Kinetic modelling of isomerization and anaerobic degradation of n- and i-butyrate

Very clear experimental evidence of isomerization between n- and i-butyrate during their anaerobic degradation was presented. A first experiment in the presence of bromoethane sulfonic acid (BESA), an inhibitor of methanogenesis, allowed the equilibrium distribution of n-, i-butyrate and acetate to be determined. To elucidate the mechanism of the isomerization process, a kinetic analysis was employed. The results suggested that i-butyrate was isomerized into n-butyrate prior to being oxidized to acetate. A mechanism for butyrate degradation, based on the values of the kinetic parameters obtained, was proposed.     

Aerobic thermophilic waste sludge biotreatment: carboxylic acid production and utilization during biodegradation of bacterial cells under oxygen limitation

The production and utilization of carboxylic acids during aerobic thermophilic treatment of a model sludge composed of bacterial cells were examined in a laboratory treatment system. Operation under a limited supply of O₂, typical for such treatment systems, resulted in a distinct pattern of production and simultaneous utilization of low molecular weight carboxylic acids. Pulse-addition of a mixture of carboxylic acids at the end of a fed-batch cycle indicated that simultaneous utilization of acetate, propionate, isobutyrate, n-butyrate and isovalerate could occur, but only after a lag phase during which only acetate was utilized. In an attempt to differentiate between production and utilization of the carboxylic acids, a series of pulse experiments were performed using ¹⁴C-labelled acetate. The results indicated that production continued late into the fed-batch cycle whereas utilization could occur during the entire cycle. When acetate was pulsed to the process, only 11% of the acetate carbon was incorporated into new biomass, whereas 75% was converted into CO₂. However, 14% of the original radioactivity persisted in the supernatant despite complete acetate utilization. This suggested that some of the acetate was metabolized into more slowly biodegradable products. Correspondence to: G. Hamer     

Isomerization ofn- andi-butyrate in anaerobic methanogenic systems

Studies of the degradation of the two isomeric forms of butyrate in different anaerobic environments showed isomerization betweenn- andi-butyrate. Degradation rates were similar for the different examined systems and degradation rates forn-butyrate degradation were generally higher than fori-butyrate. Degradation rates forn-butyrate ranged from 0.52 to 1.39 day^–1, while the rates fori-butyrate were from 0.46 to 1.15 day^–1. Production of isomers was not observed when the volatile fatty acid degradation was inhibited by addition of bromoethane sulfonic acid, indicating that isomerization was coupled to the methanogenic degradation of the acid. The degree of isomerization observed duringn-butyrate degradation was similar to the degree duringi-butyrate degradation. Experiments indicated that the isomerization degree was higher for the thermophilic than for the mesophilic inocula.     

Preparation of High Yields of Algal Protoplasts Using Buccal Juice of Sea Hare and Commercial Cellulase

Buccal juice of the sea hare Aplysia juliana was found to degrade algal polysaccharides. The optimal enzyme composition for protoplast preparation from Undaria pinnatifida was protein at 48 μg/ml buccal juice from sea hare, 10 mg/ml cellulase Onozuka-RS, 0.4 M NaCl, 0.8 M sorbitol, 2 mg/ml dextran sulfate sodium salt, and 1 μl/ml 2-mercaptoethanol in 10 mM MES buffer (pH 6.0). Protoplasts of Eisenia bicyclis, Endarachne binghamiae (Phaeophyta), and Ulva pertusa (Chlorophyta) could also be prepared in a similar manner. Yields of these protoplasts were about 10⁷ cells per gram of fresh weight alga. Received January 26, 1998; accepted September 17, 1998.     

Process development for high-level secretory production of carboxypeptidase Y by Saccharomyces cerevisiae

In order to develop a production process for carboxypeptidase Y (CPY, yeast vacuolar protease) secreted by Saccharomyces cerevisiae KS58-2D, medium composition, culture conditions, and expression systems were investigated. We found that the addition of histidine to thiamine-free medium, in which CPY production was almost negligible, raised the intracellular thiamine level, resulting in the increase of CPY production. On the basis of the choice of an expression system that uses an inducible GAL10 promoter, reassessment of histidine concentration in the medium, and optimization of the pH level during cultivation (pH 6.5), active CPY was secreted in a quantity of over 400 mg/l, which was more than tenfold that higher than that previously reported. The process developed could be easily scaled-up to industrial-scale fermentation. Received: 16 January 1998 / Received revision: 16 February 1998 / Accepted: 27 February 1998     

Degradation of cyanide in agroindustrial or industrial wastewater in an acidification reactor or in a single-step methane reactor by bacteria enriched from soil and peels of cassava

During cassava starch production, large amounts of cyanoglycosides were released and hydrolysed by plant-borne enzymes, leading to cyanide concentrations in the wastewater as high as 200 mg/l. For anaerobic degradation of the cyanide during pre-acidification or single-step methane fermentation, anaerobic cultures were enriched from soil residues of cassava roots and sewage sludge. In a pre-acidification reactor this culture was able to remove up to 4 g potassium cyanide/l of wastewater at a hydraulic retention time (t _HR) of 4 days, equivalent to a maximal cyanide space loading of 400 mg CN⁻ l⁻¹ day⁻¹. The residual cyanide concentration was 0.2–0.5 mg/l. Concentrated cell suspensions of the mixed culture formed ammonia and formate in almost equimolar amounts from cyanide. Little formamide was generated by chemical decay. A concentration of up to 100 mmol ammonia/l had no inhibitory effect on cyanide degradation. The optimal pH for cyanide degradation was 6–7.5, the optimal temperature 25–37 °C. At a pH of 5 or lower, cyanide accumulated in the reactor and pre-acidification failed. The minimal t _HR for continuous cyanide removal was 1.5 days. The enriched mixed culture was also able to degrade cyanide in purely mineralic wastewater from metal deburring, either in a pre-acidification reactor with a two-step process or in a one-step methanogenic reactor. It was necessary to supplement the wastewater with a carbon source (e.g. starch) to keep the population active enough to cope with any possible inhibiting effect of cyanide. Received: 29 April 1998 / Received revision: 8 June 1998 / Accepted: 14 June 1998     

Plant regeneration and multiplication of the emergent wetland monocot Juncus accuminatus

A reliable callus regeneration and shoot multiplication system for wetland monocot Juncus accuminatus has been established. Callus was induced from 6-day-old seedlings on Murashige and Skoog medium supplemented with 5 mg/l picloram. The callus differentiated into shoots upon transfer to 5 mg/l benzyladenine (BA)-supplemented medium. Effects of medium pH (3.8–7.8) and source of callus (grown in the dark or continuous light) on regeneration were determined. Both parameters significantly influenced regeneration. Regenerated shoots were multiplied by subculturing shoots onto 5 mg/l BA medium at 4-week intervals. The regenerated shoots were rooted on 0.1 mg/l naphthaleneacetic-acid-supplemented medium. The rooted plants were transferred to pots containing a commercial potting mix and established in the greenhouse. Plants covered with plastic grew faster and flowered earlier than uncovered plants. All plants set viable seeds. Received: 21 July 1997 / Revision received: 23 December 1997 / Accepted: 19 January 1998     

Effect of feeding strategy on Zymomonas mobilis CP4 fed-batch fermentations and mathematical modeling of the system

In this work, the effect of the feeding strategy in Zymomonas mobilis CP4 fed-batch fermentations on the final biomass and ethanol concentrations was studied. Highest glucose yields to biomass (0.018 g/g) and to ethanol (0.188 g/g) were obtained in fed-batch fermentations carried out using different feeding rates with a glucose concentration in the feed equal to 100 g/l. Lower values (0.0102 g biomass/g glucose and 0.085 g ethanol/g glucose) were obtained when glucose accumulated to levels higher than 60 g/l. On the other hand, the highest biomass (5 g/l) and ethanol (39 g/l) concentrations were obtained using a glucose concentration in the feed equal to 220 g/l and exponentially varied feeding rates. Experimental data were used to validate the mathematical model of the system. The prediction errors of the model are 0.39, 14.36 and 3.24 g/l for the biomass, glucose and ethanol concentrations, respectively. Due to the complex relationship for describing the specific growth rate, a fed-batch culture in which glucose concentration is constant would not optimize the process. Received: 30 November 1999 / Received revision: 24 March 2000 / Accepted: 7 April 2000     

In vitro plant regeneration of fig (Ficus carica L. cv. gular) using apical buds from mature trees

A reliable procedure for multiple-shoot induction and plantlet regeneration was developed with apical buds collected from 7- to 8-year-old trees of Ficus carica L. using Murashige and Skoog's (MS) medium supplemented with 2.0 mg/l 6-benzylaminopurine and 0.2 mg/l 1-naphthaleneacetic acid. The in-vitro-regenerated shoots were further multiplied on MS medium supplemented with 2.0 mg/l 6-benzylaminopurine and 0.2 mg/l 1-naphthaleneacetic acid and an average multiplication rate of four per subculture was established with 90% success. Excised shoots were rooted in liquid half strength MS medium supplemented with 2.0 mg/l indole-3-butyric acid and 0.2% activated charcoal. Regenerated plantlets were successfully established in soil, with a success rate of 68%. Received: 28 July 1997 / Revision received: 15 January 1998 / Accepted: 7 February 1998     

Degradation of phenol and phenolic compounds by a defined denitrifying bacterial culture

Phenol, a major pollutant in several industrial waste waters is often used as a model compound for studies on biodegradation. This study investigated the anoxic degradation of phenol and other phenolic compounds by a defined mixed culture of Alcaligenes faecalis and Enterobacter species. The culture was capable of degrading high concentrations of phenol (up to 600 mg/l) under anoxic conditions in a simple minimal mineral medium at an initial cell mass of 8 mg/l. However, the lag phase in growth and phenol removal increased with increase in phenol concentration. Dissolved CO₂ was an absolute requirement for phenol degradation. In addition to nitrate, nitrite and oxygen could be used as electron acceptors. The kinetic constants, maximum specific growth rate _max; inhibition constant, K _i and saturation constant, K _s were determined to be 0.206 h^–1, 113 and 15 mg phenol/l respectively. p-Hydroxybenzoic acid was identified as an intermediate during phenol degradation. Apart from phenol, the culture utilized few other monocyclic aromatic compounds as growth substrates. The defined culture has remained stable with consistent phenol-degrading ability for more than 3 years and thus shows promise for its application in anoxic treatment of industrial waste waters containing phenolic compounds.     

Kinetic models for astaxanthin production by high cell density mixotrophic culture of the microalga Haematococcus pluvialis

High cell density cultivation of Haematococcus pluvialis for astaxanthin production was carried out in batch and fed-batch modes in 3.7-L bioreactors with stepwise increased light intensity control mode. A high cell density of 2.65 g L⁻¹ (batch culture) or 2.74 g L⁻¹ (fed-batch culture) was obtained, and total astaxanthin production in the fed-batch culture (64.36 mg L⁻¹) was about 20.5% higher than in the batch culture (53.43 mg L⁻¹). An unstructured kinetic model to describe the microalga culture system including cell growth, astaxanthin formation, as well as sodium acetate consumption was proposed. Good agreement was found between the model predictions and experimental data. The models demonstrated that the optimal light intensity for mixotrophic growth of H. pluvialis in batch or fed-batch cultures in a 3.7-L bioreactor was 90–360 μmol m⁻² s⁻¹, and that the stepwise increased light intensity mode could be replaced by a constant light intensity mode. Received 24 December 1998/ Accepted in revised form 23 April 1999     

Fed-batch cultivation for the production of clavulanic acid by an immobilized Streptomyces clavuligerus mutant

In order to obtain high productivity of clavulanic acid, a newly-introduced carrier, polyurethane pellet (PUP) Z97-020 was used for the immobilization process. In a stirred-tank bioreactor, batch cultivation by Streptomyces clavuligerus KK immobilized on PUP Z97-020 gave about 3100 mg of clavulanic acid per litre, representing an increase of 200% in productivity compared with that by fed-batch cultivation of free cells (1500 mg/l). However, the clavulanic acid produced rapidly decomposed due to the pH change during batch cultivation. Fed-batch cultivation by immobilized S. clavuligerus KK gave an excellent level of clavulanic acid up to 3250 mg/l, a productivity increase of 220% compared with that by fed-batch cultivation of free cells. These results suggest that immobilization with PUP Z97-020 is a more effective process for the production of clavulanic acid and that the maintenance of pH by fed-batch cultivation with glycerol as a limiting substrate prevents the clavulanic acid from decomposing during the fermentation.     

Biodegradation of phenol by arthrobacter and modelling of the kinetics

The toxic effects of phenol, a common constituent of many industrial effluents, necessitates treatment of the polluted streams. Biodegradation is a popular technique and enjoys many advantages. The degradation of phenol with Arthrobacter species is studied in batch cultures and it is observed that the substrate is inhibiting. The fit of various models, including the model proposed earlier by us [17], to the experimental data is studied. The model is used to fit available data in literature, which unfortunately is very sparse. In all the cases the present model fits the data best.List of Symbols S mg/l substrate concentration - S ₀ mg/l threshold substrate concentration - K _I mg/l inhibition constant - K _m , K _s mg/l half saturation constant of growth kinetics - m, n constants - 1/h specific growth rate - _m 1/h maximal specific growth rate - X mg/l biomass concentration at time t - X ₀ mg/l initial biomass concentration Abbreviations MTCC Microbial Type Culture Collection - IMTECH Institute of Microbial Technology The cooperation of the staff of the Biosciences and Biotechnology Center, I.I.T. Madras is greatly appreciated.     

Modeling of the pyruvate production with Escherichia coli: comparison of mechanistic and neural networks-based models

Three different models: the unstructured mechanistic black-box model, the input–output neural network-based model and the externally recurrent neural network model were used to describe the pyruvate production process from glucose and acetate using the genetically modified Escherichia coli YYC202 ldhA::Kan strain. The experimental data were used from the recently described batch and fed-batch experiments [ Zelić B, Study of the process development for Escherichia coli-based pyruvate production. PhD Thesis, University of Zagreb, Faculty of Chemical Engineering and Technology, Zagreb, Croatia, July 2003. (In English); Zelić et al. Bioproc Biosyst Eng 26:249–258 (2004); Zelić et al. Eng Life Sci 3:299–305 (2003); Zelić et al Biotechnol Bioeng 85:638–646 (2004)]. The neural networks were built out of the experimental data obtained in the fed-batch pyruvate production experiments with the constant glucose feed rate. The model validation was performed using the experimental results obtained from the batch and fed-batch pyruvate production experiments with the constant acetate feed rate. Dynamics of the substrate and product concentration changes was estimated using two neural network-based models for biomass and pyruvate. It was shown that neural networks could be used for the modeling of complex microbial fermentation processes, even in conditions in which mechanistic unstructured models cannot be applied.     

Plant regeneration from meristem-derived callus protoplasts of apple (Malus3domestica cv. `Fuji')

A procedure has been established for regeneration from meristem-derived callus protoplasts of scion cultivars of apple that have been difficult to regenerate from leaf protoplasts. Calli were induced from the meristem of apples, Malus×domestica cvs `Fuji' and `Jonagold' and Malus prunifolia var `ringo Asami Mo84-A', cultured on MS medium (2 mg/l 2,4-D, 1 mg/l BA, 0.8% agar) and subcultured in a liquid medium. The ability to regenerate plants from suspension calli was studied under eight different combinations with respect to IAA, ABA, and TDZ concentrations. With the materials studied here, two combinations, one with 0.1 mg/l IAA, 0.1 mg/l ABA, and 2.0 mg/l TDZ and another with 0.1 mg/l IAA, 1.0 mg/l ABA, and 2.0 mg/l TDZ, were effective for plant regeneration. Protoplasts were isolated from the above suspension cultures and then cultured in KM8P medium containing IBA (2 mg/l), BA (1 mg/l), 2,4-D (0.4 mg/l), and MES (5 mM, pH 5.7). Shoot formation of protoplast-derived calli was studied in the above-mentioned regeneration media. The high concentration of Gelrite (0.5% and 0.7%) was also shown to be important for shoot formation of protoplast-derived calli. Shoot primordia were formed in the medium containing IAA (0.1 mg/l), ABA (1.0 mg/l), and TDZ (2.0 mg/l). Ultimately, five regenerants of `Fuji' protoplasts were obtained from 200 protoplast-derived calli. Received: 19 June 1998 / Revision received: 9 October 1998 / Accepted: 27 October 1998     

Selective inhibition of methanogenesis to enhance ethanol and n-butyrate production through acetate reduction in mixed culture fermentation

Acetate reduction is an alternative digestion process to convert organic waste into ethanol. Using acetate for fuel ethanol production offers the opportunity to use organic waste materials instead of sugar-containing feedstock. Methanogenesis, however, competes with acetate reduction for acetate and hydrogen and lowers the final efficiency. The aim of this research is to selectively inhibit methanogenesis and to enhance acetate reduction. Acetate reduction was stimulated in batch tests at pH between 4.5 and 8; and at pH 6 with and without thermal pre-treatment. It was found that methanogenesis was selectively inhibited while acetate reduction was enhanced after thermal pre-treatment incubated at pH 6. Initially the acetate reduction yielded 7.7 ± 3.2 mM ethanol with an efficiency of 60.2 ± 8.7%, but later on it was consumed to form 7.02 ± 0.85 mM n-butyrate with an efficiency of 76.2 ± 14.0%. It was the first time demonstrated that n-butyrate can be produced by mixed cultures from only acetate and hydrogen.     

Activation of an indigenous microbial consortium for bioaugmentation of pentachlorophenol/creosate contaminated soils

Soil activation, a concept based on the cultivation of biomass from a fraction of a comtaminated soil for subsequent use as an inoculum for bioaugmentation of the same soil, was studied as a method for the aerobic biodegradation of pentachlorophenol (PCP) and polycyclic hydrocarbons (PAH) in contaminated soils. A microbial consortium able to degrade PCP and PAH in contaminated soil from wood-preserving facilities was isolated and characterized for PCP degradation and resistance. To obtain an active consortium from the contaminated soil in a fed-batch bioreactor, the presence of soil as a support or source of nutrients was found to be essential. During the 35 days of bioreactor operation, residual PCP in solution remained near zero up to a loading rate of 700mg/l per day. The PCP meneralization rate increased from 70 mg/l per day when no PCP was added to the bioreactor to 700 mg/l per day at the maximum loading rate. The consortium tolerated a PCP concentration of 400 mg/l in batch experiments. Production of a PCP-degrading consortium in a fed-batch slurry bioreactor enhanced the activity of PCP biodegradation by a factor of ten. PAH biodegradation increased, during the same time period, by a factor of 30 and 81 for phenanthrene and pyrene, respectively. Preliminary laboratory-scale results indicated that a significant reduction in the time required for degradation of PCP and PAH in contaminated soil could be achieved using activated soil as an inoculum.Issued as NRC 33861 correspondence to: R. Samson     

Microflora of dissimilative nitrate reduction in a denitrifying reactor

The predominant denitrifiers and ammonifiers from methanogenic, aerobic and denitrifying reactor sludge were isolated and characterised. The population of ammonifiers increased by three orders of magnitude during the operation of the denitrifying reactor treating landfill leachate. The predominant ammonifiers were enterobacteria, and the predominant denitrifiers belonged to the genera Alcaligenes and Pseudomonas. Studies in pure culture showed that ammonia production by ammonifiers was favoured by fermentable substrates and by high C/N ratios. For acetate, only nitrite was obtained as the reduction product of nitrate, even at high C/N ratios. Furthermore, for glucose, nitrite addition caused a shift in fermentation products, with an increase in the acetate/ethanol ratio, with no significant differences in growth rates. Received: 14 January 1998 / Received revision: 11 May 1998 / Accepted: 21 May 1998     

On-line monitoring and modelling based process control of high rate nitrification — lab scale experimental results

This paper presents a new concept for the control of nitrification in highly polluted waste waters. The approach is based on mathematical modelling. To determine the substrate degradation rates of the microorganisms involved, a mathematical model using gas measurement is used. A fuzzy-controller maximises the capacity utilisation efficiencies. The experiments carried out in a lab-scale reactor demonstrate that even with highly varying ammonia concentrations in the influent, the nitrogen concentrations in the effluent can be kept within legal limits.List of Symbols c mg/l concentration - c mg/l gas concentration - H ₂ Henry-coefficient - k _L a 1/h mass transfer coefficient - mol/l dissociation constant - K _iS mg/l substrate inhibitor constant - k _iH mg/l inhibitor constant - k _S mg/l saturation constant - K _O2 mg/l oxygen saturation constant - r(B) mg/lh growth rate - r(S) mg/lh degradation reaction rate - t _v h retention time - T °C temperature - V 1 volume - V 1/h flow rate - Y g/g yield coefficient - k _b capacity utilisation efficiency - 1/h specific growth rate     

Micropropagation of Bambusa edulis through nodal explants of field-grown culms and flowering of regenerated plantlets

Nodal explants obtained from 10-year-old field-grown culms of Bambusa edulis produced multiple shoots on a Murashige-and-Skoog-based medium supplemented with 0.1 mg/l of thidiazuron (TDZ). Hundreds of regenerated shoots rooted well on a medium supplemented with 0.01 mg/l TDZ and 0.5 mg/l 2,4-dichlorophenoxyacetic acid and were successfully transferred to soil for field trials. Albinism occurred at the rate of about 30% among the regenerated shoots, and isolated albino shoots also proliferated on the medium containing TDZ. Some of the green and albino shoots also flowered on the medium containing TDZ. A potted plant also flowered and survived after flowering. Received: 20 August 1997 / Revision received: 12 December 1997 / Accepted: 12 January 1998