Itaconic acid production by Aspergillus terreus on raw starchy materials

Starchy materials such as corn starch, soft wheat flour, potato flour, cassava flour, sorghum starch, sweet potato and industrial potato flours, either acid or enzymatically hydrolysed, were used as substrates for itaconic acid production by Aspergillus terreus NRRL 1960. Both production and yield were highest on corn starch (18·4 g l⁻¹ and 34·0%, respectively). The degree of hydrolysis had a great influence on acid production which was highest when corn starch was saccharified at 85 DE (dextrose equivalent). In a 3 litre benchtop fermenter, itaconic acid production and productivity were 19·8 g l⁻¹ and 0·13 g l⁻¹ h⁻¹, respectively.     

Water‐based measurement of rainbow trout melatonin

Melatonin (MLT) was isolated from water samples using solid phase extraction cartridges and measured by radioimmunoassay. In tanks of rainbow trout Oncorhynchus mykiss , concentrations of MLT in water increased rapidly from <0·1 ng l⁻¹ in the light phase to 0·7 ng l⁻¹ in the dark giving calculated release rates of <1 ng kg⁻¹ h⁻¹ and 15 ng kg⁻¹ h⁻¹, respectively. This cycle in water MLT values corresponds to reported changes in plasma MLT concentrations. The colour of the tanks and a daytime acute handling stress did not affect concentrations of MLT in either the light or dark phase.     

Structural stability of Sclerotium rolfsii ATCC 201126 β-glucan with fermentation time: a chemical, infrared spectroscopic and enzymatic approach

Aims:  Sclerotium rolfsii ATCC 201126 exopolysaccharides (EPSs) recovered at 48 h (EPS I) and 72 h (EPS II) of fermentation, with differences in rheological parameters, hydrogel topography, salt tolerance, antisyneresis, emulsifying and suspending properties, were subjected to a polyphasic characterization in order to detect structural divergences.
Methods and Results:  Fermenter-scale production led to productivity ( P _r) and yield ( Y _P/C) values higher at 48 h ( P _r = 0·542 g l⁻¹ h⁻¹; Y _P/C = 0·74) than at 72 h ( P _r = 0·336 g l⁻¹ h⁻¹; Y _P/C = 0·50). Both EPSs were neutral glucose-homopolysaccharides with a β-(1,3)-glycosidic backbone and single β-(1,6)-glucopyranosyl sidechains regularly attached every three residues in the main chain, as revealed by chemical analyses. The infra-red diagnostic peak at 890 cm⁻¹ confirmed β-glycosidic linkages, while gentiobiose released by β-(1,3)-glucanases confirmed single β-1,6-glycosidic branching for both EPSs.
Conclusions:  The true modular repeating unit of S. rolfsii ATCC 201126 scleroglucan could be resolved. Structural stability was corroborated and no structural differences could be detected as to account for the variations in EPSs behaviour.
Significance and Impact of the Study:  Recovery of S. rolfsii ATCC 201126 scleroglucan at 48 h might be considered based on better fermentation kinetic parameters and no detrimental effects on EPS structural features.     

Lactic acid production by Lactobacillus delbrueckii in a dual reactor system using packed bed biofilm reactor

Aims:  An integrated dual reactor system for continuous production of lactic acid by Lactobacillus delbrueckii using biofilms developed on reticulated polyurethane foam (PUF) is demonstrated.
Methods and Results:  Lactobacillus delbrueckii was immobilized on PUF, packed in a bioreactor and used in lactic acid fermentation. The rate of lactic acid production was significantly high with a volumetric productivity of 5 g l⁻¹ h⁻¹ over extended period of time. When coupled to a bioreactor, the system could be operated as dual reactor for over 1000 h continuously without augmentation of inoculum and no compromise on productivity.
Conclusions:  Polyurethane foams offer an excellent support for biofilm formation.
Significance and Impact of the Study:  The system was very robust and could be operated for prolonged period at a volumetric productivity of 4–6 g l⁻¹ h⁻¹.     

Ammonium ion affecting tylosin production by Streptomyces fradiae NRRL 2702 in continuous culture

Nitrogen regulation in tylosin production by Streptomyces fradiae NRRL 2702 was studied in chemostat culture using a soluble synthetic medium. The maximum value of specific tylosin formation rate ( q _TYL) was 1·13 mg g⁻¹ h⁻¹ at the specific growth rate (μ) of 0·05 h⁻¹, and q _TYL decreased with increasing levels of the specific growth rate after reaching a rate of 0·1 h⁻¹. The optimum conditions for tylosin formation were that the specific ammonium ion uptake rate ( q _N) and μ were 0·13 mmol g⁻¹ h⁻¹ and 0·05 h⁻¹, respectively. The specific formation rates of threonine dehydratase (TDT) and tylosin were repressed by high levels of specific ammonium ion uptake rate. This study showed the adaptation to chemostat cultures of the nitrogen regulation of tylosin fermentations.     

Effects of feeding and induction strategy on the production of BmR1 antigen in recombinant E. coli

Aim:  To investigate the effects of feeding and induction strategies on the production of Bm R1 recombinant antigen.
Methods and Results:  Fed-batch fermentation was studied with respect to the specific growth rate and mode of induction to assess the growth potential of the bacteria in a bioreactor and to produce high yield of Bm R1 recombinant antigen. Cells were grown at a controlled specific growth rate (μ_set) during pre-induction, followed by constant feeding postinduction. The highest biomass (24·3 g l⁻¹) was obtained during fed-batch process operated at μ_set of 0·15 h⁻¹, whereby lower μ_set (0·075 h⁻¹) gave the highest protein production (9·82 mg l⁻¹). The yield of Bm R1 was increased by 1·2-fold upon induction with 1 mmol l⁻¹ IPTG (isopropyl-β- d -thiogalactoside) compared to using 5 mmol l⁻¹ and showed a further 3·5-fold increase when the culture was induced twice at the late log phase.
Conclusions:  Combination of feeding at a lower μ_set and twice induction with 1 mmol l⁻¹ IPTG yielded the best result of all variables tested, promising an improved method for Bm R1 production .
Significance and Impact of the Study:  This method can be used to increase the production scale of the Bm R1 recombinant antigen to meet the increasing demand for Brugia Rapid^™, a commercial diagnostic test for detection of brugian filariasis.     

Formation and microbial community analysis of chloroanilines-degrading aerobic granules in the sequencing airlift bioreactor

Aims:  This paper investigates a selection-based acclimation strategy for improving the performance and stability of aerobic granules at a high chloroanilines loading.
Methods and Results:  The experiments were conducted in a sequencing airlift bioreactor (SABR) to develop aerobic granules fed with chloroanilines (ClA). The evolution of aerobic granulation was monitored using image analysis and scanning electron microscopy, and PCR–DGGE analysis of microbial community was performed. The sludge granulation was apparently developed by decreased settling time and gradual increased ClA loading to 0·8 kg m⁻³ day⁻¹. A steady-state performance of the granular SABR was reached at last, as evidenced by biomass concentration of 6·3 g l⁻¹ and constant ClA removal efficiency of 99·9%. The mature granules had a mean size of 1·55 mm, minimal settling velocity of 68·4 m h⁻¹, specific ClA degradation rate of 0·181 g gVSS⁻¹ day⁻¹. Phylogenetic analysis of aerobic ClA-degrading granules confirmed the dominance of β - , γ -Proteobacteria and Flavobacteria.
Conclusions:  The chosen operating strategy involving step increase in ClA loading and enhancement of major selection pressures was successful in cultivating the aerobic ClA-degrading granules.
Significance and Impact of the Study:  This research could be helpful for improving the stability of aerobic granules via optimizing operating conditions and developing economic feasible full-scale granular bioreactor.     

Response surface methodology to optimize the nutritional parameters for enhanced production of jasmonic acid by Lasiodiplodia theobromae

Aims:  To find out the cumulative effect of the nutritional parameters and to enhance the production of jasmonic acid (JA) in static fermentation by Lasiodiplodia theobromae using response surface methodology (RSM).
Method and Results:  Malt extract, sucrose, NaNO₃ and MgSO₄.7H₂O were analysed by a 30-trial central composite design using RSM for optimizing their concentrations in the medium and the effect of their mutual interaction on JA production. Sucrose and NaNO₃ were found highly significant in influencing the JA production. Malt extract and MgSO₄.7H₂O showed an effect on the JA production in interaction with other variables. When the optimum values of the parameters obtained through RSM (19·95 g l⁻¹ malt extract, 50 g l⁻¹ sucrose, 7·5 g l⁻¹ NaNO₃ and 3·51 g l⁻¹ MgSO₄.7H₂O) were applied, 32% increase in JA production (299 mg l⁻¹) was observed in comparison with 225 mg l⁻¹ of JA produced with same media components not analysed by RSM and subsequently validated the statistical model.
Conclusions:  Increase in JA production was achieved by optimizing the nutritional parameters.
Significance and Impact of the Study:  This is the first report of using RSM for optimizing a medium for JA production. It resulted in an increase in JA production without augmentation of costly additives.     

Isolation of a kojic acid-producing fungus capable of using starch as a carbon source

A fungal strain (S33-2), able to grow on cooked starch and produce a substantially high level of kojic acid, was isolated from morning glory flower ( Bixa orellana ). The fungus was characterized and identified as Aspergillus flavus. The effect of different types of starch (sago, potato and corn starch) on growth of strain S33-2 and kojic acid production was examined using shake flasks. It was found that strain S33-2 grew well on all types of starch investigated. However, kojic acid production was highest when corn starch was used, with the maximum kojic acid obtained being comparable to fermentation using glucose. The highest kojic acid production (19·2 g l⁻¹) was obtained when 75 g l⁻¹ corn starch was used. This gave a yield, based on starch consumed, and an overall productivity of 0·256 g g⁻¹ and 0·04 g l⁻¹ h⁻¹, respectively.     

Survey of wound-induced ethylene production by excised root segments

Ethylene production was measured from excised 10-mm apical and subapical root segments from 50 cultivars in 19 species of 7 families. Monocotyledonous species tended to have much lower rates of ethylene production than dicotyledonous species. Ethylene production was generally higher in apical root segments than in subapical segments within 1 h of wounding. However, cultivars of Cucumis melo , C. sativus , Helianthus annuus , Hibiscus esculentus , and Zea mays had higher rates of ethylene production from subapical segments. In apical root segments, Phaseolus aureus cv. Berken had the highest ethylene production rate (76.7 ηl g⁻¹ h⁻¹), while Zea mays cv. Silver Queen had the lowest rate (0.6 ηl g⁻¹ h⁻¹). In subapical root segments, Cucumis sativus cv. Armenian had the highest rate (55.7 ηl g⁻¹ h⁻¹), while Zea mays cv. Silver Queen again had the lowest rate (0.6 ηl g⁻¹ h⁻¹). The many different responses in magnitude and kinetics of wound-induced ethylene production among the species, cultivars and tissues should provide interesting and useful systems with which to study wound responses and induced ethylene production.     

Effect of feeding level and feeding frequency on specific dynamic action in Silurus meridionalis

The effect of feeding level ( F _L; 0·5 to 4% dry diet mass per wet fish body mass) and feeding frequency (once every 4 days to twice per day) on postprandial metabolic response was investigated in southern catfish Silurus meridionalis at 27·5° C. The results showed that there was no significant difference in the specific dynamic action (SDA) coefficient among the groups of different feeding levels ( P  > 0·05). The duration increased from 26·0 to 40·0 h and the peak metabolic rate increased from 207·8 to 378·8 mg O₂ kg⁻¹ h⁻¹ when the feeding level was increased from 0·5 to 4%. The relationship between the peak metabolic rate ( R _P, mg O₂ kg⁻¹ h⁻¹) and F _L could be described as: R _P = 175·4 + 47·3 F _L( r ² = 0·943, n  = 40, P  < 0·001). The relationship between the SDA duration ( D , h) and F _L could be described as D =30·97 F _L^0·248 ( r ²=0·729, n =40, P  < 0·001).     

The content of prostaglandins and their precursors in Mortierella and Cunninghamella species

Five strains of filamentous fungi belonging to the genera Mortierella and Cunninghamella were examined for the content of dihomo-γ-linolenic, arachidonic, eicosapentaenoic acids and prostaglandins (type E₂ and F _2α). Prostaglandins were detected using an ELISA method in mycelia of all tested strains (range 50–4800 ng g⁻¹ of PGE₂ and 6–30 ng g⁻¹ of PG F _2α). Several micro-organisms also produced prostaglandins in the culture medium (2·2–137·6 μg l⁻¹ for PGE₂ and 0·4–7·8 μg l⁻¹ for PG F _2α).     

Temporal and spatial variation in growth of juvenile Atlantic salmon

Spatial and temporal variation in length‐at‐age and environmental factors affecting variation in growth rate of juvenile Atlantic salmon Salmo salar were studied using data from a long‐term study in the River Stjørdalselva, central Norway. Mean annual instantaneous growth rate among 1+ and 2+year juvenile Atlantic salmon varied between 0·59 and 1·50 g g⁻¹ year⁻¹ and mean instantaneous daily growth rate of young‐of‐the‐year (YOY) varied between 0·013 and 0·033 g g⁻¹ day⁻¹. Between year variation in growth was larger than the within year intra‐watercourse spatial variation. For YOY and 1+year Atlantic salmon, a major part of the observed between year variation in growth rates was explained by variation in mean daily water discharge and spring temperature. For 2+year juvenile Atlantic salmon, mean daily water discharge and cohort density were the only variables to significantly explain variation in growth rates. A large part of the within water‐course spatial variation could not be explained by temperature variations and juvenile Atlantic salmon in the uppermost areas of the river, experiencing the lowest ambient temperatures during the growth period, displayed the highest growth rates. Within the baselines set by temperature, biotic and abiotic factors connected to water flow regime and variation in food availability are suggested to be a major determinants of the temporal and spatial variation in juvenile Atlantic salmon growth rates.     

Physiological impact of sea lice on swimming performance of Atlantic salmon

Atlantic salmon Salmo salar were infected with two levels of sea lice Lepeophtheirus salmonis (0·13 ± 0·02 and 0·02 ± 0·00 sea lice g⁻¹). Once sea lice became adults, the ventral aorta of each fish was fitted with a Doppler cuff to measure cardiac output ( ̇ ), heart rate ( f _H) and stroke volume ( V _S) during swimming. Critical swimming speeds ( U _crit) of fish with higher sea lice numbers [2·1 ± 0·1 BL (body lengths) s⁻¹] were significantly lower ( P  < 0·05) than fish with lower numbers (2·4 ± 0·1 BL s⁻¹) and controls (sham infected, 2·6 ± 0·1 BL s⁻¹). After swimming, chloride levels in fish with higher sea lice numbers (184·4 ± 11·3 mmol l⁻¹) increased significantly (54%) from levels at rest and were significantly higher than fish with fewer lice (142·0 ± 3·7 mmol l⁻¹) or control fish (159·5 ± 3·5 mmol l⁻¹). The f _H of fish with more lice was 9% slower than the other two groups at U _crit. This decrease resulted in ̇ not increasing from resting levels. Sublethal infection by sea lice compromised the overall fitness of Atlantic salmon. The level of sea lice infection used in the present study was lower than has previously been reported to be detrimental to wild Atlantic salmon.     

Isolation and characterization of alginate-degrading bacteria for disposal of seaweed wastes

Aims:  Isolation of novel alginate degrading bacteria for the disposal of seaweed waste in composting process.
Methods and Results:  Decomposition of alginate polymers was checked by the 3,5-dinitrosalicylic acid (DNS) method for reducing sugar, and absorbance at 235 nm for unsaturated sugar. A bacterium A7 was isolated from wakame compost and confirmed to belong to the genus Gracilibacillus by partial 16S rDNA analysis. The optimum condition for the growth of A7 in a medium containing 5 g l⁻¹ of sodium alginate is as follows: pH, 8·5–9·5; NaCl, 0·5 mol l⁻¹; temperature, 30°C and polypeptone as nutrient content, 2–5 g l⁻¹. In a laboratory-scale composting experiment, the alginate content in wakame compost decreased to 14·3% after 72 h of composting from an initial value of 36%, indicating the effectiveness of alginate decomposition of A7 in wakame composting.
Conclusions:  The bacterium A7 was found to be alginate lyase-producing in genus Gracilibacillus and effective in degrading alginate to oligosaccharides in wakame during composting process.
Significance and Impact of the Study:  Development of new methods for the disposal of marine wastes and production of functional products.     

Culture conditions affect the chemical composition of the exopolysaccharide synthesized by the fungus Aureobasidium pullulans

Aims:  To identify if culture conditions affect the chemical composition of exopolysaccharide (EPS) produced by Aureobasidium pullulans .
Methods and Results:  In batch airlift and continuously stirred tank (CSTR) reactors the EPS produced with low (0·13 g l⁻¹ N) initial NaNO₃ or (NH₄)₂SO₄ levels contained pullulan, with maltotriose as its major component, similar to that synthesized in the airlift reactor with high (0·78 g l⁻¹ N) initial NaNO₃ levels. EPS produced by CSTR grown cultures with high (NH₄)₂SO₄ levels contained little pullulan, possibly because of a population shift from unicells to mycelium. This chemical difference may explain why total EPS yields did not fall as they did with cultures grown under identical conditions with high NaNO₃ levels, where the pullulan component of the EPS disappeared. EPS synthesized in N-limiting chemostat cultures of A. pullulans changed little with growth rate or N source, being predominantly pullulan consisting of maltotriose units.
Conclusions:  While the EPS chemical composition changed little under N-limiting conditions, high initial medium N levels determined maltotriose content and/or pullulan content possibly by dictating culture morphology.
Significance and Impact of the Study:  These results emphasize the requirement of all studies to determine EPS chemical composition when examining the influence of culture conditions on EPS yields.     

Automated concentration and recovery of micro-organisms from drinking water using dead-end ultrafiltration

Aims:  Concentration of pathogens diluted in large volumes of water is necessary for their detection. An automated concentration system placed online in drinking water distribution systems would facilitate detection and mitigate the risk to public health.
Methods and Results:  A prototype concentrator based on dead-end hollow fibre ultrafiltration was used to concentrate Bacillus atrophaeus spores directly from tap water. Backflush was used to recover accumulated particulates for analysis. In field tests conducted on a water utility distribution system, 3·2 × 10⁴–1·4 × 10⁶ CFU ml⁻¹ (6·1 × 10⁶–3·0 × 10⁸ CFU) were recovered from the filter when 2·9 × 10⁷–1·0 × 10⁹ CFU were spiked into the system. Per cent recovery ranged from 21% to 68% for flow volumes of 15–21 l. Tests using spore influent levels <10 CFU l⁻¹ (spike < 1000 CFU) yielded 23–40% recovery for volumes >100 l.
Conclusions:  B. atrophaeus spores at levels <10 CFU l⁻¹ were concentrated directly from tap water using an automated dead-end hollow-fibre ultrafiltration system.
Significance and Impact of the Study:  The prototype concentrator represents a critical step towards an autonomous system that could be installed in drinking water distribution lines or other critical water lines to facilitate monitoring. Recovered samples can be analysed using standard or rapid biosensor methods.     

3-Methylindole production is regulated in Clostridium scatologenes ATCC 25775

Aims:  3-Methylindole (3-MI) is a degradation product of l -tryptophan and is both an animal waste malodorant and threat to ruminant health. Culture conditions influencing 3-MI production in Clostridium scatologenes ATCC 25775 were investigated.
Methods and Results:  Extracellular 3-MI levels in cells cultured in brain heart infusion (BHI) medium (pH 7·0) at 33°C and 37°C for 72 h were 907 ± 38 and 834 ± 121  μ mol l⁻¹, respectively. Cells cultured in tryptone-yeast (TY) extract medium at 37°C for 48 h produced 104 ± 86  μ mol l⁻¹ 3-MI; however, addition of 1 mmol l⁻¹  l -tryptophan failed to increase extracellular levels (113 ± 50  μ mol l⁻¹ 3-MI). Specific activity of indole acetic acid decarboxylase measured in BHI, TY and TY plus 1 mmol l⁻¹ tryptophan-grown cells displayed 35-, 33- and 76-fold higher levels than in semi-defined medium-grown cells.
Conclusions:  When cultured in rich medium, at 33°C or 37°C and pH 7·0, Cl. scatologenes ATCC 25775 optimally produced 3-MI. Addition of l- tryptophan to medium did not lead to significant increases in extracellular 3-MI levels. Whole cell assays indicate growth in rich medium significantly up-regulated 3-MI production.
Significance and Impact of the Study:  Information presented here may prove useful in understanding what factors influence 3-MI production in malodorous animal wastes.     

Inhibition of the tempe mould, Rhizopus oligosporus, by ammonia

The hyphal extension rate of Rhizopus oligosporus NRRL 2710 was slowed in the presence of 0·42 and 0·84 mmol NH3 l⁻¹ and inhibited by 1·3 mmol l⁻¹. Sporulation was prevented at NH3 concentrations of 0·42 mmol l⁻¹ andabove. There was no evidence of toxicity due to NH⁺₄ at concentrations up to 300 mmol l⁻¹.Independent of the concentrations of NH3 or NH⁺₄, the lower the pH value, in therange 6·0–9·0, the higher was the rate of hyphal extension. It is suggested that accumulationof toxic levels of NH3 could be responsible for the cessation of mould growth in tempe.     

Impact of clay particles on the cutaneous exchange of oxygen across the chorion of Atlantic salmon eggs

Rates of oxygen consumption for Atlantic salmon Salmo salar embryos approaching hatching were determined. Values were recorded using a 'closed system' experimental set‐up. A magnetic stirrer was used to ensure that zones of oxygen depletion did not develop in the micro‐environment surrounding the respiring eggs. Recorded values of oxygen consumption ranged from 0·0024 to 0·0038 mg O₂ egg⁻¹ h⁻¹, with a mean consumption rate of 0·0032 mg O₂ egg⁻¹ h⁻¹. The values of oxygen consumption were similar to those reported in other studies using a closed system experimental set‐up, however, they were lower than those reported in a study adopting a flow‐through system. The introduction of clay‐sized sediment to the incubation chamber created a thin film (<1 mm) of sediment on the egg surface, and resulted in reduced rates of oxygen consumption. The additional 0·3 g of clay sediment reduced oxygen consumption by an average of 41% and the addition of a further 0·2 g of clay sediment reduced consumption by an average of 98%. Two explanations for the recorded reduction in consumption were proposed: (i) the creation of a low permeability seal around the eggs restricted the availability of oxygen to the incubating embryos and (ii) the clay‐sized fine sediment physically blocked the micro‐pore canals in the egg membrane, thereby restricting oxygen uptake.