. phosphovorus, it can be concluded that this organism could be a good candidate for microbial PHA production because of its advantages of easy growth, high biomass and PHB yield on substrate and no significant production of fermentative byproducts.     

Poly(3-hydroxybutyrate) production by Bacillus cereus SPV using sugarcane molasses as the main carbon source

The main hindrance in the use of polyhydroxyalkanoates (PHAs) as a replacement for existing petroleum-based plastics is their high production cost. The carbon source accounts for 50% of the cost for PHA production. Thus, increasing the yield and productivity of PHAs on cheap substrates is an important challenge for biotechnologists to support the commercialization and further applications of these polymers. In this study, we have investigated the use of an agricultural raw material, sugarcane molasses, as the main carbon source for poly(3-hydroxybutyrate) (P(3HB)) production by Bacillus cereus SPV. These studies were carried out in both shaken flasks and 2 L bioreactors. Various conditions were evaluated for their effects on biomass and P(3HB) accumulation. A high polymer yield was obtained, 61.07% dry cell weight (DCW) in a 1 L shaken flask study and 51.37% DCW in a 2 L fermenter study. These yields are 50% higher than previously observed with Bacillus cereus SPV. Hence, the results are encouraging and show that sugarcane molasses are a promising carbon source for an economical and commercially viable production of P(3HB).     

Production and characterization of poly‐3‐hydroxybutyrate from biodiesel‐glycerol by Burkholderia cepacia ATCC 17759

Glycerol, a byproduct of the biodiesel industry, can be used by bacteria as an inexpensive carbon source for the production of value‐added biodegradable polyhydroxyalkanoates (PHAs). Burkholderia cepacia ATCC 17759 synthesized poly‐3‐hydroxybutyrate (PHB) from glycerol concentrations ranging from 3% to 9% (v/v). Increasing the glycerol concentration results in a gradual reduction of biomass, PHA yield, and molecular mass (M_n and M_w) of PHB. The molecular mass of PHB produced utilizing xylose as a carbon source is also decreased by the addition of glycerol as a secondary carbon source dependent on the time and concentration of the addition. ¹H‐NMR revealed that molecular masses decreased due to the esterification of glycerol with PHB resulting in chain termination (end‐capping). However, melting temperature and glass transition temperature of the end‐capped polymers showed no significant difference when compared to the xylose‐based PHB. The fermentation was successfully scaled up to 200 L for PHB production and the yield of dry biomass and PHB were 23.6 g/L and 7.4 g/L, respectively. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Short Communication: Optimization of bioprocess conditions for exopolysaccharide production by Klebsiella oxytoca

Optimization of bioprocess conditions increased exopolysaccharide production by a strain of Klebsiella oxytoca from 6g/l to 15g/l; this corresponded to an increase in medium viscosity from 36cP at 12s–1 to 20,000 cP at 0.6 s–1. A combination of equal proportions of tryptone nitrogen and urea nitrogen proved to be the best nitrogen source. Lactose was shown to be the preferred carbon source. At an optimum temperature of 25°C, a pH of 7 was found to be the best for exopolysaccharide production. The concentration of exopolysaccharide produced on whey, enriched whey, enriched whey permeate and lactose-rich medium was comparable.     

Isolation of a thermotolerant bacterium producing medium-chain-length polyhydroxyalkanoate

Aims:  The aim of this study was to isolate a thermotolerant micro‐organism that produces polyhydroxyalkanoates (PHAs) composed of medium‐chain‐length (mcl) HA units from a biodiesel fuel (BDF) by‐product as a carbon source. Methods and Results:  We successfully isolated a thermotolerant micro‐organism, strain SG4502, capable to accumulate mcl‐PHA from a BDF by‐product as a carbon source at a cultivation temperature of 45°C. The strain could also produce mcl‐PHA from acetate, octanoate and dodecanoate as sole carbon sources at cultivation temperatures up to 55°C. Taxonomic studies and 16S rRNA gene sequence analysis revealed that strain SG4502 was phylogenetically affiliated with species of the genus Pseudomonas. This study is the first report of PHA synthesis by a thermotolerant Pseudomonas. Conclusions:  A novel thermotolerant bacterium capable to accumulate mcl‐PHA from a BDF by‐product was successfully isolated. Significance and Impact of the Study:  A major issue regarding industrial production of microbial PHAs is their much higher production cost compared with conventional petrochemical‐based plastic materials. Especially significant are the cost of a fermentative substrate and the running cost to maintain a temperature suitable for microbial growth. Thus, strain SG4502, isolated in this study, which assimilates BDF by‐product and produces PHA at high temperature, would be very useful for practical application in industry.     

Production of medium-chain-length polyhydroxyalkanoates by high-cell-density cultivation of Pseudomonas putida under phosphorus limitation

High-cell-density fed-batch cultures of Pseudomonas putida were carried out for the production of medium-chain-length polyhydroxyalkanoates (PHAs) using oleic acid as a carbon source. By employing an optimal feeding strategy without the limitation of any nutrient, a high cell concentration of 173 g/L was achieved, but the PHA concentration and PHA content were only 32.3 g/L and 18.7 wt%, respectively. To increase the PHA concentration and content, phosphorus limitation was applied during fed-bath culture by reducing the initial KH(2)PO(4) concentration. When the initial KH(2)PO(4) concentration was reduced to 4 g/L, cell concentration, PHA concentration, and PHA content obtained in 38 h were 141 g/L, 72. 6 g/L, and 51.4 wt%, respectively, resulting in a high productivity of 1.91 g PHA/L per hour.     

Utilization of residues from agro-forest industries in the production of high value bacterial cellulose

Bacterial cellulose (BC), a very peculiar form of cellulose, is gaining considerable importance due to its unique properties. In this study, several residues, from agro-forestry industries, namely grape skins aqueous extract, cheese whey, crude glycerol and sulfite pulping liquor were evaluated as economic carbon and nutrient sources for the production of BC. The most relevant BC amounts attained with the residues from the wine and pulp industries were 0.6 and 0.3 g/L, respectively, followed by biodiesel crude residue and cheese whey with productions of about, 0.1 g/L after 96 h of incubation. Preliminary results on the addition of other nutrient sources (yeast extract, nitrogen and phosphate) to the residues-based culture media indicated that, in general, these BC productions could be increased by ∼200% and ∼100% for the crude glycerol and grape skins, respectively, after the addition organic or inorganic nitrogen.