Characterization of two-substrate fermentation processes for xylitol production using recombinant Saccharomyces cerevisiae containing xylose reductase gene

Fermentation characteristics of recombinant Saccharomyces cerevisiae containing a xylose reductase gene from Pichia stipitis were investigated in an attempt to convert xylose to xylitol, a natural five-carbon sugar alcohol used as a sweetener. Xylitol was produced with a maximum yield of 0.95 g g⁻¹ xylitol xylose consumed in the presence of glucose used as a co-substrate for co-factor regeneration. Addition of glucose caused inhibition of xylose transport and accumulation of ethanol. Such problems were solved by adopting glucose-limited fed-batch fermentations where a high ratio of xylose to glucose was maintained during the bioconversion phase. The optimized two-substrate fed-batch fermentation carried out with S. cerevisiae EH13.15:pY2XR at 30°C resulted in 105.2 g l⁻¹ xylitol concentration with 1.69 g l⁻¹ h⁻¹ productivity.     

Continuous production of lactic acid from whey permeate by Lactobacillus helveticus in two chemostats in series

The effect of dilution rate on the production of lactic acid from whey permeate by Lactobacillus helveticus has been investigated. In the first chemostat of a two-stage system, total conversion (98.1%) and maximum lactic acid concentration (43.7 g l⁻¹) were obtained at a dilution rate (D_Itot) of 0.06 h⁻¹. Maximum volumetric productivities of lactic acid (8.27 g l⁻¹ h⁻¹) and biomass (1.90 g l⁻¹ h⁻¹) occurred at D_Itot of 0.40 h⁻¹. The fraction of -lactate in the product was found to increase with dilution rate and reached a maximum of 66% at the same dilution rate. The maximum specific growth rate (μ_max) on this medium was 0.7 h⁻¹. A Y_ATP (max) value of 22.4 g dry weight (mol ATP)⁻¹ and a maintenance coefficient of 8.0 mmol ATP (g dry weight h)⁻¹ were determined. The second stage, in series with the first, confirmed these results and further showed that the total residence time could be reduced by 50%, compared with a single chemostat for the same nearly complete level of substrate conversion.     

A coupled two-stage continuous fermentation for solvent production by Clostridium acetobutylicum

The effects of nitrogen and phosphate in batch and continuous AEB fermentations were tested. Both nitrogen- and phosphate-limited fermentations favored acid formation but not solvent production. A coupled two-stage continuous fermentation was performed for 30 days with a nitrogen-limited first stage fermentation for enhanced acid production. The bacteria from the acidogenic phase (first stage) fermentation were continuously pumped into a 14-l second stage fermentor with supplemental glucose and nitrogen for solvent production. The second stage fermentor had a maximum butanol productivity of 0.4 g l⁻¹ h⁻¹ (total solvent production was 0.6 g l⁻¹ h⁻¹) at a dilution rate of 0.06 h⁻¹.     

Effects of medium glucose concentration and pH on docosahexaenoic acid content of heterotrophic Crypthecodinium cohnii

The effects of medium glucose concentration and pH on growth and docosahexaenoic acid (DHA, C22:6 ω-3) content of Crypthecodinium cohnii were investigated. Over a range of glucose concentrations (5–40 g l⁻¹) investigated, the highest specific growth rate (0.12 h⁻¹), highest cell dry weight concentration (3.13 g l⁻¹) and highest growth yield on glucose (0.6 g g⁻¹) were obtained at 20 g l⁻¹ glucose. However, the highest degree of fatty acid unsaturation (3.2) and highest DHA proportion (53.4% of total fatty acids) were achieved at 5 g l⁻¹ glucose. Low glucose concentrations enhanced the degree of fatty acid unsaturation and DHA formation. Medium pH also affected cell growth, fatty acid unsaturation and DHA proportion. When medium pH was 7.2, the highest specific growth rate (0.089 h⁻¹), highest cell dry weight concentration (2.73 g l⁻¹), highest growth yield on glucose (0.564 g g⁻¹), highest degree of fatty acid unsaturation (3.4) and highest DHA proportion (56.8% of total fatty acids) were obtained. Results suggest that glucose concentration and pH value could be effectively manipulated to achieve maximum DHA production by C. cohnii.     

Efficient plant regeneration from seedling explants of two commercially important temperate eucalypt species–Eucalyptus nitens and E. globulus

A reliable and reproducible method for plant regeneration in vitro of two important temperate eucalypts, Eucalyptus nitens and E. globulus, has been developed which utilises seedling explants. Highly regenerative callus was obtained from individual cotyledon and hypocotyledon explants of both species following cultivation on Murashige and Skoog’s (MS) basal nutrient medium supplemented with 30 g l⁻¹ sucrose, 5–10% (v/v) coconut water, 0.8% agar, 1 mg l⁻¹ -naphthalene-acetic acid (NAA) and 0.5 mg l⁻¹ N⁶ benzylaminopurine (BAP). Shoot differentiation was observed 7–8 weeks after transfer of callus onto regeneration medium containing 0.5 mg l⁻¹ NAA and 1 mg l⁻¹ BAP. In a few instances, direct shoot regeneration occurred without an intervening callus phase in both species. The frequency of plant regeneration was higher for callus derived from hypocotyl segments (30–35%) compared to cotyledonary explants (20–25%) though the average number of shoots per cotyledonary explant was generally higher than for hypocotyl explants. Somatic embryos were observed occasionally in E. nitens, arising from the surface of organogenic callus. Organised structures closely resembling somatic embryos were also observed in E. globulus. Regenerated shoots (30–40%) of both species could be rooted in modified MS media containing indole-3-butyric acid (IBA) and plantlets were successfully transferred to soil.     

Arachidonic acid production by Mortierella alpina with growth-coupled lipid synthesis

The fungus Mortierella alpina LPM 301, a producer of arachidonic acid (ARA), was found to possess a unique property of a growth-coupled lipid synthesis. An increase in specific growth rate (μ) from 0.03 to 0.05 h⁻¹ resulted in a two-fold increase in the specific rate of lipid synthesis (milligram lipid (gram per lipid-free biomass) per hour). Under batch cultivation in glucose-containing media with urea or potassium nitrate as nitrogen sources, the ARA content was 46.0 and 60.4% of lipid; 16.4 and 18.8% of dry biomass; and 4.2 and 4.5 g l⁻¹, respectively. Under continuous cultivation of the strain, the productivity of ARA synthesis was 16.2 and 19.2 mg l⁻¹ h⁻¹ at μ=0.05 and 0.03 h⁻¹, respectively.     

Immobilization of Acidithiobacillus ferrooxidans by a PVA–boric acid method for ferrous sulphate oxidation

Acidithiobacillus ferrooxidans was immobilized in poly(vinyl alcohol) (PVA) by a PVA–boric acid method, and spherical beads of uniform size were produced. Biooxidation of ferrous iron by immobilized cells was investigated in repeated batch culture and continuous operation in a laboratory scale packed-bed bioreactor. During repeated batch culture, the cell-immobilized gels were stable and showed high constant iron-oxidizing activity. In continuous operation in a packed-bed bioreactor, biooxidation of ferrous iron fits a plug-flow reaction model well. A maximum Fe²⁺ oxidation rate of 1.89 g l⁻¹ h⁻¹ was achieved at the dilution rate of 0.38 h⁻¹ or higher, while no obvious precipitate was detected in the bioreactor.     

Immobilization in polyelectrolyte complex capsules: Encapsulation of a gluconate-oxidizing Serratia marcescens strain

In previous papers, it was shown that eukaryotic microbial systems can be encapsulated in polyelectrolyte complexes (PEC) prepared from sodium cellulose sulfate and poly(dimethyldiallylammonium chloride) with maintainance of vitality. In the present study, prokaryotic cells were successfully encapsulated in these PEC. Serratia marcescens B345 (IMET 11312) was chosen as a model organism. This strain converts gluconic acid to 2-ketogluconic acid. Since the 2-ketogluconic acid produced has very strong complexing properties, the number of applicable immobilization methods is restricted. Due to the high stability of PEC towards complexing agents, these problems can be overcome by the described method.

As already described in previous papers, a preimmobilization of cells in a PEC coprecipitate prior to capsule formation proved to be advantageous also for encapsulation of bacilli. The mean productivity of the encapsulated S. marcescens cells was 1–4.4 g l⁻¹ h⁻¹ in comparison to 5 g l⁻¹ h⁻¹ for free cells. The productivity was highly dependent on the flow rate of the reactor. The encapsulated cells were used for 1,200 h in a continuous biotransformation process for the production of 2-ketogluconic acid.     

Rapid production of thermostable cellulase-free xylanase by a strain of Bacillus subtilis and its properties

A Bacillus subtilis strain isolated from a hot-spring was shown to produce xylanolytic enzymes. Their associative/synergistic effect was studied using a culture medium with oat spelts xylan as xylanase inducer. Optimal xylanase production of about 12 U ml⁻¹ was achieved at pH 6.0 and 50°C, within 18 h fermentation. At 50°C, xylanase productivity obtained after 11 h in shake-flasks, 96,000 U l⁻¹ h⁻¹, and in reactor, 104,000 U l⁻¹ h⁻¹ was similar. Increasing temperature to 55°C a higher productivity was obtained in the batch reactor 45,000 U l⁻¹ h⁻¹, compared to shake-flask fermentations, 12,000 U l⁻¹ h⁻¹. Optimal xylanolytic activity was reached at 60°C on phosphate buffer, at pH 6.0. The xylanase is thermostable, presenting full stability at 60°C during 3 h. Further increase in the temperature caused a correspondent decrease in the residual activity. At 90°C, 20% relative activity remains after 14 min. Under optimised fermentation conditions, no cellulolytic activity was detected on the extract. Protein disulphide reducing agents, such as DTT, enhanced xylanolytic activity about 2.5-fold. When is used xylan as substrate, xylanase production decreased as function of time in contrast, with trehalose as carbon source, xylanase production in maintained constant for at least 80 h fermentation.     

Photosynthetic response of Myriophyllum salsugineum A.E. orchard to photon irradiance, temperature and external free CO2

The photosynthetic capacity of Myriophyllum salsugineum A.E. Orchard was measured, using plants collected from Lake Wendouree, Ballarat, Victoria and grown subsequently in a glasshouse pond at Griffith, New South Wales. At pH 7.00, under conditions of constant total alkalinity of 1.0 meq dm⁻³ and saturating photon irradiance, the temperature optimum was found to be 30–35°C with rates of 140 μmol mg⁻¹ chlorophyll a h⁻¹ for oxygen production and 149 μmol mg⁻¹ chlorophyll a h⁻¹ for consumption of CO₂. These rates are generally higher than those measured by other workers for the noxious Eurasian water milfoil, Myriophyllum spicatum L., of which Myriophyllum salsugineum is a close relative. The light-compensation point and the photon irradiance required to saturate photosynthetic oxygen production were exponentially dependent on water temperature. Over the temperature range 15–35°C the light-compensation point increased from 2.4 to 16.9 μmol (PAR) m⁻² s⁻¹ for oxygen production while saturation photon irradiance increased from 41.5 to 138 μmol (PAR) m⁻² s⁻¹ for oxygen production and from 42.0 to 174 μmol (PAR) m⁻² s⁻¹ for CO₂ consumption. Respiration rates increased from 27.1 to 112.3 μmol (oxygen consumed) g⁻¹ dry weight h⁻¹ as temperature was increased from 15 to 35°C. The optimum temperature for productivity is 30°C.     

Continuous sucrose hydrolysis by an immobilized whole yeast cell biocatalyst

An immobilized biocatalyst with invertase activity prepared by immobilization of whole yeast cells without use of any insoluble carrier was tested in tubular fixed-bed reactors from the point of view of possible application for continuous full-scale sucrose hydrolysis. At inlet sucrose concentration above 60% (w/w) and reaction temperature 60–70°C, total sucrose hydrolysis was achieved at a flow rate of 0.6–1.5 bed volumes per hour. At a flow rate about 10 bed volumes per hour, the conversion was still 0.5. The specific productivity of the biocatalyst was 3–25 h⁻¹; the productivity of the reactor was 1–9 kg l⁻¹ h⁻¹. The half-life of the biocatalyst invertase activity was 815 h at 70°C. The specific pressure drop over the biocatalyst bed was less than 23 kPa m⁻¹. The biocatalyst was proved to be fully capable of continuous sucrose hydrolysis in fixed-bed reactors.     

Antimicrobial activity of steady-state cultures of Bacillus sp. CCMI 1051 against wood contaminant fungi

The effect of changing dilution rate (D) on Bacillus sp. CCMI 1051 at dilution rates between 0.1 and 0.55 h⁻¹ in a glucose-limited medium was studied. Biomass values varied between 0.88 and 1.1 g L⁻¹ at D values of 0.15–0.35 h⁻¹. Maximal biomass productivity was found to be 0.39 g L⁻¹ h⁻¹, obtained at D = 0.35 h⁻¹ and corresponding to a 54.4% conversion of the carbon into cell mass. The highest rate of glucose consumption was 4.45 mmol g⁻¹ h⁻¹ occurring at D = 0.4 h⁻¹. The glucose concentration inside the chemostat was below the detection level starting to accumulate around 0.4 h⁻¹. Growth inhibition of fifteen strains of fungi by the broth of the steady-state cell-free supernatants was assessed. Results showed that the relative inhibition differ among the target species but was not influenced by the dilution rate changing.     

A study in UF-membrane reactor on activity and stability of nitrile hydratase from Microbacterium imperiale CBS 498-74 resting cells for propionamide production

The bioconversion of propionitrile to propionamide was catalysed by nitrile hydratase (NHase) using resting cells of Microbacterium imperiale CBS 498-74 (formerly, Brevibacterium imperiale). This microorganism, cultivated in a shake flask, at 28 °C, presented a specific NHase activity of 34.4 U mg_DCW⁻¹ (dry cell weight). The kinetic parameters, K_m and V_max, tested in 50 mM sodium phosphate buffer, pH 7.0, in the propionitrile bioconversion was evaluated in batch reactor at 10 °C and resulted 21.6 mM and 11.04 μmol min⁻¹ mg_DCW⁻¹, respectively. The measured apparent activation energy, 25.54 kJ mol⁻¹, indicated a partial control by mass transport, more likely through the cell wall.

UF-membrane reactors were used for kinetic characterisation of the NHase catalysed reaction. The time dependence of enzyme deactivation on reaction temperature (from 5 to 25 °C), on substrate concentrations (from 100 to 800 mM), and on resting cell loading (from 1.5 to 200 μg  ml⁻¹) indicated: lower diffusional control (E_a=37.73 kJ mol⁻¹); and NHase irreversible damage caused by high substrate concentration. Finally, it is noteworthy that in an integral reactor continuously operating for 30 h, at 10 °C, 100% conversion of propionitrile (200 mM) was attained using 200 μg  ml⁻¹ of resting cells, with a maximum volumetric productivity of 0.5 g l⁻¹ h⁻¹.     

Actual and potential dark respiration rates and different electron transport pathways in freshwater aquatic plants

The rates of respiratory O₂ uptake have been studied in leaves, stems and whole shoots of several freshwater plants: 6 angiosperms, 2 bryophytes and one alga. For angiosperm leaves, rates varied widely with species (30–142 μmol O₂ (gDW)⁻¹ h⁻¹), were correlated with chlorophyll content and were higher than those of the stems (13–71 μmol O₂ (gDQ)⁻¹ h⁻¹). The rates for the shoots of bryophytes (53–66 μmol O₂ (gDW)⁻¹ h⁻¹) and for the alga Cladophora glomerata (L.) Kütz. (96 μmol O₂ (gDW)⁻¹ h⁻¹) were slightly higher than those of most angiosperm stems, but lower than those for most leaves.

These plants had a significant cyanide-resistant respiration, suggesting the existence of an alternative pathway to the “classic” cytochrome system. This pathway was found to be active in all the species studied, as judged by responses to a specific inhibitor, SHAM (salicylhydroxamic acid). Measurement of electron-transport system (ETS) activity showed that there is a large electron-transport capacity which is not normally used by respiration in vivo.     

Simultaneous production of anthocyanin and triterpenoids in suspension cultures of Perilla frutescens

When cultivated in Murashige & Skoog medium supplemented with 0.2 mg l⁻¹ 2,4-dichlorophenoxy acetic acid and 0.5 mg l⁻¹ 6-benzyladenine, Perilla frutescens cells in suspension culture grew rapidly reaching about 13.6 g dry wt l⁻¹ after 12 days. The cell line produced both anthocyanin 0.9 g l⁻¹ and triterpenoids: 16 mg l⁻¹ oleanolic acid (OA), 25 mg l⁻¹ ursolic acid (UA) and 14 mg l⁻¹ tormentic acid (TA). When P. frutescens cells of 7-day-old cultures were exposed to a yeast elicitor at 0.5–5% (v/v) for 7 days, it was found that anthocyanin content peaked at 10.2% of dry weight with yeast elicitor at 1% (v/v) whereas the maximum production of oleanolic acid and ursolic acid in cultures treated with 2% (v/v) yeast elicitor was 19 and 27 mg l⁻¹, a 46 and 24% increase over the control, respectively. This is the first report of simultaneous production of both anthocyanin and triterpenoids in a single culture system.     

Semicontinuous production of lactic acid in a bioreactor coupled with nanofiltration membranes

The advantages of nanofiltration membranes coupled with a CSTR were demonstrated for the semicontinuous production of lactic acid from whey permeate. Lactic acid was removed from the growth medium while lactose was kept in the bioreactor with the bacterial cells; moreover, Mg²⁺ ions were also recycled in the bioreactor at 96% and the nanofiltrate color was greatly reduced. The highest volumetric productivity achieved with this device was 7.1 g l⁻¹ h⁻¹ and the lactate concentration was 55 g l⁻¹. The specific productivity was 3.54 h⁻¹. More than 99% of the membrane fouling after 44 h of fermentation was reversible. The initial permeate flux was restored easily by a water rinse. The performance of this type of membrane bioreactor was discussed.     

Batch and continuous fermentation of succinic acid from wood hydrolysate by Mannheimia succiniciproducens MBEL55E

Batch and continuous cultures of Mannheimia succiniciproducens MBEL55E were carried out in a complex medium containing a NaOH-treated wood hydrolysate for the production of succinic acid. The wood hydrolysate based medium was treated with NaOH before sterilization to reduce the formation of inhibitory compounds. M. succiniciproducens MBEL55E utilized xylose as well as glucose in the wood hydrolysate based medium as a carbon source for the succinic acid production. In batch cultures, the final succinic acid concentration of 11.73 g l⁻¹ was obtained from the pre-treated wood hydrolysate based medium, resulting in a succinic acid yield of 56% and a succinic acid productivity of 1.17 g l⁻¹ h⁻¹, while the corresponding continuous cultures gave the succinic acid yield and productivity of 55% and 3.19 g l⁻¹ h⁻¹, respectively. These results suggest that succinic acid can be produced economically and efficiently by the fermentation of M. succiniciproducens MBEL55E from an inexpensive biomass-based wood hydrolysate.     

Nutrient removal by thermophilic Fischerella (Mastigocladus laminosus) in a simulated algaculture process

A novel nutrient removal/waste heat utilization process was simulated using semicontinuous cultures of the thermophilic cyanobacterium Fischerella. Dissolved inorganic carbon (DIC)-enriched cultures, maintained with 10 mg l⁻¹ daily productivity, diurnally varying temperature (from 55°C to 26–28°C), a 12:12 light cycle (200 μE sec⁻¹ m⁻²) and 50% biomass recycling into heated effluent at the beginning of each light period, removed > 95% of NO₃⁻ + NO₂⁻−N, 71% of NH₃-N, 82% of PO₄³⁻ −P, and 70% of total P from effluent water samples containing approximately 400 μg l⁻¹ combined N and 60 μg l⁻¹ P. Nutrient removal was not severely impaired by an altered temperature gradient, doubled light intensity, or DIC limitation. Recycling 75% of the biomass at the end of each light period resulted in unimpaired NO₃⁻ + NO₂⁻ removal, 38–45% P removal and no net NH₃ removal. Diurnally varying P removal, averaging 50–60%, and nearly constant > 80% N removal, are therefore projected for a full-scale process with continuous biomass recycling.     

Zooplankton feeding ecology: bacterivory by metazoan microzooplankton

Bacterivory by the rotifer Brachionus plicatilis Müller, nauplii and copepodites of the copepods Centropages Krøyer sp. and Acartia tonsa Dana, and the tintinnid Favella panamensis Kofoid & Campbell was examined using fluorescently labelled bacteria (FLB) and epifluorescence microscopy. FLB were < 1 μm in diameter, and were offered at environmental concentrations (1.47−9.08 × 10⁶ cells·ml⁻¹). FLB were visible within rotifers, nauplii, copepodites, and tintinnids, confirming ingestion. Rotifer clearance rates (32–418 μl·animal⁻¹·h⁻¹) exhibited no relation with FLB concentration. In some cases rates of clearance of FLB by rotifers were different with alternative phytoplankton food (Nanochloris Naumann sp.) than in replicates with FLB alone, whereas in other cases presence of alternative food exhibited no clear effects on rates of ingestion of FLB. Clearance rates for all six naupliar stages of A. tonsa nauplii (0–320 μl·animal⁻¹·h⁻¹) were stage-related, with higher rates by NIII-VI nauplii than NI-II nauplii. Nauplii had higher rates of clearance of FLB in the absence of alternative phytoplankton food (Isochrysis Parke sp.). Clearance rates of FLB by a single stage of Centropages sp. nauplii, A. tonsa CI copepodites and F. panamensis (each obtained at only a single food concentration of either 1.5 or 5.0 × 10⁶ cells·ml⁻¹) were within the range of 85–142 μl·animal⁻¹·h⁻¹. These ranges were similar to those of rotifers and A. tonsa nauplii. This is the first report of FLB ingestion by metazoan marine microzooplankton. Although rotifers and ciliates might be expected to ingest small particles such as FLB using ciliary induced feeding currents, the means by which nauplii and copepodites eat FLB is less clear. We propose that they may “eat” bacteria as they “drink” to osmoregulate.     

Optimization of androstenedione production in an organic–aqueous two-liquid phase system

A systematic evaluation of the effect of key operational parameters on the selective cleavage of sitosterol to 4-androstene-3,17-dione (AD) with Mycobacterium sp. NRRL B-3805 in a dioctyl phthalate: aqueous buffer two-liquid phase system was performed. Of the parameters assessed, buffer composition, biomedium pH, temperature, and biomass and substrate concentration were those that mostly affected overall bioconversion rate. The optimum pH was 7.5 with Tris buffer. The highest bioconversion rate was observed at 35 °C, although at 40 °C bioconversion activity was virtually lost. Michaelis–Menten type kinetics adequately described the bioconversion system. Increasing biomass concentration from 10 to 70 g_{wet cell weight} l⁻¹ favored AD final yield, although the specific AD yield slightly decreased.