Kinetics of growth and chemical composition ofFusarium moniliforme cultivated on carob aqueous extract for microbial protein production

Summary The kinetics of growth and the chemical composition ofFusarium moniliforme cultivated on aqueous carob pod extract were investigated. The extract was adjusted to provide 0.5, 1.0, 2.0 and 4.0% carob sugars supplemented with inorganic salts at the ratio: carob sugar: NH₄H₂PO₄: MgSO₄.7H₂O=1:0.6:0.012. The extract contained 16 mg tannic acid (Folin-Dennis) per g of carob sugar.The phase of vigorous growth was exponential. Tannins were not observed to depress growth. The maximum value of 0.22 h^–1 for a specific growth rate corresponding to a generation time of 3.15 h was obtained when the fungus was cultivated on a 4% carob sugar medium. The dry mycelium produced per g of consumed carob sugar was then 0.515 g.The protein and purine content was affected by the composition of the growth medium. Protein values up to 37.7% true (Lowry) and 53.1% crude (NX6.25) of dry mycelium were recorded. Mean purine contents were 89 and 116 mol/g, corresponding to nucleic acid levels of 5.7 and 7.5% for mycelium grown on 0.5 and 4.0% carob sugar respectively.These findings linked with those previously reported regarding the good appearance and nutritional quality ofF. moniliforme (Drouliscos et al., 1976) make this fungus worthy of consideration for the production of protein.     

Energetics of tobacco cells,Nicotiana tabacum L., growing on sucrose medium

Summary Batch cultures of tobacco cells induced from Nicotiana tabacum L. cv. Bright Yellow-2 were carried out under oxygen-limited conditions using sucrose as the sole carbon source. Maintenance coefficients for sugar, m, and for oxygen, m_O, were 0.02 mmol glucose/g cell dry weight/h and 0.09 mmol O₂/g cell dry weight/h, and true growth yields for sugar, Y_G, and for oxygen, Y_GO, were 107 g cell dry weight/mol glucose and 61 g cell dry weight/mol O₂, respectively.Balance equations based on electrons available from the culture suggested that the carbon-substrate consumed by the cells might be metabolized mainly in biosynthetic processes without the excretion of extracellular products.     

Sugarcane-pressmud as a novel substrate for production of citric acid by solid-state fermentation

Sugarcane-pressmud, a by-product of cane-sugar manufacture, was used as a substrate for production of citric acid by Aspergillus niger CFTRI 30, in a solid-state fermentation system. Of the 170 g of sugar supplied, 131 g were consumed, with a 79% yield of citric acid over 120 h. Potassium ferrocyanide improved the conversion to about 88% and lowered the fermentation time by 24 h. Enrichment with sugar and NH₄NO₃ was essential to improve productivity. About 174 g citric acid/kg dry sugarcane-pressmud were produced after 120 h in ferrocyanid-treated medium which initially contained 12.5% (w/w) effective sugar and 0.1% (w/w) NH₄NO₃. About 3% (w/w) of the original sugar present in the sugarcane-pressmud was non-utilizable. This is the first report on the potential of sugarcane-pressmud for citric acid production.V.S. Shankaranand and B.K. Lonsane are with the Fermentation Technology and Bioengineering Discipline, Central Food Technological Research Institute, Mysore-570 013, India     

Coffee husk: an inexpensive substrate for production of citric acid by Aspergillus niger in a solid-state fermentation system

Aspergillus niger CFTRI 30 produced 1.3 g citric acid/10 g dry coffee husk in 72 h solid-state fermentation when the substrate was moistened with 0.075 M NaOH solution. Production was increased by 17% by adding a mixture of iron, copper and zinc to the medium but enrichment of the moist solid medium with (NH₄)₂SO₄, sucrose or any of four enzymes did not improve production. The production of about 1.5 g citric acid/10 g dry coffee husk at a conversion of 82% (based on sugar consumed) under standardized conditions demonstrates the commercial potential of using the husk in this way.The authors are with the Department of Microbiology and Bioengineering, Central Food Technological Research Institute, Mysore-570 013, India;     

Carbon assimilation pathways in sulfate-reducing bacteria II. Enzymes of a reductive citric acid cycle in the autotrophic Desulfobacter hydrogenophilus

The strict anaerobe Desulfobacter hydrogenophilus is able to grow autotrophically with CO₂, H₂, and sulfate as sole carbon and energy sources. The generation time at 30°C under autotrophic conditions in a pure mineral medium was 15 h, the growth yield was 8 g cell dry mass per mol sulfate reduced to H₂S. Enzymes of the autotrophic CO₂ assimilation pathway were investigated. Key enzymes of the Calvin cycle and of the acetyl CoA pathway could not be found. All enzymes of a reductive citric acid cycle were present at specific activities sufficient to account for the observed growth rate. Notably, an ATP-citrate lyase (1.3 mol · min^-1 · mg cell protein^-1) was present both in autotrophically and in heterotrophically grown cells, which was rapidly inactivated in the absence of ATP. The data indicate that in D. hydrogenophilus a reductive citric acid cycle is operating in autotrophic CO₂ fixation. Since other autotrophic sulfate reducers possess an acetyl CoA pathway for CO₂ fixation, two different autotrophic pathways occur in the same physiological group.Dedicated to Prof. H. G. Wood on the occasion of his 80th birthday     

Continuous fermentation to produce fungal protein. Effect of growth rate on the biomass yield and chemical composition of Fusarium moniliforme

Fusarium moniliforme was grown on a carob aqueous extract in a chemostat for fungal protein production. The substrate was adjusted to provide 0.5% carob sugars supplemented with inorganic salts. The dilution rate varied from 0.086 to 0.227 hr^?1 under constant conditions of temperature (30°C), pH (4.5), and oxygen saturation (60–80%). A yield of 0.709 g dry mycelium/g consumed carob sugar and a productivity value of 0.687 g dry mycelium/liter hr^?1 were obtained at μ = 0.205 hr^?1. The maintenance coefficient was 0.077 g carob sugar/g dry mycelium hr^?1. While the carbohydrate and purine content of dry mycelium increased at μ values from 0.114 to 0.205 hr^?1 both true (Lowry) and crude (N × 6.25) protein contents decreased at the same μ range. Maximum values of 36.3% true and 47.9% crude protein of dry mycelium were obtained at μ = 0.114 hr^?1, whereas a minimum purine content of 99.8 μmol/g corresponding to 6.42% nucleic acids was recorded at μ = 0.086 hr^?1. It was concluded that a continuous fermentation of carob aqueous extract using F. moniliforme should be operated at growth rates of approximately 0.205 hr^?1 in order to maximize protein production.     

The effect of carbon dioxide on the growth kinetics of fructose-limited chemostat cultures of Acetobacterium woodii DSM 1030

The growth of the anaerobic acetogenic bacterium Acetobacterium woodii DSM 1030 was investigated in fructose-limited chemostat cultures. A defined medium was developed which contained fructose, mineral salts, cysteine · HCl and Ca pantothenate (1 mg · 1^–1) supplied in a vitamin supplement. Growth at high dilution rates was dependent on the presence of CO₂ in the gas phase. The ^max was found to be 0.16 h^–1 and the fructose maintenance requirement was 0.1 to 0.13 mmol fructose · (g dry wt)^–1 · h^–1. A growth yield of 61 g dry wt · (mol fructose)^–1, corrected for the cell maintenance requirement and for incorporation of fructose carbon into cell biomass, was determined from the fructose consumption. A corresponding growth yield of 69 g dry wt · (mol fructose)^–1 was calculated from the acetate production assuming that fructose fermentation was homoacetogenic. A Y_ATP of 12.2 to 13.8 g dry wt · (mol ATP)^–1 was calculated from these growth yields using a value of 5 mol ATP · (mol fructose)^–1 as an estimate of the amount of ATP synthesised from fructose fermentation. The addition of yeast extract (0.5 g · 1^–1) to the medium did not influence the ^max or cell yield. After prolonged growth under fructose-limited conditions the requirement of the culture for CO₂ in the gas phase was reduced.Abbreviations YE yeast extract - IC inorganic carbon - D fermenter dilution rate : h^–1 - M_X maintenance requirement for X: mmol X · (g dry wt)^–1 · h^–1 - X may be fructose (Fruct), fructose consumed in energy metabolism (Fruct [E]), acetate (Ac) - ATP CO₂, NH _inf4 ^sup+ or Pi - qX specific rate of utilisation or consumption of X: mmol X · (g dry wt)^–1 · h^–1 - V fermenter volume: litre - rC · Cell, fermenter cell carbon production: mmol C · h^–1 - Y_X yield of cells on X: g dry wt · (mol X)^–1 - Y _infx ^supmax the yield corrected for cell maintenance: g dry wt · (mol X)^–1 - S_ATP stoichiometry of ATP synthesis from fructose: mol ATP · (mol frucose)^–1 - x cell concentration: g dry wt · 1^–1 - specific growth rate : h^–1 - ^max maximum specific growth rate: h^–1     

Utilization of Brewery Spent Grain Liquor by Aspergillus niger

Aspergillus niger was found capable of rapidly converting about 97% of the sugar from brewery spent grain liquor to fungal mass. The yield of dry mycelium, based on the sugar consumed, was approximately 57%. This fungus produced 1.10% titratable acid calculated as citric acid and reduced the biochemical oxygen demand by 96%.     

Effects of methanol on cell growth and lipid production from mixotrophic cultivation of Chlorella sp.

The marine microalga Chlorella sp. was cultivated under mixotrophic conditions using methanol as an organic carbon source, which may also act to maintain the sterility of the medium for long-term outdoor cultivation. The optimal methanol concentration was determined to be 1% (v/v) for both cell growth and lipid production when supplying 5% CO₂ with 450 μE/m²/sec of continuous illumination. Under these conditions, the maximal cell biomass and total lipid production were 4.2 g dry wt/L and 17.5% (w/w), respectively, compared to 2.2 g dry wt/L and 12.5% (w/w) from autotrophic growth. Cell growth was inhibited at methanol concentrations above 1% (v/v) due to increased toxicity, whereas 1% methanol alone sustained 1.0 g dry wt/L and 4.8% total lipid production. We found that methanol was preferentially consumed during the initial period of cultivation, and carbon dioxide was consumed when the methanol was depleted. A 12:12 h (light:dark) cyclic illumination period produced favorable cell growth (3.6 g dry wt/L). Higher lipid production was observed with cyclic illumination than with continuous illumination (18.6% (w/w) vs 17.5% (w/w)), and better lipid production was also obtained under mixotrophic rather than autotrophic conditions. Interestingly, under mixotrophic conditions with 12:12 (h) cyclic illumination, high proportions of C_16:0, C_18:0, and C_18:1 were observed, which are beneficial for biodiesel production. These results strongly indicate that the carbon source is important for controlling both lipid composition and cell growth under mixotrophic conditions, and they suggest that methanol could be utilized to scale up production to an open pond type system for outdoor cultivation where light illumination changes periodically.     

Carbon assimilation by the autotrophic thermophilic archaebacterium Thermoproteus neutrophilus

Growth of Thermoproteus neutrophilus at 85°C was studied using an improved mineral medium with CO₂, CO₂ plus acetate, CO₂ plus propionate, or CO₂ plus succinate as carbon sources; sulfur reduction with H₂ to H₂S was the sole source of energy. None of the carbon compounds added was oxidized to CO₂. The organism grew autotrophically with a generation time of 9–14 h, up to a cell density of 0.5 g dry weight per liter (2×10⁹ cells/ml). Propionate did not stimulate, succinate slightly stimulated the growth rate. Acetate, even at low concentrations (0.5 mM), stimulated the growth rate, the generation time being shortened to 3–4 h. Acetate provided 70% of the cell carbon, which shows that Thermoproteus neutrophilus is a facultative autotroph. The path of these carbon precursors into cell compounds was studied by ¹⁴C long-term labelling and investigation of enzyme activities. Propionate could not be used as a major carbon source and was incorporated only into isoleucine, probably via the citramalate pathway. Acetate was a preferred carbon source which suppressed autotrophic CO₂ fixation: acetate grown cells exhibited an incomplete citric acid cycle in which 2-oxoglutarate dehydrogenase was present, but fumarate reductase was repressed. The succinate incorporation pattern and enzyme pattern indicated that autotrophic CO₂ fixation proceeded via a yet to be defined reductive citric acid cycle.     

Photosynthesis by carrot tissue cultures

Summary ¹⁴CO₂-fixation rates in green carrot callus cultres (about 35 g chlorophyll/g fresh wt) were determined in gaseous and liquid media using a range of light intensities and CO₂ concentrations. Main products of light-dependent CO₂-fixation were sucrose, alanine, glutamine, serine/glycine and malic acid. In darkness, glutamine and malic acid were formed.Light CO₂-fixation rates were about ten times higher than dark fixation rates and reached 50–90 mol/mg chlorophyll/h in 10000 lux, 1% CO₂ in air. Net O₂-evolution by the tissue was demonstrated polarographically under these conditions. Light CO₂-fixation rates were linearly related to chlorophyll levels while dark fixation was independent of chlorophyll content. Lowered O₂ partial pressures in gaseous conditions increased ¹⁴CO₂-fixation rates. Ribulose diphosphate carboxylase and phosphoenol pyruvate carboxylase activities and their distribution in subcellular fractions were examined.When carrot tissue cultures were grown for two or four weeks on agar media lacking a carbohydrate source, in 10000 lux and 1% CO₂ in either air or N₂, dry weight increases were obtained although chlorophyll levels eventually declined.     

Dynamics of Photosynthesis in Pine Stands

The CO₂ exchange of pine (Pinus sylvestris L.) shoots was continuously monitored over several years. The spatial and temporal variability of gas exchange was thoroughly investigated for three forest types. The net uptake of CO₂ by current-season shoots in a bilberry pine stand equaled 5.4 g CO₂ per g dry wt for the growing season. The net CO₂ assimilation by one-year-old shoots over a growing season constituted 9.9 and 2.4 g CO₂/(g dry wt year) in the upper and lower crown parts, respectively. The nighttime respiration of the current-season shoots released 0.7 g CO₂/(g dry wt year), and the respiration of one-year-old shoots in the upper and lower parts of the canopy released 0.45 and 0.36 g CO₂/(g dry wt year), respectively. Recalculation of three-year data per entire crown yielded an annual average carbon assimilation of 1.54 g C/(g dry wt year). Water relations markedly affected CO₂ fixation. Nevertheless, the daily average values of photosynthesis in the summer were similar for pine stands with bilberry, heather, and dwarf shrub–polytric as understory dominants. It is shown that the realization of changes in photosynthetic function is related in time to growth processes.     

Influence of Glucose and Saturated Free-Fatty Acid Mixtures on Citric Acid and Lipid Production by Yarrowia lipolytica

In the present report, the effect of glucose and stearin (substrate composed by saturated free-fatty acids) on the production of biomass, reserve lipid, and citric acid by Yarrowia lipolytica ACA-DC 50109 was investigated in nitrogen-limited cultures. Numerical models that were used in order to quantify the kinetic behavior of the above Yarrowia lipolytica strain showed successful simulation, while the optimized parameter values were similar to those experimentally measured and the predictive ability of the models was satisfactory. In nitrogen-limited cultures in which glucose was used as the sole substrate, satisfactory growth and no glucose inhibition occurred, although in some cases the initial concentration of glucose was significantly high (150 g/l). Citric acid production was observed in all trials, which was in some cases notable (final concentration 42.9 g/l, yield 0.56 g per g of sugar consumed). The concentration of unsaturated cellular fatty acids was slightly lower when the quantity of sugar in the medium was elevated. In the cases in which stearin and glucose were used as co-substrates, in spite of the fact that the quantity of cellular lipid inside the yeast cells varied remarkably (from 0.3 to 2.0 g/l – 4 to 20% wt/wt), de novo fatty acid biosynthesis was observed. This activity increased when the yeast cells assimilated higher sugar quantities. The citric acid produced was mainly derived from the catabolism of sugar. Nevertheless, citric acid yield on sugar consumed and citrate specific production rate, as evaluated by the numerical model, presented substantially higher values in the fermentation in which no fat was used as glucose co-substrate compared with the cultures with stearin used as co-substrate.     

A High Performance Fermentation Process for Citric Acid Production by Aspergillus niger NGGCB-101, Using Vermiculite as an Additive in a Stirred Bioreactor

The present study describes the use of vermiculite for enhanced citric acid productivity by a mutant strain of Aspergillus niger NG^GCB-101 in a stirred bioreactor of 15.0 l capacity. The maximum amount of citric acid (96.10 g/l) was obtained with the control 144 h after mycelial inoculation. To enhance citric acid production, varying levels of vermiculite were added as an additive into the fermentation medium. The best results were observed when 0.20 g/l vermiculite was added into the medium 24 h after inoculation resulting in the production of 146.88 g citric acid monohydrate/l. The dry cell mass and residual sugar were 11.75 and 55.90 g/l, respectively. Mixed mycelial pellets (1.08–1.28 mm, dia) were observed in the fermented culture broth. When the culture grown at different vermiculite levels was monitored for Q _p , Q _s and q _p , there was a significant enhancement (P 0.05) in these variables over the control (vermiculite-free). Based on these results, it is concluded that vermiculite might affect mycelial morphology and subsequent TCA cycle performance to improve carbon source utilization by the mould, basic parameters for high performance citric acid fermentation.     

Citric acid production from glucose. I. Growth and excretion kinetics in a stirred fermentor

The growth and citric acid production kinetics of Saccharomycopsis lipolytica on glucose are investigated in an aerated stirred fermentor. Cellular growth first proceeds exponentially until exhaustion of ammonia in the fermentation medium. Cells then continue to grow at a reduced rate with a concomitant decrease in intracellular nitrogen content. Citric and isocitric acid production starts at the end of the growth phase. During about 80 hr excretion proceeds at a constant rate of 0.7 g/liter/hr for citric acid and 0.1 g/liter/hr for isocitric acid. The final citric and isocitric acid concentrations are 95 and 10g/liter, respectively. During acid excretion cellular respiration accounts for 60 and 35% of consumed oxygen and glucose. Both acid and CO₂ production rates follow a Michaelis–Menten-type dependence on oxygen concentration with Michaelis–Menten constants of 0.9 and 0.15 mg/liter for acid and CO₂ productions, respectively.     

The influence of type and concentration of the carbon source on production of citric acid by Aspergillus niger

Summary The influence of various carbon sources and their concentration on the production of citrate by Aspergillus niger has been investigated. The sugars maltose, sucrose, glucose, mannose and fructose (in the given order) were carbon sources giving high yields of citric acid. Optimal yields were observed at sugar concentrations of 10% (w/v), with the exception of glucose (7.5%). No citric acid was produced on media containing less than 2.5% sugar. Precultivation of A. niger on 1% sucrose and transference to a 14% concentration of various other sugars induced citrate accumulation. This could be blocked by the addition of cycloheximide, an inhibitor of de novo protein synthesis. This induction was achieved using maltose, sucrose, glucose, mannose and fructose, and also by some other carbon sources (e.g. glycerol) that gave no citric acid accumulation in direct fermentation. Precultivation of A. niger at high (14%) sucrose concentrations and subsequent transfer to the same concentrations of various other carbohydrates, normally not leading to citric acid production, led to formation of citrate. Endogenous carbon sources were also converted to citrate under these conditions. A 14%-sucrose precultivated mycelium continued producing some citrate upon transfer to 1% sugar. These results indicate that high concentrations of certain carbon sources are required for high citrate yields, because they induce the appropriate metabolic imbalance required for acidogenesis.     

Selection of a strain of Aspergillus for the production of citric acid from pineapple waste in solid-state fermentation

Aspergillus foetidus ACM 3996 (=FRR 3558) and three strains of Aspergillus niger ACM 4992 (=ATCC 9142), ACM 4993 (=ATCC 10577), ACM 4994 (=ATCC 12846) were compared for the production of citric acid from pineapple peel in solid-state fermentation. A. niger ACM 4992 produced the highest amount of citric acid, with a yield of 19.4g of citric acid per 100g of dry fermented pineapple waste under optimum conditions, representing a yield of 0.74g citric acid/g sugar consumed. Optimal conditions were 65% (w/w) initial moisture content, 3% (v/w) methanol, 30°C, an unadjusted initial pH of 3.4, a particle size of 2mm and 5ppm Fe²⁺. Citric acid production was best in flasks, with lower yields being obtained in tray and rotating drum bioreactors.     

Malonomonas rubra gen. nov. sp. nov., a microaerotolerant anaerobic bacterium growing by decarboxylation of malonate

From anoxic marine sediment samples, new anaerobic, microaerotolerant, Gram-negative, non-sporeforming bacteria were isolated which grew in mineral medium with malonate as sole source of carbon and energy. Cells were motile thin rods, often forming large aggregates. Malonate was decarboxylated to acetate with concomitant growth yields of 1.9–2.1 g dry cell matter per mol malonate degraded. Fumarate and malate were fermented to succinate and CO₂. No other substrates were used. No inorganic electron acceptors were reduced. At least 150 mM NaCl was required for growth with either substrate. High amounts of a periplasmic cytochrome c were detected, as well as small amounts of a membrane-bound cytochrome b. All enzymes of the citric acid cycle were found to be present. The DNA base ratio was 48.3 mol% guanine plus cytosine. Since this new bacterium cannot be affiliated with any of the known genera and species, a new genus and species, Malonomonas rubra is proposed.     

Accumulation and partial re-consumption of polyols during citric acid fermentation by Aspergillus niger

Summary Quantitative balances have been made for sugar and oxygen uptake rates during citric acid accumulation by Aspergillus niger: during the first phase of citric acid accumulation (up to 130 h) more sugar is taken up than the production of biomass, CO₂ and citric acid account for. In contrast, during later phases of fermentation more citric acid, CO₂ and biomass are formed than sugar uptake would theoretically allow. A similar pattern is obtained for oxygen uptake, where less uptake occurs during the early phase of fermentation than needed for complete balance, and the reverse is observed during the late stage of fermentation. It could subsequently be shown that this is caused by the intermediate accumulation and partial re-consumption of a number of polyhydric alcohols (glycerol, arabitol, erythritol and mannitol) during citric acid fermentation.Dedicated to Professor H. J. Rehm on the occasion of his 60th birthday with kind regards     

Anaerobic degradation of toluene in denitrifying Pseudomonas sp.: indication for toluene methylhydroxylation and benzoyl-CoA as central aromatic intermediate

The anaerobic degradation of toluene has been studied with whole cells and by measuring enzyme activities. Cultures of Pseudomonas strain K 172 were grown in mineral medium up to a cell density of 0.5 g of dry cells per liter in fed-batch culture with toluene and nitrate as the sole carbon and energy sources. A molar growth yield of 57 g of cell dry matter formed per mol toluene totally consumed was determined. The mean generation time was 24 h. The redox balance between toluene consumed (oxidation and cell material synthesis) and nitrate consumed (reduction to nitrogen gas and assimilation as NH₃) was 77% of expectation if toluene was completely oxidized; this indicated that the major amount of toluene was mineralized to CO₂. It was tested whether the initial reaction in anaerobic toluene degradation was a carboxylation or a dehydrogenation (anaerobic hydroxylation); the hypothetical carboxylated or hydroxylated intermediates were tested with whole cells applying the method of simultanous adaptation: cells pregrown on toluene degraded benzyl alcohol, benzaldehyde, and benzoic acid without lag, 4-hydroxybenzoate and p-cresol with a 90 min lag phase and phenylacetate after a 200 min lag phase. The cells were not at all adapted to degrade 2-methylbenzoate, 4-methylbenzoate, o-cresol, and m-cresol, nor did these compounds support growth within a few days after inoculation with cells grown on toluene. In extracts of cells anaerobically grown on toluene, benzyl alcohol dehydrogenase, benzaldehyde dehydrogenase, and benzoyl-CoA synthetase (AMP forming) activities were present. The data (1) conclusively show anaerobic growth of a pure culture on tolucne; (2) suggest that toluene is anaerobically degraded via benzoyl-CoA; (3) imply that water functions as the source of the hydroxyl group in a toluene methylhydroxylase reaction.