Production of Candida utilis protein from peat extracts

Sphagnum peat extracts or hydrolysates have been obtained and used as a culture medium for the production of Candida utilis biomass as single cell proteins. Acid hydrolysis of ground peat (4–60 mesh) in an autoclave operated under a set of conditions for acid strength (0.3-1.5 (v/v) H₂SO₄), holding time (1–4 hr), temperature (100–165°C), and weight ratio of dry peat to solution (3.3–16.7 g dry peat/100 g solution) yielded carbohydrate-rich extracts of different concentrations (1–34g/liter). The best yield (mg total carbohydrate/g dry peat) was obtained for a holding time of I hr and a temperature of 152°C. Low peat concentratio (4.1 g dry peat/100 g solution)resulted in high yield(280mg total carbohydrate/gdry peat) with a corresponding low carbohydrate content in hydrolysate (13 g/liter), while a lower yield with a higher carbohydrate content (34 g/liter)in hydrolysate were found when increasing peat concentration (16.7 g dry peat/100 g solution). Shake-fladk experiments using peat hydrolysates as the culture medium together with NH₄OH (～4.8 g/liter) and K₂HPO₄(5 g/liter) as nitrogen and phosphate supplement, respectively, gave a maximum biomass concentration of 7.5 g/liter after 60 hr at 30°C and 200rpm. Batch cultivation in a fermentor under controlled conditions for aeration (4.2 liter/min), agitation (500rpm), temperature (30°C), and pH (5.0) produced a maximum biomass of 10 g/liter after 20 hr with a specific growth rate of 0.13 hr^?1. For the continuous cultivation, a maximal biomass productivity of 1.24 g/gliter-he was obtained at a dilution rate of 0.125 hr ^?1. Monod's equation's equation has been used for the estimation of the coefficients μ_Max, K_s, and Y. It was found that the yield coefficient Y is not constant during the progress of batch cultivation.     

Identification of Flavone Compounds in Eighteen Gramineae Species

Conditions for tryptophan synthesis from pyruvic acid, indole and NH₄Cl by Enterobacter aerogenes AHU 1540 having a high tryptophanase activity, were investigated using a reaction mixture containing 1.7% of pyruvic acid. Under optimum conditions, 16.4g/liter of tryptophan was accumulated after 24 hr of incubation.

Agaricus campestris AHU 9382 produced pyruvic acid in amounts of 22 ~ 26.5 g/liter from 5% of glucose after 3-days shaking culture. When E. aerogenes was added to this fermentation broth together with indole and NH₄Cl, pyruvic acid produced was rapidly converted to tryptophan and yields of tryptophan as high as 15 g/liter were obtained after 12 hr of incubation. Furthermore, pyruvic acid fermentation by Saccharomyces exiguus AHU 3110 or Corynebacterium sp. 37-3A could also be used as a pyruvic acid source for subsequent tryptophan production.     

Production of microbial protein from tree bark by Phanerochaete chrysosporium

Phanerochaete chrysosporium was grown in fermentors on NaOH-extracted maple, pine, and cedar barks at the optimum substrate concentration of 1% (w/v). The yields (mg protein/liter) on maple, pine, and cedar were 1500, 1200, and 880, respectively, which are probably due to the different lignin contents of the barks. Lignin is not utilized. The productivities at 30°C obtained for pine (4.07 × 10^?2 g protein/liter hr) and cedar (2.63 × 10^?2 g protein/liter hr) barks were greater than for maple (2.63 × 10^?2 g protein/liter hr). The substrate (bark) was the limiting component of the fermentation. Over the 26–38°C temperature range protein productivity increased by a factor of three (1.55 × 10^?2 vs. 4.61 × 10^?2 g protein/liter hr) for maple bark. Low agitation rates resulted in an overproduction of cellulase and reduced levels of microbial protein.     

Increased production of cellulase of Trichoderma sp. by pH cycling and temperature profiling

Cultivation of Trichoderma reesei QM 9414 on 3% (w/v) cellulose medium (C/N ratio = 8.5) produced 4.5 IU/ml celulase 180 hr at a cell growth of 8.0 g/liter (0.266 g cell/g cellulose). It corresponded to an average cellulase productivity 25.0 IU/liter/hr (3.5 IU/g cell/hr). In the same medium 9.5 g/liter cell mass (0.316 g cell/g cellulose), 6.2 IU/ml cellulase, and 38.75 IU/liter/hr (4.0 IU/g cell/hr) cellulase productivity could be obtained using pH cycling condition during cultivation. Cell mass, cellulase yield, and productivity were further increased to 10.0 g/liter, 7.2 IU/ml, and 44.0 IU/liter/hr (4.5 IU/g cell/hr), respectively, by simultaneous pH cycling and temperature profiling strategy. Results are described.     

A SEAWATER MEDIUM FOR DINOFLAGELLATES AND THE NUTRITION OF CACHONINA NIEI1

Two media have been devised: an enriched seawater medium for culture of dinoflagellates and a defined medium for rapid growth of the dinoflagellate Cachonina niei. A wide range in salinity (10.23–42.38 g/liter NaCl) is tolerated by C. niei. Below 0.6 g/liter MgSO₄, 0.19 g/liter KCl, and 0.22 g/liter CaCl₂, the generation time greatly increases. Increase in MgSO₄ to 7.22 g/liter, KCl to 1.12 g/liter or CaCl₂ to 2.22 g/liter has little effect on generation time. The temperature optimum is 19–23 C. Saturating light intensity for growth is 1000 ft-c and for photosynthesis (determined manometrically) is slightly less than 2000 ft-c. Cachonina niei requires B₁₂ and thiamin. Neither silicate nor its competitive inhibitor germanate affects generation time or cell yield indicating silicon is not required. Of a variety of buffers tested, Tris is the best. Optimal growth occurs at pHs of 7.5–8.3. Glycerol is inhibitory and does not support dark growth.     

Formation of higher alcohols and phenol by strains of zymomonas sp.

Strains of the bacteria Zymomonas sp. were studied for their ability to form higher alcohols. In a complex growth medium, six strains were shown to produce significant amounts of 1-propanol, 1-butanol, 2-methyl-1-butanol, 3-methyl-1-butanol, 2-methyl-2-butanol, pentanols, secondary hexyl-alcohols, and trace amounts of n-hexanol. When resting cells of these organisms were placed into a fermentation medium containing glucose and Tris-buffer, Z. mobilis 8938 produced increased levels of 1-butanol, and secondary hexyl-alcohols at concentrations of 13.5 mg/liter and 5.8 mg/liter, respectively. Another strain, Z. mobilis subsp. mobilis B 806, stimulated the formation of 1-propanol and 1-butanol at concentrations of 14.9 mg/liter and 23.52 mg/liter, respectively. Amino acids or amino acid precursors were then added to the fermentation medium. The presence of threonine and α-ketobutyric acid stimulated Z. mobilis 8938 to produce 82.6 mg/liter secondary hexyl-alcohols and 8.0 mg/liter n-hexanol, respectively. Isoleucine and valine increased the production of 2-methyl-1-butanol (394.0 mg/liter) and 3-methyl-1-butanol (113.4 mg/liter), respectively, by Z. mobilis subsp. mobilis B 806. Glutamine enhanced the formation of 2-methyl-2-butanol production to concentrations 38.8 mg/liter in Zymomonas strain B 806. Additional experiments suggested that higher alcohol production could also be accomplished in the absence of glucose when cells were allowed to metabolize the precursors only. The effect of aromatic amino acids on phenol production was determined using resting cells of Zymomonas sp. The maximum yield of phenol (111.6 mg/liter) was found by Zymomonas strain 8938 in the presence of tyrosine. The addition of phenylalanine also stimulated this strain to form 71.4 mg/liter of phenol.     

Biomethanation: Anaerobic fermentation of CO2, H2 and CO to methane

Studies to examine the microbial fermentation of coal gasification products (CO₂, H₂ and CO) to methane have been done with a mixed culture of anaerobic bacteria selected from an anaerobic sewage digestor. The specific rate of methane production at 37°C reached 25 mmol/g cell hr. The stoichiometry for methane production was 4 mmol H₂/mol CO₂. Cell recycle was used to increase the cell concentration from 2.5 to 8.3 g/liter; the volumetric rate of methane production ran from 1.3 to 4 liter/liter hr. The biogasification was also examined at elevated pressure (450 psi) and temperature to facilitate interfacing with a coal gasifier. At 60°C, the specific rate of methane production reached 50 mmol/g cell hr. Carbon monoxide utilization by the mixed culture of anaerobes and by a Rhodopseudomonas species was examined. Both cultures are able to carry out the shift conversion of CO and water to CO₂ and hydrogen.     

Effect of Electrical Conductivity and Silicate on Infection of Basil with Colletotrichum gloeosporioides in Soilless Culture

The aim of this study was to evaluate the effect of potassium silicate administration and of electrical conductivity of nutrient solution in three experiments against Colletotrichum gloeosporioides infection on basil (Ocimum basilicum L. cv Genovese Gigante) grown in a closed soilless system. Potassium silicate was added at 100 mg/l of nutrient solution at three different levels of electrical conductivity: 1.5–1.6 mS/cm (E.C.1), 3–3.2 mS/cm (E.C.2, 0.70 g/l NaCl) and 4–4.2 mS/cm (E.C.3, 0.95 g/l NaCl). Basil plants were inoculated with C. gloeosporioides spores 21–31 days after sowing or placing the pots on the channels, applying 5 ml of conidial suspension to each treatment. The increased electrical conductivity of the nutrient solution generally reduced the incidence and severity of the disease, with the highest electrical conductivity (E.C.3) providing the best results. The addition of potassium silicate to the different nutrient solutions showed a significant reduction in both incidence and severity of the disease compared to a solution without silicate and the best results were given by the addition of silicate with the highest electrical conductivity (E.C.3) in all the trials carried out. The combination of high electrical conductivity and potassium silicate supplied gave good results. The possibility and benefits of applying Si amendments in practice are examined.     

IN VITRO DEVELOPMENT OF THE ISOLATED SHOOT APICAL MERISTEM OF ANGIOSPERMS

Development of complete plants was achieved from isolated shoot apical meristems of Nicotiana tabacum L., Daucus carota L., Nicotiana glauca Grah., Tropaeolum majus L., and Coleus blumei Benth. The explants consisted of only meristematic dome tissue with no visible leaf primordia. A simple nutrient medium composed of the Murashige and Skoog salt mixture, 100 mg/liter myo-inositol, 0.4 mg/liter thiamin-HCl, 1-2 mg/liter IAA, 30 g/liter sucrose, and 1% agar was adequate. Histologically there occurred principally tissue enlargement during the first 3-6 days, followed by appearance of bipolar organization in 6-9 days and formation of a well-defined root apex and initiation of first leaf primordium by 12 days.     

High efficiency plant regeneration by somatic embryogenesis from callus of mature embryo explants of bread wheat (Triticum aestivum) and grain sorghum (Sorghum bicolor)

Summary Tissue culture methods were developed for reproducible induction and maintenance of embryogenic (E) callus established from developmentally mature embryo explants of bread wheat (Triticum aestivum) and grain sorghum (Sorghum bicolor). Embryogenic callus was obtained by culturing seeds and mature embryos of wheat on Linsmaier and Skoog’s (LS) medium containing 5 or 2 mg/liter 2,4-dichlorophenoxyacetic acid (2,4-D), respectively, and for sorghum mature embryos on LS medium containing 2 mg/1 2,4-D plus 0.5 mg/liter kinetin. Plant regeneration from E callus was achieved for several months and quantified on a fresh-weight basis of E callus. Phenotypically normal plants were regenerated from E callus cultured on LS medium supplemented with 0.1 mg/liter IAA plus 0.5 mg/liter benzyladenine (BA) for wheat and 1.0 mg/liter IAA plus 0.5 mg/1BA for sorghum. Wheat research was funded by the United States Agency for International Development, Washington, DC, cooperative agreement DNA-4137-A-00-4-53-00. Sorghum research was supported by the Gas Research Institute, Chicago, IL, contract 5084-260-0973. Expert technical asistance was provided by Nitschka S. ter Kuile, Barbara J. Ashton, Laurie Osborne, Erin Scott, and Kathleen M. Petersen.     

Subcellular organization of N2-fixing nodules of cowpea (Vigna unguiculata) supplied with silicon

Summary Provision of silicon (0, 0.048, 0.096, 0.24, 0.48, and 0.96 g/1) in the form of silicic acid (H₄SiO₄) to nodulated cowpea plants(Vignia unguiculata [L.] Walp.) grown in liquid culture resulted in considerable changes in the internal organization of nodule structure. Compared to the control plants which received no added silicate, bacteroid numbers increased significantly (P ≤ 0.05) at silicate concentrations of both 0.096 and 0.48 g/1. The number of symbiosomes also increased by 3.2-fold at the silicate concentration of 0.96 g/1 compared to the control. In contrast, the size of bacteroids and symbiosomes decreased significantly (P ≤ 0.05) inside nodules of silicate-treated plants. The peribacteroid space was also decreased considerably (P ≤ 0.05) with the application of 0.096 and 0.96 g of silicate per liter to plants. However, the size of intercellular spaces adjacent to infected and uninfected interstitial cells within the nodule medulla increased significantly (P ≤ 0.05) at 0.096 g of silicate per liter followed by a sharply marked (P ≤ 0.05) decrease with each subsequent increase in silicate application. The result was a large decrease (P≤0.05) in the area of bacteria-infected tissue occupied by intercellular space at the highest silicate concentration, which was caused by a significant (P ≤ 0.05) increase in cell wall thickness. Our findings show that the positive effects of silicon on N₂ fixation might actually be due to an increased number of bacteroids and symbiosomes.     

Effect of silicate on the growth and arsenate uptake by rice (Oryza sativa L.) seedlings in solution culture

A solution culture experiment was conducted to investigate the effect of silicate on the yield and arsenate uptake by rice. Rice seedlings (Oryza sativaL. cv. Weiyou 77) were cultured in modified Hoagland nutrient solution containing three arsenate levels (0, 0.5 and 1.0 mg L ^–1 As) and four silicate levels (0, 14, 28 and 56 mg L ^–1 Si). Addition of Si significantly increased shoot dry weight (P=0.001) but had little effect on root dry weight (P=0.43). Addition of As had no significant effect on shoot dry weight (P=0.43) but significantly increased root dry weight (P=0.01). Silicon concentrations in shoots and roots increased proportionally to increasing amounts of externally supplied Si (P < 0.001). The presence of As in the nutrient solution had little effect on shoot Si concentration (P=0.16) but significantly decreased root Si concentration (P=0.005). Increasing external Si concentration significantly decreased shoot and root As concentrations and total As uptake by rice seedlings (P <0.001). In addition, Si significantly decreased shoot P concentration and shoot P uptake (P <0.001). The data clearly demonstrate a beneficial effect of Si on the growth of rice seedlings. Addition of Si to the growth medium also inhibited the uptake of arsenate and phosphate by the rice seedlings.     

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 application of constant recycle solids concentration in completely mixed oxygen activated sludge process

For better operational control of the completely mixed oxygen activated sludge process (CMOAS), a study concerning the kinetics, performance, and operational stability of the Ramanathan-Gaudy model was conducted. Short-term experiments were conducted at various dilution rates (1/9, 1/6, 1/3, 1/1.5, and 1/1.0 hr^?1) by using two recycle solids concentration values (5000 and 10,000 mg/liter). The influent substrate was an actual industrial organic wastewater (soft drink waste) and its concentration was maintained at 1000 mg/liter COD. The hydraulic recycle ratio, α, was maintained at 0.30. It was found that for CMOAS system with constant recycle cell concentration, a “steady state” with respect to reactor biological solids and effluent COD at different dilution rates could be attained. No appreciable dilute-out of reactor biological solids and substrate was observed up to the dilution rate of 1 hr^?1 for both systems of different X_R (5000 and 10,000 mg/liter). For the system of X_R = 5000 mg/liter, except the dilution rate of hr^?1, the effluent filtrate COD was lower than 100 mg/liter, the aerator biological solids concentration was about 1550 mg/liter, and the COD removal efficiency was higher than 90% for all dilution rates. For the system of X_R = 10,000 mg/liter, the effluent filtrate COD was lower than 71 mg/liter, the aerator biological solids concentration was about 2750 mg/liter, and the COD removal efficiency was higher than 90% throughout all the dilution rates selection in the present study. The value of the Sludge Volume Index (SVI) was the range of 37.0 to 58.5 and provided good settleability of sludge. The sludge yield was 0.53 for the system of X_R = 5000 mg/liter and 0.57 for the system of X_R = 10,000 mg/liter. The carbohydrate and the protein content of the cells were 10.1–21.6% and 35.6–50.6%, respectively. For predicting the reactor biological solid and effluent COD of the CMOAS system by using the Ramanathan-Gaudy model, two sets of values for the biological kinetic constants should be considered since it provided the best fit of predicted values of the observed values. In the present study, μ_m = 0.4 hr^?1, k_s = 92 mg/liter for 1/3 ? D ? 1, and μ_m = 0.05 hr^?1, k_s = 11.1 mg/liter for 1/9 ? D < 1/3 were used to calculate the predicted values of reactor biological solid and effluent filtrate COD.     

Production of high-quality edible protein from Candida yeast grown in continuous culture

Candida utilis NRRL Y-900 was grown in aerobic continuous culture with cane molasses as the source of the growth-limiting carbon. At 1% reducing sugar in the chemostal (10 liter working volume) feed medium, addition of Zn (25μM) to a minimal salts medium resulted in an increase in the biomass productivity of the chemostat from 1.7 to 2.6 g/liter/hr with a growth yield of 0.55 g dry biomass/g reducing sugar utilized at D_max. On the average, the yeast biomass was 50–55% protein. At S_R > 2% sugar, the biomass productivity was limited by the oxygen supply. With O₂-supplemented aeration (at S_R = 4.2%)the maximum biomass productivity Was 7.25 g/liter/hr. Aerobic ethanol production was not observed. A highquality undenatured protein fraction was isolate from the yeast homogenate by isoelectric precipitation at pH 4.5. Contaminating nucleic acid was removed as an insoluble complex by chelation with an organic cation (cetavlon). The final protein product contained about 3% RNA (DWB) and was suitable for use as a food additive.     

Sublethal Cytotoxic Effects of Mercuric Chloride on the Ciliate Tetrahymena pyriformis*

SYNOPSIS. The behavior and ultrastructure of Tetrahymena pyriformis was assessed after exposure to dosages of 8 and 16% of the lethal concentration of HgCl² (TLm 96 hr). The lower dosage caused no abnormal changes in cell motility, activity of the water explusion vesicles, or cell shape; the higher dosage caused deleterious changes in these parameters. The higher sublethal HgCl² concentration (0.50 mg/liter) elicited damage of several cell structures. This damage persisted and accumulated with time up to 24 hr. At the lower HgCl² dosage (0.25 mg liter) there were extensive changes after 1-hr exposure involving primarily mitochondria; however, all major changes were repaired after 24 hr of constant exposure to the HgCl², indicating adaptation to the toxicant. Based solely on cytotoxic evidence an attempt is made to apply the findings defining what constitutes a “safe'’concentration of HgCl₂ in the cell's environment.     

THE HETEROTROPHIC CAPABILITIES OF CYCLOTELLA MENEGHINIANA1

Cyclotella meneghiniana grew heterotrophically in darkness when glucose in concentrations from 5 mg/liter to 10 g/liter was provided. The other compounds tested did not support growth. However, in continuous light (300 ft-c) growth wax not enhanced if glucose wax provided. Under diurnal conditions of light (300 ft-c) approximately 12–14 hr of darkness were required to observe the enhancement effects of glucose. Uptake studies with labeled glucose indicated that uptake is not dependent on glucose, but that it occurs only at low light intensities. Cells required 12–14 hr of darkness to develop the uptake system.     

Regeneration of garlic plants (allium sativum L., CV. “Chonan”) via cell culture in liquid medium

Summary Aiming at the genetic improvement of garlic cultivars, a cell suspension protocol was established which includes the induction of friable callus, establishment of cells in liquid medium, plating, regeneration, and bulb formation. Calluses of various textures from compact to friable and from green to yellowish were obtained by culturing explants excised from inner leaves of garlic bulbs on Marashig-Shoog (MS) medium with 2,4 dichlorophenoxy acetic acid (2,4-D), (1.1 mg/liter [5.0 μM]), picloram (1.2 mg/liter [5.0 μM]), and kinetin (2.1 mg/liter [10 μM]). Friable callus occurred on MS-A contained 2,4-D alone (1.0 mg/liter [4.52 μM]) and this callus was used to develop cell suspension cultures, which were maintained in liquid MS-B medium with a 2,4-D/benzyl adenine (BA) (0.5 mg/liter [2.25 μM]: 0.5 mg/liter [2.22 μM]) ratio. High plating efficiency was obtained on MS-C medium with different naphthalene acetic acid/BA combinations. Regeneration occurred after transfer of the caulogenic mass to MS-C medium containing 10 mg/liter (74.02 μM) and 20 mg/liter (148.04 μM) adenine for 60 days, followed by transfer to adenine-free medium. Plantlets transplanted to soil showed normal phenology. Shoots grown on modified MS medium supplemented with indolylbutryic acid (3.0 mg/liter [14.7 μM]) stimulated bulb formation by 30 days in culture.     

ENDOGENOUS LEVELS OF INDOLE-3-ACETIC ACID IN SYNCHRONOUS CULTURES OF CHLORELLA PYRENOIDOSA12

Endogenous levels of indole-3-acetic acid were mesaured in synchronous cultures of Chlorella pyrenoidosa (TX-7-11-05). The cultures were synchronized by alternating light:dark periods of 15:9 hr at a temperature of 40 ± 1 C. After 2 synchronous cycles the cultures were exposed to a low light treatment of 350 ± 100 ft-c. The time to incipient cell division under these conditions was 6 hr and 15 min. Samples were taken at 3 sampling periods during the low light treatment period:low light 0 hr (LL0); low light 3 hr (LL3); and low light 6:15 hr (LL6:15). The algal extracts were analyzed by a fluorometric procedure which measured the indole-α-pyrone product formed by the action of the trifluoracetic acid-acetic anhydride reagent with IAA. The IAA levels increased gradually from the autospore stage (5.19 μg × 10^?4/mg dry wt) to the adolescent stage (7.13 μg × 10^?4/mg dry wt) and more rapidly when approaching the ripened adult stage (14.55 μg × 10^?4/mg dry wt). The mean percentage increase from autospore to adolescent was 36.9%, and from adolescent to ripened adult 104.6%. The total percentage increase from autospore to adult was 180.3%. Levels of IAA were 2 times higher just prior to division than in the autospore stage.     

Si amelioration of Al toxicity in barley (Hordeum vulgare L.) growing in two Andosols

The influence of silicon on aluminium toxicity in barley (Hordeum vulgare L. cv. Shunrai) was studied in two Andosols. Silicon sources were a solution of sodium metasilicate with pH adjusted to 5.0, silica gel, and an industrial waste, porous hydrated calcium silicate. The waste is produced in large amounts in the manufacturing processes of autoclaved light concrete, and has been used as a silicon source for rice plants. The addition of the waste increased the concentration of Si in the soil solution, soil pH and amelioration of aluminium toxicity was observed. The addition of silica gel and sodium metasilicate solution to both soils increased significantly (p<0.05) the Si concentration of the soil solutions, but no amelioration of aluminium toxicity was observed. An amelioration of aluminium toxicity by the waste porous hydrated calcium silicate was probably due to the increase in soil pH rather than to the increase of silicon concentration in the soil solution.