Evaluation of sugar cane hemicellulose hydrolyzate for cultivation of yeasts and filamentous fungi

Sugar cane bagasse hemicellulosic fraction submitted to hydrolytic treatment with 100 mg of sulfuric acid per gram of dry mass, at 140°C for 20 min, was employed as a substrate for microbial protein production. Among the 22 species of microorganisms evaluated, Candida tropicalis IZ 1824 showed TRS consumption rate of 89.8%, net cell mass of 11.8 g L⁻¹ and yield coefficient (Y_x/s) of 0.50 g g⁻¹. The hydrolyzate supplemented with rice bran (20.0 g L⁻¹), P₂O₅ (2.0 g L⁻¹) and urea (2.0 g L⁻¹) provided a TRS consumption rate of 86.3% and a cell mass of 8.4 g L⁻¹. At pH 4.0 cellular metabolism was inhibited, whereas at pH 6.0 the highest yield was obtained. The presence of furfural (2.0 g L⁻¹) hydroxymethylfurfural (0.08 g L⁻¹) and acetic acid (3.7 g L⁻¹) in the hydrolyzate did not interfere with cultivation at pH 6.0. Received 25 October 1996/ Accepted in revised form 10 March 1997     

Mineralization of pentachlorophenol in a contaminated soil by Pseudomonas sp UG30 cells encapsulated in κ-carrageenan

A fermentation process in Escherichia coli for production of supercoiled plasmid DNA for use as a DNA vaccine was developed using an automated feed-back control nutrient feeding strategy based on dissolved oxygen (DO) and pH. The process was further automated through a computer-aided data processing system to regulate the cell growth rate by controlling interactively both the nutrient feed rate and agitation speed based on DO. The process increased the total yield of the plasmid DNA by approximately 10-fold as compared to a manual fed-batch culture. The final cell yield from the automated process reached 60 g L⁻¹ of dry cell weight (OD₆₀₀ = 120) within 24 h. A plasmid DNA yield of 100 mg L⁻¹ (1.7 mg g⁻¹ cell weight) was achieved by using an alkaline cell lysis method. Plasmid yield was confirmed using High Performance Liquid Chromatography (HPLC) analysis. Because cells had been grown under carbon-limiting conditions in the automated process, acetic acid production was minimal (below 0.01 g L⁻¹) throughout the fed-batch stage. In contrast, in the manual process, an acid accumulation rate as high as 0.36 g L⁻¹ was observed, presumably due to the high nutrient feed rates used to maintain a maximum growth rate. The manual fed-batch process produced a low cell density averaging 10–12 g L⁻¹ (OD₆₀₀ = 25–30) and plasmid yields of 5–8 mg L⁻¹ (approximately 0.7 mg g⁻¹ cells). The improved plasmid DNA yields in the DO- and pH-based feed-back controlled process were assumed to be a result of a combination of increased cell density, reduced growth rate (μ) from 0.69 h⁻¹ to 0.13 h⁻¹ and the carbon/nitrogen limitation in the fed-batch stage. The DO- and pH-based feed-back control, fed-batch process has proven itself to be advantageous in regulating cell growth rate to achieve both high cell density and plasmid yield without having to use pure oxygen. The process was reproducible in triplicate fermentations at both 7-L and 80-L scales. Received 22 March 1996/ Accepted in revised form 20 September 1996     

Production of curdlan using sucrose or sugar cane molasses by two-step fed-batch cultivation of Agrobacterium species

Maltose and sucrose were efficient carbon sources for the production of curdlan by a strain of Agrobacterium sp. A two-step, fed-batch operation was designed in which biomass was first produced, followed by curdlan production which was stimulated by nitrogen limitation. There exists an optimal timing for nitrogen limitation for curdlan production in the two-step, fed-batch operation. Maximum curdlan production (60 g L⁻¹) was obtained from sucrose with a productivity of 0.2 g L⁻¹ h⁻¹ when nitrogen was limited at a cell concentration of 16.0 g L⁻¹. It was also noted that the curdlan yield from sucrose was as high as 0.45 g curdlan g⁻¹ sucrose, and the highest specific production rate was 1.0 g curdlan g⁻¹ cells h⁻¹ right after nitrogen limitation. Of particular importance was the use of molasses as a cheap carbon source to produce curdlan in the two-step, fed-batch cultivation. As high as 42 g L⁻¹ of curdlan with a yield of 0.35 g curdlan g⁻¹ total sugar was obtained after 120 h of fed-batch cultivation. Received 20 August 1996/ Accepted in revised form 26 November 1996     

Rapid atrazine mineralisation in soil slurry and moist soil by inoculation of an atrazine-degrading Pseudomonas sp. strain

The evaluation of pesticide-mineralising microorganisms to clean-up contaminated soils was studied with the widely applied and easily detectable compound atrazine, which is rapidly mineralised by several microorganisms including the Pseudomonas sp. strain Yaya 6. The rate of atrazine removal was proportional to the water content of the soil and the amount of bacteria added to the soil. In soil slurry, 6 mg atrazine kg soil⁻¹ was eliminated within 1 day after application of 0.3 g dry weight inoculant biomass kg soil⁻¹ and within 5 days when 0.003 g kg soil⁻¹ was used. In partially saturated soil (60% of the maximal water-holding capacity) 15 mg atrazine kg soil⁻¹ was eliminated within 2 days by 1 g biomass kg soil⁻¹ and within 25 days when 0.01 g biomass kg soil⁻¹ was used. In unsaturated soil, about 60% [U-ring-¹⁴C]atrazine was converted to ¹⁴CO₂ within 14 days. Atrazine was very efficiently removed by the inoculant biomass, not only in soil that was freshly contaminated but also in soil aged with atrazine for up to 260 days. The bacteria exposed to atrazine in unsaturated sterile soil were still active after a starvation period of 240 days: 15 mg newly added atrazine kg soil⁻¹ was eliminated within 5 days. Received: 31 October 1997 / Received revision: 16 January 1998 / Accepted: 18 January 1998     

Heavy metal chelation by non-living biomass of three color forms of Kappaphycus alvarezii (Doty) Doty

Water pollution by toxic heavy metals is a burning environmental problem and has presented a challenge to humans. Removal of heavy metals using non-living biomass of seaweeds could be a potential solution to this problem. In the present investigation, biomass of three color forms of Kappaphycus alvarezii, viz. brown, green and pale yellow, were studied in the laboratory for their heavy metal chelating capacity using cadmium, cobalt, chromium and copper. Amongst the four concentrations used (25, 50, 75 and 100 mg L⁻¹) maximum chelation of Cd, Co and Cu was recorded at 25 mg L⁻¹ concentration. The highest amount of Cr was chelated at 100 mg L⁻¹ by all the three color forms. The pale yellow form showed maximum chelation for all four metals studied. Further, chelation in all the color forms was found to be: Cd 5.37 ± 0.59–15.84 ± 0.32 %, Co 21.19 ± 0.13–32.32 ± 0.62 %, Cr 65.38 ± 0.27–88.09 ± 0.51 % and Cu 59.53 ± 0.37–90.28 ± 0.89 %. All the three color forms of K. alvarezii serve as an excellent biodetoxifier as they all chelated considerable amounts of heavy metals.     

Metal resistance and removal by two strains of the green alga, <Emphasis Type="Italic">Chlorella vulgaris</Emphasis> Beijerinck,isolated from Laguna de Bay,Philippines

Two strains of Chlorella vulgaris Beijerinck isolated from two different sites in Laguna de Bay, Philippines, were studied for their resistance and ability to remove four metal ions, i.e., Cu²⁺, Cr⁶⁺, Pb²⁺, and Cd²⁺ added separately in BG-11 growth medium. The growth of the two strains was severely inhibited at 2 mg.L⁻¹ of Cu²⁺, 5 mg.L⁻¹ of Cr⁶⁺, 8 mg.L⁻¹ of Pb²⁺, and 10 mg.L⁻¹ of Cd²⁺. However, the two strains exhibited different EC₅₀ values for the same metal ion. The WB strain had a significantly higher resistance (p < 0.01) for Cd²⁺ and Cr⁶⁺ compared with the SB strain, while the SB strain had significantly higher resistance (p < 0.01) for Cu²⁺ compared with the WB strain. On the other hand, the two strains behaved differently in their capacity to remove the metal ions in BG-11 medium containing 1.0 mg.L⁻¹ of the three metal ions, except for Cu²⁺, which was added at 0.1 mg.L⁻¹. The WB strain showed the highest removal of Cd²⁺ at 70.3% of total, followed by Pb²⁺ at 32%, while the SB strain exhibited the highest removal of Pb²⁺ at 48.7% followed by Cd²⁺ at 40.7% of the total. Both strains showed the least removal of Cr⁶⁺ at 28% and 20.8% of the total for the WB and SB strains respectively. The percentage removal for Cu²⁺ was 50.7% and 60.8% for the WB and SB strains respectively. After 12 days of incubation, both strains showed that a greater percentage of the metal ions removed were accumulated intracellularly than adsorbed at a ratio of at least 2:1. Both strains manifested the same cytological deformities, like a loss of pyrenoids at 10 mg.L⁻¹ in all four metal ions. Discoloration and disintegration of chloroplasts were observed at 1.0 mg.L⁻¹ in Cu²⁺ and 5 mg.L⁻¹ in Cr⁶⁺. The nonrelease of autospores from the mother cells was observed at 10 mg.L⁻¹ in Cu²⁺ and Cr⁶⁺. Presented at the 6th Meeting of the Asian Pacific Society of Applied Phycology, Manila, Philippines.     

Docosapentaenoic acid (C22:5, ω-3) production by Pythium acanthicum

The physiological roles of omega-3 fatty acids, eicosapentaenoic acid and docosahexaenoic acid have been investigated in detail and microbial strains producing these polyunsaturated fatty acids have been characterised. It has recently been suggested that docosapentaenoic acid may have an important role, especially in infant nutrition, and that its positive health effects have been overlooked. Docosapentaenoic acid (C22:5, ω-3) production by a strain of Pythium acanthicum ATCC 18660 was thus investigated. Optimum conditions for growth of P. acanthicum ATCC 18660 and docosapentaenoic acid production were: pH 6.0, temperature 20°C and incubation time, 10 days. Among different saccharides and complex nitrogen sources tested, glucose and sodium glutamate were preferred carbon and nitrogen sources, respectively. Maximum biomass content (10.4 g L⁻¹) and docosapentaenoic acid yield (49.9 mg L⁻¹) were obtained in 10 days. An increase in docosapentaenoic acid volumetric yields to 108–110 mg L⁻¹ was obtained when linseed oil was used to supplement glucose or soy flour-containing medium. Batch feeding of additional glucose or linseed oil further enhanced the docosapentaenoic acid volumetric yield to 132 mg L⁻¹ and 125 mg L⁻¹, respectively, in 14 days. The specific production of docosapentaenoic acid in preliminary experiments ranged from 1.0–5.0 mg g⁻¹ biomass. As conditions were optimised, docosapentaenoic acid specific production titers were generally in the range of 4.0–5.5 mg g⁻¹ and increases in docosapentaenoic acid volumetric production could be attributed to increased biomass production. The limited improvement obtained by modifying culture conditions indicates that increasing volumetric yields of docosapentaenoic acid by modifying culture conditions appears to represent a significant barrier to commercialisation of such a process and suggests a more fundamental manipulation of metabolism and physiology is required. Received 06 November 1997/ Accepted in revised form 10 January 1998     

Effect of flow rate on heavy metal accumulation by rotating biological contactor (RBC) biofilms

Immobilized biofilms are effective in heavy metal removal. The current studies investigated the use of rotating biological contactor (RBC) biofilms in treatment of a wastewater containing cadmium, copper and zinc, each at a concentration of 100 mg L⁻¹. In particular, the influence of hydraulic retention time (HRT) on metal accumulation was studied. Longer HRTs (>12 h) were associated with greater metal removal than short HRTs, particularly with regard to cadmium and zinc. The system was also shown to operate successfully over an extended period of time, at an HRT of 24 h, with removal efficiencies of approximately 34%, 85% and 57% for Cd²⁺, Cu²⁺ and Zn²⁺ respectively after 5–8 weeks contact. Journal of Industrial Microbiology & Biotechnology (2000) 24, 244–250. Received 28 July 1999/ Accepted in revised form 21 December 1999     

Treatment of cyanide-containing wastewater from the food industry in a laboratory-scale fixed-bed methanogenic reactor

During the process of producing cassava starch from Manihot esculenta roots, large amounts of cyanoglycosides were released, which rapidly decayed to CN⁻ following enzymatic hydrolysis. Depending on the varying cyanoglycoside content of the cassava varieties, the cyanide concentration in the wastewater was as high as 200 mg/l. To simulate anaerobic stabilization, a wastewater with a chemical oxygen demand (COD) of about 20 g/l was prepared from cassava roots and was fermented in a fixed-bed methanogenic reactor. The start-up phase for a 99% degradation of low concentrations of cyanide (10 mg/l) required about 6 months. After establishment of the biofilm, a cyanide concentration of up to 150 mg CN⁻/l in the fresh wastewater was degraded during anaerobic treatment at a hydraulic retention time of 3 days. All nitrogen from the degraded cyanide was converted to organic nitrogen by the biomass of the effluent. The cyanide-degrading biocoenosis of the anaerobic reactor could tolerate shock concentrations of cyanide up to 240 mg CN⁻/l for a short time. Up to 5 mmol/l NH₄Cl (i.e. 70 mg N/l = 265 mg NH₄Cl/l) in the fresh wastewater did not affect cyanide degradation. The bleaching agent sulphite, however, had a negative effect on COD and cyanide removal. For anaerobic treatment, the maximum COD space loading was 12 g l⁻¹ day⁻¹, equivalent to a hydraulic retention time of 1.8 days. The COD removal efficiency was around 90%. The maximum permanent cyanide space loading was 50 mg CN⁻ l⁻¹ day⁻¹, with tolerable shock loadings up to 75 mg CN⁻ l⁻¹day⁻¹. Under steady-state conditions, the cyanide concentration of the effluent was lower than 0.5 mg/l. Received: 15 August 1997 / Received revision: 10 October 1997 / Accepted: 14 October 1997     

Pullulan production from deproteinized whey by Aureobasidium pullulans

Aureobasidium pullulans P56 was investigated using an adaptation technique and a mixed culture system. The adaptation of A. pullulans and the mixed cultures of A. pullulans and/or Lactobacillus brevisX20, Debaryomyces hansenii 194 and Aspergillus niger did not increase the production of polysaccharide. Enzymic hydrolysis of lactose in deproteinized whey gave a higher polysaccharide concentration and polysaccharide yield than acidic hydrolysed lactose. Maximum polysaccharide concentration (11.0 ± 0.5 g L⁻¹), biomass dry weight (10.5 ± 0.4 g L⁻¹), polysaccharide yield (47.2 ± 1.8%) and sugar utilization (93.2 ± 2.8%) were achieved using enzyme-hydrolysed whey (pH 6.5) containing 25 g L⁻¹ lactose and supplemented with K₂HPO₄ 0.5%, L-glutamic acid 1%, olive oil 2.5%, and Tween 80 0.5%. In this case the pullulan content of the crude polysaccharide was 40%. Received 16 December 1997/ Accepted in revised form 12 March 1999     

Mercury and cadmium concentrations in the tissues of three species of southern albatrosses

Cadmium and mercury concentrations were measured in the tissues of 64 individual albatrosses [23 wandering albatrosses (Diomedea exulans), 9 royal albatrosses (Diomedea epomophora) and 32 shy albatrosses (Thalassarche cauta)] which were killed as by-catch in longline fishing activities between 1991 and 1994. Mercury concentrations were also determined for 33 shy albatross eggs (excluding shells). The birds were all sexed and assigned to one of two age classes (immature and adult). The three species exhibited differences both in overall concentrations of cadmium and mercury, and also in the pattern of accumulation of metals with age and sex. Wandering albatrosses exhibited the highest mercury concentrations with a mean concentration in adult liver samples of 920.0 ± 794.1 μg g⁻¹ dry weight. Shy albatrosses had the lowest mercury concentrations with mean concentrations in adult livers of 36.3 ± 21.4 mg g⁻¹ dry weight. The highest mercury concentration was 1800 μg g⁻¹ for an adult female wandering albatross. Cadmium concentrations were less variable, with adult royal albatrosses having the highest average concentrations (180.0 ± 165.0 in adult kidneys) and adult shy albatrosses the lowest (40.1 ± 20.0 in adult kidney). The highest individual cadmium concentration was 287 μg g⁻¹ for a juvenile wandering albatross. There was no evidence of increased accumulation of cadmium with age in any of the species, but wandering albatrosses showed higher mercury concentrations in adults than juveniles. Female wandering albatrosses also had significantly higher mercury concentrations than males. The mercury contents of the shy albatross eggs were very low, with a maximum concentration of 5.4 μg g⁻¹. The results of this study are consistent with the findings of previous work on albatrosses and support the notion that the life-history strategy of these species (i.e. long-lived with low reproductive output) may be an important determinant in the concentrations of some metals found in their tissues. Accepted: 15 February 1999     

Estimation of fungal biomass in the decaying cones ofPinus densiflora

Fungal biomass in the decaying cones ofPinus densiflora was investigated. Leaching, immobilization and mobilization phases were recognized in the decomposition process of cones. Fungal biomass was estimated by the agar-film technique, using a conversion factor of 0.62 mg dry wt. mm⁻³ of hyphal volume to biomass and a factor of 2.5 for in-efficiencency of homogenization. The fungal biomass was 4.9±2.1 (mean±S.D.) mg dry wt. g⁻¹ dry matter in the cones on the tree, 11±6 mg g⁻¹ in the leaching phase, 19±7 mg g⁻¹ in the immobilization phase and 30±15 mg g⁻¹ in the mobilization phase. It significantly increased after cones had lain on the forest floor, and also in the immobilization phase. The latter result suggests that the fungal biomass contributed to the immobilization of nitrogen in the decomposition process. The ratio of ergosterol content to fungal biomass in the cones was 2.9–8.8 μg mg⁻¹ dry wt., lying in the range of 2–16 μg mg⁻¹ reported for mycelia. This suggested that the estimate of fungal biomass was reasonable. Reduction in this ratio with the dry weight loss in the cones suggested that the proportion of relatively active fungal biomass decreased with the progress of decomposition.     

Production of xylitol by Candida peltata

Candida peltata NRRL Y-6888 to ferment xylose to xylitol was evaluated under different fermentation conditions such as pH, temperature, aeration, substrate concentration and in the presence of glucose, arabinose, ethanol, methanol and organic acids. Maximum xylitol yield of 0.56 g g⁻¹ xylose was obtained when the yeast was cultivated at pH 6.0, 28°C and 200 rpm on 50 g L⁻¹ xylose. The yeast produced ethanol (0.41 g g⁻¹ in 40 h) from glucose (50 g L⁻¹) and arabitol (0.55 g g⁻¹ in 87 h) from arabinose (50 g L⁻¹). It preferentially utilized glucose > xylose > arabinose from mixed substrates. Glucose (10 g L⁻¹), ethanol (7.5 g L⁻¹) and acetate (5 g L⁻¹) inhibited xylitol production by 61, 84 and 68%, respectively. Arabinose (10 g L⁻¹) had no inhibitory effect on xylitol production. Received 24 December 1998/ Accepted in revised form 18 March 1999     

Detection of Sphingomonas spp in soil by PCR and sphingolipid biomarker analysis

Sphingomonas spp possess unique abilities to degrade refractory contaminants and are found ubiquitously in the environment. We developed Sphingomonas genus-specific PCR primers (SPf-190 and SPr1-852) which showed specific amplification of a 627-bp 16S rDNA fragment from Sphingomonas spp. A PCR assay using these Sphingomonas specific primers was developed to detect Sphingomonas aromaticivorans B0695R in three texturally distinct soil types, showing detection limits between 1.3–2.2 × 10³ CFU g⁻¹ dry soil. A sphingolipid extraction protocol was also developed to monitor Sphingomonas populations in soil quantitatively. The detection limit of the assay was 20 pmol g⁻¹ dry soil, equivalent to about 3 × 10⁵ cells g⁻¹ dry soil. Survival of S. aromaticivorans B0695R was monitored in the three different soils by antibiotic selective plate counting, PCR and sphingolipid analysis. All three approaches showed that the B0695R cells persisted in the low biomass Sequatchie sub-soil at about 3–5 × 10⁷cells g⁻¹ dry soil. In comparison to the plate counting assay, both the PCR and sphingolipid analysis detected a significantly higher level of B0695R cells in the clay soil and Sequatchie top-soil, indicating the possibility of the presence of viable but non-culturable B0695R cells in the soils. The combination of PCR and sphingolipid analysis may provide a more realistic estimation of Sphingomonas population in the environment. Received 17 March 1999/ Accepted in revised form 07 April 1999     

Kinetics of CO conversion into H<Subscript>2</Subscript> by <Emphasis Type="Italic">Carboxydothermus hydrogenoformans</Emphasis>

The objective of this study was to improve the biological water–gas shift reaction for producing hydrogen (H₂) by conversion of carbon monoxide (CO) using an anaerobic thermophilic pure strain, Carboxydothermus hydrogenoformans. Specific hydrogen production rates and yields were investigated at initial biomass densities varying from 5 to 20 mg volatile suspended solid (VSS) L⁻¹. Results showed that the gas–liquid mass transfer limits the CO conversion rate at high biomass concentrations. At 100-rpm agitation and at CO partial pressure of 1 atm, the optimal substrate/biomass ratio must exceed 5 mol CO g⁻¹ biomass VSS in order to avoid gas–liquid substrate transfer limitation. An average H₂ yield of 94 ± 3% and a specific hydrogen production rate of ca. 3 mol g⁻¹ VSS day⁻¹ were obtained at initial biomass densities between 5 and 8 mg VSS⁻¹. In addition, CO bioconversion kinetics was assessed at CO partial pressure from 0.16 to 2 atm, corresponding to a dissolved CO concentration at 70°C from 0.09 to 1.1 mM. Specific bioactivity was maximal at 3.5 mol CO g⁻¹ VSS day⁻¹ for a dissolved CO concentration of 0.55 mM in the culture. This optimal concentration is higher than with most other hydrogenogenic carboxydotrophic species.     

Kinetic analysis on the two-step processes of AOB and NOB in aerobic nitrifying granules

Complete granulation of nitrifying sludge was achieved in a sequencing batch reactor. For the granular sludge, batch experiments were conducted to characterize the kinetic features of ammonia oxidizers (AOB) and nitrite oxidizers (NOB) in the granules using the respirometric method. A two-step nitrification model was established to determine the kinetic parameters of both AOB and NOB. In addition to nitrification reactions, the new model also took into account biomass maintenance and mass transfer through the granules. The yield coefficient, maximum specific growth rate, and affinity constant for ammonium for AOB were 0.21 g chemical oxygen demand (COD) g⁻¹ N, 0.09 h⁻¹, and 9.1 mg N L⁻¹, respectively, whereas the corresponding values for NOB were 0.05 g COD g⁻¹ N, 0.11 h⁻¹, and 4.85 mg N L⁻¹, respectively. The model developed in this study performed well in simulating the oxygen uptake rate and nitrogen conversion kinetics and in predicting the oxygen consumption of the AOB and NOB in aerobic granules.     

Immobilization of blue green microalgae on loofa sponge to biosorb cadmium in repeated shake flask batch and continuous flow fixed bed column reactor system

Summary An indigenous strain of blue green microalga, Synechococcus sp., isolated from wastewater, was immobilized onto loofa sponge discs and investigated as a potential biosorbent for the removal of cadmium from aqueous solutions. Immobilization has enhanced the sorption of cadmium and an increase of biosorption (21%) at equilibrium was noted as compared to free biomass. The kinetics of cadmium biosorption was extremely rapid, with (96%) of adsorption within the first 5 min and equilibrium reached at 15 min. Increasing initial pH or initial cadmium concentration resulted in an increase in cadmium uptake. The maximum biosorption capacity of free and loofa immobilized biomass of Synechococcus sp. was found to be 47.73 and 57.76 mg g⁻¹ biomass respectively. The biosorption equilibrium was well described by Langmuir adsorption isotherm model. The biosorbed cadmium was desorbed by washing the immobilized biomass with dilute HCl (0.1 M) and desorbed biomass was reused in five biosorption–desorption cycles without an apparent decrease in its metal biosorption capacity. The metal removing capacity of loofa immobilized biomass was also tested in a continuous flow fixed-bed column bioreactor and was found to be highly effective in removing cadmium from aqueous solution. The results suggested that the loofa sponge-immobilized biomass of Synechococcus sp. could be used as a biosorbent for an efficient removal of heavy metal ions from aqueous solution.     

Caroteno-protein and exopolysaccharide production by co-cultures of Rhodotorula glutinis and Lactobacillus helveticus

The lactose-negative yeast Rhodotorula glutinis 22P and the homofermentative lactic acid bacterium Lactobacillus helveticus 12A were cultured together in a cheese whey ultrafiltrate containing 42 g L⁻¹ lactose. The chemical composition of the caroteno-protein has been determined. The carotenoid and protein contents are 248  μ g g⁻¹ dry cells and 48.2% dry weight. Carotenoids produced by Rhodotorula glutinis 22P have been identified as β-carotene 15%, torulene 10%, and torularhodin 69%. After separating the cell mass from the microbial association, the exopolysaccharides synthesized by Rhodotorula glutinis 22P were isolated from the supernatant medium in a yield of 9.2 g L⁻¹. The monosaccharide composition of the synthesized biopolymer was predominantly D-mannose (57.5%). Received 08 July 1996/ Accepted in revised form 11 December 1996     

Large-scale cultivation of adventitious roots of Echinacea purpurea in airlift bioreactors for the production of chichoric acid, chlorogenic acid and caftaric acid

Adventitious roots of Echinacea purpurea were cultured in airlift bioreactors (20 l, 500 l balloon-type, bubble bioreactors and 1,000 l drum-type bubble bioreactor) using Murashige and Skoog (MS) medium with 2 mg indole butyric acid l⁻¹ and 50 g sucrose l⁻¹ for the production of chichoric acid, chlorogenic acid and caftaric acid. In the 20 l bioreactor (containing 14 l MS medium) a maximum yield of 11 g dry biomass l⁻¹ was achieved after 60 days. However, the amount of total phenolics (57 mg g⁻¹ DW), flavonoids (34 mg g⁻¹ DW) and caffeic acid derivatives (38 mg g⁻¹ DW) were highest after 50 days. Based on these studies, pilot-scale cultures were established and 3.6 kg and 5.1 kg dry biomass were achieved in the 500 l and 1,000 l bioreactors, respectively. The accumulation of 5 mg chlorogenic acid g⁻¹ DW, 22 mg chichoric acid g⁻¹ DW and 4 mg caftaric acids g⁻¹ DW were achieved with adventitious roots grown in 1,000 l bioreactors.     

Effects of carbon concentration and carbon-to-nitrogen ratio on growth, conidiation, spore germination and efficacy of the potential bioherbicide Colletotrichum coccodes

The effect of carbon concentration and carbon-to-nitrogen ratio (C:N) as well as their interaction on Colletotrichum coccodes growth and sporulation in submerged flask culture were evaluated. When C:N ratios were held constant, both mycelial dry biomass and spore yield increased with increasing carbon concentration. The specific spore yields (spore yield g⁻¹ carbon), however, were not significantly different for the same C:N ratio in most cases. The highest spore yields (1.3 × 10⁸ spores per ml) were obtained from media containing 20 g per liter carbon with C:N ratios ranging from 5:1 to 10:1. When the C:N ratio was greater than 15:1, spore yields were significantly decreased with increasing C:N ratios. High carbon concentration (20 g L⁻¹) combined with high C:N ratios (above 15:1) reduced both mycelial growth and sporulation, and increased spore matrix production. Spores produced in medium containing 10 g L⁻¹ carbon with C:N ratios from 10:1 to 15:1 had 90% germination on potato dextrose agar after 12 h and caused extensive shoot dry weight reduction on the target weed, velvetleaf. These results suggest that C:N ratios from 10:1 to 15:1 are optimal for C. coccodes spore production. Received 9 December 1997/ Accepted in revised form 22 May 1998