Large-scale production of bacterial biomass from methanol for use as milk-replacer

Summary A mixed methanol-utilizing bacterial culture was utilized to produce bacterial biomass as milk replacer. This culture comprised three pure strains: KISRI-5 (NCIB 12135), KISRI-512 (NCIB 12137) and KISRI-5112 (NCIB 12138). Optimal concentrations of methanol (15 g 1^–1) and medium elements as well as optimal growth conditions, e.g., pH (6.8), temperature (38°C), dissolved oxygen and dilution rate, were established. The maximum biomass yield coefficient obtained under optimized conditions was 0.48 g g^–1.· Large-scale production was successfully carried out in a 1500 1 fermenter under chemostat conditions. A good product was obtained having high true protein content (59–62%) and low polysaccharides (5%) without microbial contamination.     

The use of a mixed culture of Geotrichum candidum,Candida krusei and Hansenula anomala for microbial protein production from whiskey distillery spent wash

Summary The use of a mixed culture of Geotrichum candidum, Hansenula anomala and Candida krusei for microbial protein production and treatment of whiskey distillery spent wash was investigated in both batch and continuous culture. Although capable of assimilating the same substrate constituents in pure culture the organisms used different constituents for growth in mixed continuous culture, allowing a stable mixed population to be established. The relative proportions of the three organisms in the population was dependent on the dilution rate.The mixed culture did not give superior yields or productivities, but did give proportionally greater COD reduction and would be expected to bemore stable and resistant to contamination.At a dilution rate of 0.10 h^–1 the mixed culture gave a biomass yield of 4.8 g l^–1, containing 55% crude protein, with a COD reduction of 31.5%, while in mixed batch culture a biomass yield of 12.5 g l^–1, containing 48% crude protein, was obtained with a COD reduction of 54.9%.     

Lipid accumulation inRhodotorula glutinis on sugar cane molasses in single-stage continuous culture

Microbial lipids produced byRhodotorula glutinis grown in continuous culture with molasses under nitrogen-limiting conditions were evaluated and the effects of growth rate on fatty acid composition were studied. As the growth rate decreased, cell biomass, lipid content and lipid yield gradually increased. The maximum lipid content recorded was 39% (w/w) of dry cell biomass at a dilution rate of 0.04 h^–1. The growth rate also affected fatty acid composition: oleic acid decreased with decreasing growth rate while stearic acid increased.     

Mass cultivation of the nitrogen-fixing cyanobacteriumGloeotrichia natans,indigenous to rice-fields

Gloeotrichia natans, a nitrogen fixing cyanobacterium common in rice fields in the Philippines, was used for studies to establish key features of its physiology and potential production in outdoor cultures. Under optimal growth conditions (38 °C, pH 8.0, no carbon enrichment) the specific growth rate of rice-field isolate was 0.076 h^–1. The pH of the medium (between 6.5 and 9.0) did not influence the growth rate, but it did affect phycobiliprotein content, as reflected by a change in colour. At pH 7.0 the culture was green-brown, with phycobiliproteins constituting up to 10% of the total protein, while at pH 9.0 the culture was brownish-black and the pigment content was as high as 28% of the total protein. In outdoor cultures the specific growth rate was related directly to cell density in the range of 0.7–1.5 g dry weight 1^–1 at a rate of stirring of 30 rpm, and inversely related to cell density at half this rate. At a stirring of 30 rpm, daily production of outdoor cultures harvested to maintain cell densities of 0.7, 1.15 andw 1.5 g 1^–1 were 14.7, 17.1 and 18.1 g m^–2 d^t, respectively. This rate of production was maintained for more than 45 days. Phycobiliprotein content in the culture kept at a density of 1.5 g 1^–1 reached 14% of the total biomass.     

Production of extracellular lactase fromFusarium moniliforme

Summary A strain ofFusarium moniliforme, previously used for microbial protein production, excreted lactase (-D-galactosidase, EC.3.2.1 23) when cultivated either in a whey liquid medium or on a wheat bran solid medium. The enzyme produced in both media had pH and temperature optima of 4–5 and 50–60°C respectively and was particularly suitable for processing acid whey.In the whey culture, maximum lactase yield was observed after 95 h of growth at 30°C and whey lactose concentration of 9%. The addition of ammonium, potassium and sodium ions to the growth medium considerably enhanced lactase production. A maximum enzyme yield corresponding to hydrolysis of 3 nmoles o-nitrophenyl--D-galactopyranoside sec^–1 ml^–1 of growth medium, at pH 5 and 60°C, was obtained.In the wheat bran culture, the maximum enzyme yield was obtained after 140 h of growth at 28–30°C. A marked increase in the enzyme production was observed when nitrate or phosphate was added to the growth medium. Also, the addition of certain agricultural by-products (molasses, whey) enhanced lactase production. The observed maximum yield corresponding to the hydrolysis of 182 nmoles of ONPG sec^–1 g^–1 of wheat bran, at pH 5 and 60°C, is comparable to that reported for certain microorganisms used commercially for lactase production.     

Relationships between nucleic acid,nitrogen, and growth rate of tobacco cells in suspension culture

Summary Changes in the amount of nucleic acid and nitrogen, and the relationships between these amounts and the growth rate of tobacco cells (Nicotiana tabacum L. cv. Bright Yellow-2) at different initial nitrogen concentrations in the medium, were examined in batch cultures. During culture in basal medium, the amount of intracellular nucleic acid expressed per unit of dry biomass was 36.3 mg RNA g^–1 cell and 8.1 mg DNA g^–1 cell at the beginning of batch culture. These values increased 2.5 fold for RNA and 1.5 fold for DNA during the exponential growth phase and then gradually decreased with the decline in the growth rate. Similar changes were also observed in the medium containing less nitrogen. The specific growth rate, (day^–1), of the culture corresponded to the magnitude of the intracellular RNA content (mg RNA g^–1 cell), and the linear relationship, RNA=38+23 was obtained. In addition, there were remarkable positive correlations between the total and protein nitrogen, and during the cultures. The mononucleotide composition of total RNA (AMP+UMP)/(GMP+CMP) which was suggested to be a convenient index of metabolic activity was nearly constant (0.78 to 0.80) during tobacco cell culture in the basal medium.     

Enhanced anaerobic degradation of benzene by enrichment of mixed microbial culture and optimization of the culture medium

A heterogeneous mixed culture, originally collected from two different sources, namely cow-drug and sludge from the mineral medium containing 1% glucose and then adapted on benzene as the carbon and energy source. Under anaerobic conditions benzene was degraded via benzoic acid as a major intermediate in the benzene degradation pathway. The degradation rate of benzene was improved stepwise by the number of enrichments and optimization of the culture medium. The effects of microaerobic conditions and/or physicochemical treatment with H₂O₂ prior to anaerobic degradation were studied with respect to variations in benzene degradation rate, growth of biomass and gas produced is less than the theoretical value expected and the percentage of methane in the product gas was very small (3%–3.5%). The reason for this is not well understood but it is presumed that the major group of benzene-degrading bacteria present in the culture medium are sulphate reducers and the mixed consortium is unable to degrade certain complex aromatic intermediates in the benzene degradation pathway under the experimental conditions. For an actual explanation of the situation arising in this study, further investigations must be carrie out. However, the mixed culture is capable of oxidizing benzene more rapidly to intermediate compounds and also partly into gas under the culture conditions, compared to the published data for the anaerobic degradation of benzene.     

Influence of dilution rate on the morphology and technological properties ofLactobacillus delbrueckii subsp.bulgaricus

Lactobacillus delbrueckii subsp.bulgaricus ATCC 11842 was grown in a chemostat at 45°C and pH 5.5 using glucose as the carbon source, with the aim of optimizing biomass production. Cells were grown in a complex medium under nitrogen. At dilution rates lower than 0.18h^–1, it was difficult to keep steady-state conditions and pleomorphic forms were observed. The addition of 30mM Ca²⁺ and Mn²⁺ reverted the cells to normal shape: 30mM Mg²⁺ had no effect. Increasing the dilution rate resulted in normal morphology without the addition of any cations. Under these conditions, a maximum productivity of 1.24g dry biomass 1^–1 h^–1 was obtained. The maximum growth yield, corrected for maintenance, was 30g biomass mol^–1 glucose and the maintenance energy was 0.26g glucose g^–1 biomass h^–1. Lactate was the main fermentation product at all glucose concentrations used in the fed medium. Cells grown at high dilution rates had normal technological properties (acid production and proteolysis) when tested in milk.     

A novel thermotolerant methylotrophicBacillus sp. and its potential for use in single-cell protein production

A thermotolerant methylotrophicBacillus sp. (KISRI TM1A, NCIMB 40040), isolated from the Kuwaiti environment and belonging to the group II spore-forming, bacilli, could not be correlated with any knownBacillus sp. It may, therefore, be a new species. It grew at temperatures from 37° to 58°C from pH 6.5 to 9.0 and on methanol up to 40 g l^–1. It grew well in a chemostat. Its biomass yield coefficient was improved by about 30% by optimization of medium and growth conditions, reaching a maximum of 0.44g g^–1 at 45°C pH 6.8 to 7.0, dilution rate 0.25 h^–1 with methanol at 10 g l^–1. Average crude protein and amino acid content were 84% and 60%, respectively, and maximum productivity attained under laboratory conditions was 5.06 g l^–1h^–1. It was concluded that this strain has good potential for use in single-cell protein production.     

Model aided design of repeated fed-batch penicillin fermentation

Structured models of antibiotic fermentation that quantify maturation and aging of product forming biomass are fitted to experimental data. Conditions of superiority of repeated fed batch cultivation are characterized on the basis of a performance criterion that includes penicillin productivity and costs of operation. Emphasis is placed on the relevance of such research to the model aided design of optimal cyclic operation.List of Symbols c IU/mg cost factor - D s^–1 dilution rate - J IU · cm^–3 · h^–1 net productivity - k _p IU · mg^–11 · h^–1 specific product formation rate - k _pm IU · mg^–1 · h^–1 maximum specific product formation rate - p IU/cm³ concentration of penicillin - T s final time of fermentation - t s fermentation time - X kg/m³ concentration of biomass dry weight - X ₁kg/m³ concentration of young, immature biomass - X ₂ kg/m³ concentration of mature product forming biomass - X _c kg/m³ biomass concentration of the end of growth phase - X _mkg/m³ maximum biomass concentration Greek Letters s^–1 specific maturation rate - s^–1 specific aging rate - s^–1 specific growth rate - _m s^–1 maximum specific growth rate - _p s^–1 specific growth rate during the product formation phase - s cycle time - % volume fraction of draw-off Abbreviations CC chemostat culture - RFBC repeated fed batch culture - RBC repeated batch culture     

Formation of phenylethanoid glycosides by Cistanche deserticola callus grown on solid media

The optimal growth of Cistanche deserticola callus and formation of phenylethanoid glycosides (PeG) was at 25°C with light irradiation intensity of 24 mol m^–2 s^–1 on solidified B5 media supplemented with 0.5 mg 6-benzylaminopurine l^–1, 10 mg gibberellin l^–1, 800 mg casein hydrolysate l^–1 and 20 g sucrose l^–1. After 30 d culture, the biomass reached 15.5 g dry wt callus l^–1 medium and its PEG content was 10.7% (w/w). The PeG content was 42%–127% higher than those in explants.     

Protein production from whey usingPenicillium cyclopium; growth parameters and cellular composition

Summary The growth parameters ofPenicillium cyclopium have been evaluated in a continuous culture system for the production of fungal protein from whey. Dilution rates varied from 0.05 to 0.20 h^–1 under constant conditions of temperature (28°C) and pH (3.5). The saturation coefficients in the Monod equation were 0.74 g l^–1 for lactose and 0.14 mg l^–1 for oxygen, respectively. For a wide range of dilution rates, the yield was 0.68 g g^–1 biomass per lactose and the maintenance coefficient 0.005 g g^–1 h^–1 lactose per biomass, respectively. The maximum biomass productivity achieved was 2 g l^–1 h^–1 biomass at dilution rates of 0.16–0.17 h^–1 with a lactose concentration of 20 g l^–1 in the feed. The crude protein and total nucleic acid contents increased with a dilution rate, crude protein content varied from 43% to 54% and total nucleic acids from 6 to 9% in the range of dilution rates from 0.05 to 0.2 h^–1, while the Lowry protein content was almost constant at approximately 37.5% of dry matter.Nomenclature (mg l^–1) C_o initial concentration of dissolved oxygen - (h^–1) D dilution rate - (mg l^–1) K₀₂ saturation coefficient for oxygen - (g l^–1) K_s saturation coefficient for substrate - (g g^–1 h^–1) lactose per biomass) m maintenance energy coefficient - (mM g^–1 h^–1O₂ per biomass) Q₀₂ specific oxygen uptake rate - (g l^–1) S residual substrate concentration at steady state - (g l^–1) S_o initial substrate concentration in feed - (min) t_1/2 time when C_o is equal to C_o/2 - (g l^–1) X biomass concentration - (g l^–1) X biomass concentration at steady state - (g g^–1 biomass per lactose) Y_G yield coefficient for cell growth - (g g^–1 biomass per lactose) Y_x/s overall yield coefficient - (h^–1) specific growth rate     

Biomass and icosapentaenoic acid productivities from an outdoor batch culture of Phaeodactylum tricornutum UTEX 640 in an airlift tubular photobioreactor

An outdoor tubular photobioreactor, based on an external-loop airlift, has been used to cultivate the diatom Phaeodactylum tricornutum UTEX 640, rich in icosapentaenoic acid (EPA). The system was operated in batch mode. The specific growth rate in the exponential phase, averaged over the whole day, was 0.254 day^–1 · A 25 gl^–1 maximum biomass concentration was obtained by the end of the culture period. The dissolved oxygen generated never rose over 210% of air saturation nor was there impediment to biomass generation or inhibition of photosynthesis. Furthermore, around 2 × 10¹⁷ quanta cm^–2 s^–1 light intensity seemed to saturate the photosynthetic activity. Biomass losses during the night could be reduced to less than 5% by maintaining the culture at 16°C at night. The maximum concentration and productivity of EPA were 423 mgl^–1 and 13 mgl^–1 day^–1, respectively, at the end of the culture, although a 21-mgl^–1 day^–1 net EPA generation rate was obtained at the linear growth phase.     

Production of high yields of docosahexaenoic acid byThraustochytrium roseum ATCC 28210

Culture conditions for growth and docosahexaenoic acid (DHA) production byThraustochytrium roseum ATCC 28210 were investigated with a view to increasing DHA titers. A medium was formulated (Medium 6) which produced a biomass and DHA content of 10.4 g L^–1 and 1011 mg L^–1, respectively, in a 5-day incubation. A fed-batch culture system was also developed which achieved biomass and DHA titers of 17.1 g L^–1 and 2000 mg L^–1, respectively, in 12 days.     

Quantitative physiology of Penicillium cyclopium grown on whey for production of microbial protein

Summary A filamentous fungus Penicillium cyclopium, capable of growing on deproteinized whey was isolated and characterized for the purpose of production of microbial protein.This organism has a maximum specific growth rate of 0.2 h^–1 at pH 3.0 to 4.5 and 28°C in a medium containing only ammonium nitrogen and deproteinized whey. The yield coefficients are 0.68 g biomass/g lactose, 12.0 g biomass/g nitrogen, and 2.10 g biomass/g oxygen, respectively.Crude protein and total nucleic acid contents of this organism are 47.5% and 7.4% (dry cell weight basis), respectively. The profile of essential amino acids shows that it could be a good source of animal feed or food protein.     

Bioreactor culture of heterotrophic sandalwood (Santalum album L.) cell suspensions utilizing a cell-lift impeller

Summary Growth and phenolic production by two heterotrophic suspension cultures (SW-1 and SW-2) of sandalwood cultivated in a 2.5 L bioreactor were investigated. Cultures of SW-1 cell suspensions resulted in a maximum phenolic content of 32.5 mg L^–1 compared to 12.5 mg L^–1 produced by SW-2 cell suspensions. Fresh weight doubling time (Td) was 5.8 days and the specific growth rate () was 0.12 d^–1 during exponential growth for both cell lines. The pH of the culture medium decreased from 5.5 to 3.5 during the exponential growth phase of SW-1 and SW-2 cell suspensions. The dissolved oxygen content also dropped steadily during culture and remained at 40% throughout exponential growth phase. These results should provide a basis for developing sandalwood cell cultures for bioproduction of useful compounds.Abbreviations 2,4-D: 2–4 dichlorophenoxyacetic acid - BA N⁶-benzyladenine - Eh Medium oxidation-reduction potential - K_La Oxygen transfer coefficient - MS Murashige and Skoog (1962) basal medium - SW-1 and SW-2 Sandalwood suspension lines     

Effect of growth rate on the eicosapentaenoic acid and docosahexaenoic acid content of Isochrysis galbana in chemostat culture

An isolate of Isochrysis galbana rich in eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) has been grown as a chemostat culture at 20° C and pH 8.00 controlled by CO₂ injection. From a low dilution rate (D) of 0.0024 h^–1 to 0.0377 h^–1, close to maximum growth, a decrease in EPA content from 5.21% dry weight (d.w.) to 2.80% d.w. was observed, although the percentage of EPA in the total fatty acids increased. Lipids were fractionated, EPA being the major fatty acid found in the glycolipid fraction, whereas in the neutral lipid fraction were mainly palmitic and palmitoleic acids. At the same time, the biomass concentration also decreased from 1015 mg·l^–1 to 202 mg·l^–1 over the range of Ds mentioned. Nonetheless, EPA productivity had a maximum value of 15.26 mg·l^–1·day^–1 at D=0.0208 h^–1.Correspondence to: E. Molina Grima     

Continuous culture of Streptococcus cremoris on lactose using various medium conditions

Summary Growth of a lactic streptococcus was studied in continuous cultures, under various conditions of medium richness, without carbon source limitation, and with a large range of dilution rates. Increasing the concentrations of growth factors and protein nitrogen sources resulted in increased volumetric productivities of biomass and lactic acid with maximum values in the 0.3–0.4 h^–1 dilution rate range. Growth was shown to be dependent on both the inhibitory effect of lactic acid and the availability of certain nutrients, as has previously been shown for batch cultures.Offprint requests to: A. Pareilleux     

Effect of nutrient deficiency on accumulation and relative molecular weight of poly-β-hydroxybutyric acid by methylotrophic bacterium,Pseudomonas 135

Summary Pseudomonas 135, a facultative methylotroph, was cultivated on methanol as a sole carbon and energy source for the accumulation of poly--hydroxybutyric acid (PHB). The cells grew fairly well on minimal synthetic medium containing 0.5% (v/v) of methanol at pH 7.0 and 30° C. The maximum specific growth rate was determined to be 0.26–0.28 h^–1 with a growth yield of 0.38 in the optimized growth medium. For stimulation of PHB accumulation in the cells, deficiency of nutrients such as NH _inf4 ^sup+ , Mg²⁺ and PO _inf4 ^sup3– was crucial even though cell growth was significantly suppressed. The PHB content of a 40-h culture was determined to be 37% of the total cell mass in NH _inf4 ^sup+ -limited medium, 42.5% on Mg²⁺-deficient medium, and 34.5% on PO _inf4 ^sup3– -deficient medium. The maximum content of PHB in the cells could reach 55% in NH _inf4 ^sup+ -limited fed-batch culture. The average relative molecular eight determined by gel permeation chromatography was 3.7 × 10⁵ in NH _inf4 ^sup+ -limited culture, 2.5 × 10⁵ in Mg²⁺-deficientmedium, and 3.1 × 10⁵ in PO _inf4 ^sup3– -deficient medium. Polydispersity determined in each culture was relatively high (about 10–11). The solid PHB had a melting temperature of 173° C. Correspondence to: J. M. Lebeault     

Interactions in a mixed culture composed of Cellulomonas flavigena and Xanthomonas sp. growing in continuous culture on sugar cane bagasse

A mixed culture formed by Cellulomonas flavigen and Xanthomonas sp. was able to grow in continuous culture in a medium with sugar cane bagasses as carbon source without additional growth factors. The effect of temperature, pH and dilution rate (D) upon the microbial population ratio and biomass composition was studied. Both microorganisms co-existed between 30 and 40° C, at pH 5.8–7.0 and D = 0.04–0.11 h^–1. Correspondence to: T. Ponce-Noyola