Biodegradation of shellfish wastes and production of chitosanases by a squid pen-assimilating bacterium, <Emphasis Type="Italic">Acinetobacter</Emphasis><Emphasis Type="Italic">calcoaceticus</Emphasis> TKU024

Two chitosanases (CHSA1 and CHSA2) were purified from the culture supernatant of Acinetobacter calcoaceticus TKU024 with squid pen as the sole carbon/nitrogen source. The molecular masses of CHSA1 and CHSA2 determined by SDS-PAGE were approximately 27 and 66 kDa, respectively. The optimum pH, optimum temperature, pH stability, and thermal stability of CHSA1 and CHSA2 were (pH 6, 50°C, pH 4–10, <90°C) and (pH 7, 60°C, pH 6–11, <70°C), respectively. CHSA1 and CHSA2 had broad pH and thermal stability. CHSA1 and CHSA2 were both inhibited by EDTA and were inhibited completely by 5 mM Mn²⁺. CHSA1 and CHSA2 degraded chitosan with DD ranging from 60 to 98%, and also degraded some chitin. The most susceptible substrate was 60% deacetylated chitosan. Furthermore, TKU024 culture supernatant (1.5% SPP) incubated for 5 days has the most reducing sugars (0.63 mg/ml). With this method, we have shown that shellfish wastes may have a great potential for the production of bioactive materials.     

Alginate encapsulation of shoot tips and nodal segments for short-term storage and distribution of the eucalypt Corymbia torelliana?×?C. citriodora

A protocol was developed for short-term preservation and distribution of the plantation eucalypt, Corymbia torelliana × C. citriodora, using alginate-encapsulated shoot tips and nodes as synthetic seeds. Effects of sowing medium, auxin concentration, storage temperature and planting substrate on shoot regrowth or conversion into plantlets were assessed for four different clones. High frequencies of shoot regrowth (76–100%) from encapsulated explants were consistently obtained in hormone-free half- and full-strength Murashige and Skoog (MS) sowing media. Conversion into plantlets from synthetic seeds was achieved on half-strength MS medium by treating shoot tips or nodes with 4.9–78.4 μM IBA prior to encapsulation. Pre-treatment with 19.6 μM IBA provided 62–100% conversion, and 95–100% of plantlets survived after acclimatisation under nursery conditions. Synthetic seeds containing explants pre-treated with IBA were stored for 8 weeks much more effectively at 25°C than at 4°C, with regrowth frequencies of 50–84% at 25°C compared with 0–4% at 4°C. To eliminate the in vitro culture step after encapsulation, synthetic seeds were allowed to pre-convert before sowing directly onto a range of ex vitro non-sterile planting substrates. Highest frequencies (46–90%) of plantlet formation from pre-converted synthetic seeds were obtained by transferring shoot tip-derived synthetic seeds onto an organic compost substrate. These plantlets exhibited almost 100% survival in the nursery without mist irrigation. Pre-conversion of non-embryonic synthetic seeds is a novel technique that provides a convenient alternative to somatic embryo-derived artificial seeds.     

Production of <Emphasis Type="SmallCaps">l</Emphasis>-lactic acid by <Emphasis Type="Italic">Rhizopus oryzae</Emphasis> using semicontinuous fermentation in bioreactor

Semicontinuous fermentation using pellets of Rhizopus oryzae has been recognized as a promising technology for l-lactic acid production. In this work, semicontinuous fermentation of R. oryzae AS 3.819 for l-lactic acid production has been developed with high l-lactic acid yield and volumetric productivity. The effects of factors such as inoculations, CaCO₃ addition time, and temperature on l-lactic acid yield and R. oryzae morphology were researched in detail. The results showed that optimal fermentation conditions for the first cycle were: inoculation with 4% spore suspension, CaCO₃ added to the culture medium at the beginning of culture, and culture temperature of 32–34°C. In orthogonal experiments, high l-lactic acid yield was achieved when the feeding medium was (g/l): glucose, 100; (NH₄)₂SO₄, 2; KH₂PO₄, 0.1; ZnSO₄·7H₂O, 0.33; MgSO₄·7H₂O, 0.15; CaCO₃, 50. Twenty cycles of semicontinuous fermentation were carried out in flask culture. l-lactic acid yield was 78.75% for the first cycle and 80–90% for the repeated cycles; the activities of lactate dehydrogenases (LDH) were 7.2–9.2 U/mg; fermentation was completed in 24 h for each repeated cycle. In a 7-l magnetically stirred fermentor, semicontinuous fermentation lasted for 25 cycles using pellets of R. oryzae AS 3.819 under the optimal conditions determined from flask cultures. The final l-lactic acid concentration (LLAC) reached 103.7 g/l, and the volumetric productivity was 2.16 g/(l·h) for the first cycle; in the following 19 repeated cycles, the final LLAC reached 81–95 g/l, and the volumetric productivities were 3.40–3.85 g/(l·h).     

Novel three-phase bioreactor concept for fatty acid alkyl ester production using <Emphasis Type="Italic">R. oryzae</Emphasis> as whole cell catalyst

A novel three-phase solid–gas–liquid bioreactor (SGLB) concept using gaseous alcohol and liquid rapeseed oil with immersed microorganisms overlying a nutrient agar phase (solid) is proposed for biodiesel (fatty acid alkyl esters, FAAE) production based on the high hydrophobicity and negative surface charge showed by the fungi Rhizopus oryzae. This novel bioreactor was thought to increase oil bioavailability and decrease alcohol toxicity for effective microbial growth, reaching high yields of FAAE production without any pretreatment. High growth rates were reached for R. oryzae using a SGLB simultaneously reaching a high FAAE production yield, up to 50% using methanol and up to 70% using ethanol at 144 h of incubation at 20°C. To compare the effect of gaseous alcohol, the same experiments were carried out in a three-phase solid–liquid–liquid bioreactor (SLLB), where the alcohol was added in liquid phase, showing significant R. oryzae growth but no FAAE formation. This suggests that the inhibitory effect of alcohol is more significant in lipase activity than in R. oryzae growth, and the use of alcohol in gaseous phase may decrease both of them. The experimental procedure using SGLB showed that when R. oryzae is maintained alive, it can catalyze at the same time the hydrolysis, esterification and transesterification of triglycerides from rapeseed oil, but its activity strongly depends on the used growth media. Therefore, the application of gaseous alcohol coupled with R. oryzae as immobilized whole cell catalysts may be a potential alternative to the use of commercial lipases for biodiesel production.     

Direct fermentation of potato starch in wastewater to lactic acid by<Emphasis Type="Italic">Rhizopus oryzae</Emphasis>

The fungal species ofRhizopus oryzae 2062 has the capacity to carry out a single stage fermentation process for lactic acid production from potato starch wastewater. Starch hydrolysis, reducing sugar accumulation, biomass formation, and lactic acid production were affected with variations in pH, temperature, and starch source and concentration. A growth condition with starch concentration approximately 20 g/L at pH 6.0 and 30°C was favourable for starch fermentation, resulting in a lactic acid yield of 78.3%–85.5% associated with 1.5–2.0 g/L fungal biomass produced in 36 h of fermentation.     

Thermoactive β-N-acetylhexosaminidase production by a soil isolate of Penicillium monoverticillium CFR 2 under solid state fermentation: parameter optimization and application for N-acetyl chitooligosaccharides preparation from chitin

Two fungal strains were evaluated for β-N-acetylhexosaminidase production by solid state fermentation using different agro-industrial residues such as commercial wheat bran (CWB) and shrimp shell chitin waste (SSCW), of which Penicillium monoverticillium CFR 2 a local soil isolate showed significantly (P ≤ 0.001) higher β-N-acetylhexosaminidase activity on CWB medium as compared with the activity of Fusarium oxysporum CFR 8. Fermentation parameters such as incubation temperature, incubation time, initial moisture content and inoculum concentration were optimized by statistically designed experiments, using 3**(4–1) fractional factorial design of Response Surface Methodology. The high R² (0.9512) observed during validation experiment showed the usefulness of the model. Highest level of enzyme activity (311.84 U/g IDS) was predicted at 75% (w/w) initial moisture content, 26 °C incubation temperature, 168 h incubation time and initial inoculum, at the highest concentration tested (2.95 ml spore suspension/5 g substrate). Statistical optimization yielded a 4.5 fold increase in β-N-acetylhexosaminidase activity. The crude β-N-acetylhexosaminidase showed optimum temperature of 57 ± 1 °C and pH of 3.6 and retained 50% activity after 1 h of incubation at 57 ± 1 °C. SDS–PAGE zymogram revealed crude enzyme was a monomer with an apparent molecular weight ~110 kDa. The crude enzyme formed 6.81 ± 0.03 mM/l of N-acetyl chitooligosaccharides from colloidal chitin in 24 h of incubation. HPLC analysis revealed hydrolysate contained 37.57% N-acetyl chitotriose and 62.43% N-acetyl chitohexose, indicating its potential for specific N-acetyl chitooligosaccharides production.     

Potato peel as a solid state substrate for thermostable α-amylase production by thermophilic Bacillus isolates

Summary Potato peel was found to be a superior substrate for solid state fermentation, compared to wheat bran, for the production of α-amylase by two thermophilic isolates of Bacillus licheniformis and Bacillus subtilis. Under optimal conditions, B. licheniformis produced 270 units/ml and 175 units/ml of α-amylase on potato peel and wheat bran, respectively, while the corresponding values for B. subtilis were 600 units/ml and 265 units/ml. The enzyme from B.␣licheniformis was optimally active at 90 °C and pH 9.0, while that from B. subtilis at 60 °C and pH 7.0. The nature of the experimental data permitted excellent polynomial fits, on the basis of which, two master equations, corresponding to the isolated strains, were derived for estimation of enzyme activity for any set of values of temperature, particle size, moisture, and incubation time within the indicated ranges.     

α-<Emphasis Type="SmallCaps">l</Emphasis>-Rhamnosidase of <Emphasis Type="Italic">Aspergillus terreus</Emphasis> immobilized on ferromagnetic supports

α-l-Rhamnosidase from Aspergillus terreus was covalently immobilized on the following ferromagnetic supports: polyethylene terephthalate (Dacron-hydrazide), polysiloxane/polyvinyl alcohol (POS/PVA), and chitosan. The powdered supports were magnetized by thermal coprecipitation method using ferric and ferrous chlorides, and the immobilization was carried out via glutaraldehyde. The activity of the Dacron-hydrazide (0.53 nkat/μg of protein) and POS/PVA (0.59 nkat/μg of protein) immobilized enzyme was significantly higher than that found for the chitosan derivative (0.06 nkat/μg of protein). The activity–pH and activity–temperature profiles for all immobilized enzymes did not show difference compared to the free enzyme, except the chitosan derivative that presented higher maximum temperature at 65 °C. The Dacron-hydrazide derivative thermal stability showed a similar behavior of the free enzyme in the temperature range of 40–70 °C. The POS/PVA and chitosan derivatives were stable up to 60 °C, but were completely inactivated at 70 °C. The activity of the preparations did not appreciably decrease after ten successive reuses. Apparent K _m of α-l-rhamnosidase immobilized on magnetized Dacron-hydrazide (1.05 ± 0.22 mM), POS/PVA (0.57 ± 0.09 mM), and chitosan (1.78 ± 0.24 mM) were higher than that estimated for the soluble enzyme (0.30 ± 0.03 mM). The Dacron-hydrazide enzyme derivative showed better performance than the free enzyme to hydrolyze 0.3% narigin (91% and 73% after 1 h, respectively) and synthesize rhamnosides (0.116 and 0.014 mg narirutin after 1 h, respectively).     

<Emphasis Type="Italic">Salinarchaeum laminariae</Emphasis> gen. nov., sp. nov.: a new member of the family <Emphasis Type="Italic">Halobacteriaceae</Emphasis> isolated from salted brown alga <Emphasis Type="Italic">Laminaria</Emphasis>

Halophilic archaeal strains R26^T and R22 were isolated from the brown alga Laminaria produced at Dalian, Liaoning Province, China. Cells from the two strains were pleomorphic rods and Gram negative, and colonies were red pigmented. Strains R26^T and R22 were able to grow at 20–50°C (optimum 37°C) in 1.4–5.1 M NaCl (optimum 3.1–4.3 M) at pH 5.5–9.5 (optimum pH 8.0–8.5) and neither strain required Mg²⁺ for growth. Cells lyse in distilled water and the minimum NaCl concentration required to prevent cell lysis was 8% (w/v) for strain R26^T and 12% (w/v) for strain R22. The major polar lipids of the two strains were phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester and minor phosphatidylglycerol sulfate; glycolipids were not detected. Phylogenetic analyses based on 16S rRNA genes and rpoB′ genes revealed that strains R26^T and R22 formed a distinct clade with the closest relative, Natronoarchaeum mannanilyticum. The DNA G+C content of strains R26^T and R22 was 65.8 and 66.4 mol%, respectively. The DNA–DNA hybridization value between strains R26^T and R22 was 89%. The phenotypic, chemotaxonomic and phylogenetic properties suggest that the strains R26^T and R22 represent a novel species in a new genus within the family Halobacteriaceae, for which the name Salinarchaeum laminariae gen. nov., sp. nov. is proposed. The type strain is R26^T (type strain R26^T = CGMCC 1.10590^T = JCM 17267^T, reference strain R22 = CGMCC 1.10589).     

Experimental design for the production of tensio-active agent by <Emphasis Type="Italic">Candida lipolytica</Emphasis>

The strategy of optimization using sequential factorial design was employed to enhance the tensio-active emulsifying agent produced by Candida lipolytica using soybean oil refinery residue as substrate. A full factorial design was used to evaluate the impact of three fermentation factors—amounts of refinery residue, glutamic acid and yeast extract. This allowed exclusion of the yeast extract. Full factorials designs were then sequentially used to optimize the levels of the residue and glutamic acid. The surface tension value was finally reduced to 25.29 mN/m. The maximum emulsifier activity using different substrates was within 40 h of cultivation. The surface tension of the cell-free broth containing the biosurfactant remained very stable during exposure to a wide range of pH (2–12), temperatures (0–120°C) and salinity (2–10% NaCl). The combination of an industrial waste and a cheap substrate therefore seems to be very promising for the low-cost production of potent biosurfactant.     

Encapsulation technology for short-term preservation and germplasm distribution of the African mahogany <Emphasis Type="Italic">Khaya senegalensis</Emphasis>

A protocol was developed for short-term preservation and distribution of the medicinal and timber plantation tree, Khaya senegalensis, using alginate-encapsulated shoot tips. The study assessed the effects of culture medium, storage temperature, auxin concentration and planting substrate on shoot regrowth or conversion into plantlets of four different clones. Optimal shoot growth was obtained, with high frequencies (92–100%) of shoot emergence, on Murashige and Skoog (MS) culture media containing 4.4 μM benzyladenine (BA). Encapsulated shoot tips survived longer at 25°C than at 4°C, with viability of 73–88% after 8 weeks. Conversion into plantlets was achieved on half-strength MS medium by pre-culture treatment of shoot tips with 49–490 μM indole-3-butyric acid (IBA) before encapsulation. Treatment with 245 μM IBA provided 52–98% conversion, and 90–95% of plantlets survived acclimatisation under nursery conditions. To eliminate the in vitro culture step after encapsulation, synthetic seeds were allowed to pre-convert before sowing directly onto a range of ex vitro non-sterile substrates. Highest frequencies of plantlet formation from pre-converted synthetic seeds (42–86%) were obtained by transferring synthetic seeds to organic compost, and these plantlets exhibited almost 100% survival in the nursery without mist irrigation. Pre-conversion is a novel and convenient method for producing synthetic seeds that are suitable for distribution to commercial nurseries.     

Effect of alkali treatment time and extraction time on agar from <Emphasis Type="Italic">Gracilaria vermiculophylla</Emphasis>

The effects of alkali treatment time and extraction time of native agar and alkali treated agar obtained from Gracilaria vermiculophylla were studied. The response characteristics were mainly agar yield and gel strength. Alkali treatment was carried out at 0.5, 1.0, 1.5, 2.0, 2.5, and 3.0 h. Agar yield and gel strength decreased with the increase in the time of the alkali treatment. The highest yield (15.3%) and highest gel strength (1,064 g cm⁻²) were obtained at 0.5 h, and therefore this time was used for the next experiment. The extraction of both native and alkali treated agars was carried out at 1.5, 2.0, 2.5, and 3.0 h. The best extraction time for alkali treated agar was 1.5 h, and for native agar 2.5 h. The alkali treated agar obtained with the different alkali treatment and extraction times showed higher melting (92.4–99.7°C) and gelling (35.7–39.6°C) temperatures. Native agar was lower in melting (60.2–64.1°C) and gelling (20.4–23.4°C) temperatures. The 3,6-anhydrogalactose content decreased with increasing alkali treatment time, with the opposite effect during the extraction of native and alkali treated agars.     

Identification and characterization of fermentation inhibitors formed during hydrothermal treatment and following SSF of wheat straw

A pilot plant for hydrothermal treatment of wheat straw was compared in reactor systems of two steps (first, 80°C; second, 190–205°C) and of three steps (first, 80°C; second, 170–180°C; third, 195°C). Fermentation (SSF) with Sacharomyces cerevisiae of the pretreated fibers and hydrolysate from the two-step system gave higher ethanol yield (64–75%) than that obtained from the three-step system (61–65%), due to higher enzymatic cellulose convertibility. At the optimal conditions (two steps, 195°C for 6 min), 69% of available C6-sugar could be fermented into ethanol with a high hemicellulose recovery (65%). The concentration of furfural obtained during the pretreatment process increased versus temperature from 50 mg/l at 190°C to 1,200 mg/l at 205°C as a result of xylose degradation. S. cerevisiae detoxified the hydrolysates by degradation of several toxic compounds such as 90–99% furfural and 80–100% phenolic aldehydes, which extended the lag phase to 5 h. Acetic acid concentration increased by 0.2–1 g/l during enzymatic hydrolysis and 0–3.4 g/l during fermentation due to hydrolysis of acetyl groups and minor xylose degradation. Formic acid concentration increased by 0.5–1.5 g/l probably due to degradation of furfural. Phenolic aldehydes were oxidized to the corresponding acids during fermentation reducing the inhibition level.     

Utilization of palm kernel cake for production of β-mannanase by Aspergillus niger FTCC 5003 in solid substrate fermentation using an aerated column bioreactor

The production of β-mannanase from palm kernel cake (PKC) as a substrate in solid substrate fermentation (SSF) was studied using a laboratory column bioreactor. The simultaneous effects of three independent variables, namely incubation temperature, initial moisture content of substrate and airflow rate, on β-mannanase production were evaluated by response surface methodology (RSM) on the basis of a central composite face-centered (CCF) design. Eighteen trials were conducted in which Aspergillus niger FTCC 5003 was cultivated on PKC in an aerated column bioreactor for seven days under SSF process. The highest level of β-mannanase (2117.89 U/g) was obtained when SSF process was performed at incubation temperature, initial moisture level and aeration rate of 32.5°C, 60% and 0.5 l/min, respectively. Statistical analysis revealed that the quadratic terms of incubation temperature and initial moisture content had significant effects on the production of β-mannanase (P < 0.01). A similar analysis also demonstrated that the linear effect of initial moisture level and an interaction effect between the initial moisture content and aeration rate significantly influenced the production of β-mannanase (P < 0.01). The statistical model suggested that the optimal conditions for attaining the highest level of β-mannanase were incubation temperature of 32°C, initial moisture level of 59% and aeration rate of 0.5 l/min. A β-mannanase yield of 2231.26 U/g was obtained when SSF process was carried out under the optimal conditions described above.     

Isolation of C-phycocyanin from <Emphasis Type="Italic">Synechococcus</Emphasis> sp., (<Emphasis Type="Italic">Anacystis nidulans</Emphasis> BD1)

We report a procedure for obtaining fairly pure phycocyanin from a local isolate of the cyanobacterium Synechococcus sp (Anacystis nidulans BD1). Cells were incubated with 1 mg∙mL⁻¹ of lysozyme at 37°C for 16 h with shaking. The cell-free extract was treated with activated charcoal and chitosan. The purity (A _620/280) of phycocyanin obtained after lysozyme treatment was up to 2.18, which could be improved to 4.72 after incubation with activated charcoal and chitosan. The yield of phycocyanin was 80–100 mg∙g⁻¹ dry weight of cells. The method reported here is a single-step and efficient procedure and has the potential to be adopted for large-scale production of phycocyanin.     

Effects of light and temperature on the attachment and development of <Emphasis Type="BoldItalic">Gloiopeltis tenax</Emphasis> and <Emphasis Type="BoldItalic">Gloiopeltis furcata</Emphasis> tetraspores

Two 60-day experiments were conducted to study the influence of photon flux density (PFD) and temperature on the attachment and development of Gloiopeltis tenax and Gloiopeltis furcata tetraspores. In the first experiment, tetraspores of the two Gloiopeltis species were incubated at five temperature ranges (8°C, 12°C, 16°C, 20°C, 24°C) under a constant PFD of 80 μmol photons m⁻² s⁻¹ with a photoperiod of 12:12. In a second experiment, tetraspores were incubated under five PFD gradients (30, 55, 80, 105, 130 μmol photons m⁻² s⁻¹) at a constant temperature of 16°C with a photoperiod of 12:12. Maximum density of attached tetraspores was observed at 16°C for both species. Maximum per cent of spore germinating into disc was recorded at 12–16°C for G. tenax and 8–12°C for G. furcata. Maximum per cent of discs producing erect axes for G. tenax and G. furcata were recorded at 24°C and 20°C, respectively. Light had no significant effect on tetraspore attachment and developing into disc, but it affected the growth, sprouting and survival of its discs. Under 30–55 μmol photons m⁻² s⁻¹, the discs of the two species of Gloiopeltis did not form thallus until the end of the experiment. Optimum PFD range for G. tenax discs was 80–105 μmol photons m⁻² s⁻¹, whilst it was 80–130 μmol photons m⁻² s⁻¹ for G. furcata. Results presented in this study are expected to assist the progress of artificial seeding of Gloiopeltis.     

Decline in skeletal growth of the coral Porites?lutea from the Andaman Sea, South Thailand between 1984 and 2005

Of the few studies that have examined in situ coral growth responses to recent climate change, none have done so in equatorial waters subject to relatively high sea temperatures (annual mean >27°C). This study compared the growth rate of Porites lutea from eight sites at Phuket, South Thailand between two time periods (December 1984–November 1986 and December 2003–November 2005). There was a significant decrease in coral calcification (23.5%) and linear extension rates (19.4–23.4%) between the two sampling periods at a number of sites, while skeletal bulk density remained unchanged. Over the last 46 years, sea temperatures (SST) in the area have risen at a rate of 0.161°C per decade (current seasonal temperature range 28–30°C) and regression analysis of coral growth data is consistent with a link between rising temperature and reduced linear extension in the order of 46–56% for every 1°C rise in SST. The apparent sensitivity of linear extension in P. lutea to increased SST suggests that corals in this part of the Andaman Sea may already be subjected to temperatures beyond their thermal optimum for skeletal growth. Communicated by Environment Editor Prof. Rob van Woesik     

Acid protease production by solid-state fermentation using Aspergillus oryzae MTCC 5341: optimization of process parameters

Aspergillus oryzae MTCC 5341, when grown on wheat bran as substrate, produces several extracellular acid proteases. Production of the major acid protease (constituting 34% of the total) by solid-state fermentation is optimized. Optimum operating conditions obtained are determined as pH 5, temperature of incubation of 30°C, defatted soy flour addition of 4%, and fermentation time of 120 h, resulting in acid protease production of 8.64 × 10⁵ U/g bran. Response-surface methodology is used to generate a predictive model of the combined effects of independent variables such as, pH, temperature, defatted soy flour addition, and fermentation time. The statistical design indicates that all four independent variables have significant effects on acid protease production. Optimum factor levels are pH 5.4, incubation temperature of 31°C, 4.4% defatted soy flour addition, and fermentation time of 123 h to yield a maximum activity of 8.93 × 10⁵ U/g bran. Evaluation experiments, carried out to verify the predictions, reveal that A. oryzae produces 8.47 × 10⁵ U/g bran, which corresponds to 94.8% of the predicted value. This is the highest acid protease activity reported so far, wherein the fungus produces four times higher activity than previously reported [J Bacteriol 130(1): 48–56, 1977].     

Direct fermentation of potato starch wastewater to lactic acid by Rhizopus oryzae and Rhizopus arrhizus

The biochemical kinetic of direct fermentation for lactic acid production by fungal species of Rhizopus arrhizus 3,6017 and Rhizopus oryzae 2,062 was studied with respect to growth pH, temperature and substrate. The direct fermentation was characterized by starch hydrolysis, accumulation of reducing sugar, and production of lactic acid and fungal biomass. Starch hydrolysis, reducing sugar accumulation, biomass formation and lactic acid production were affected with the variations in pH, temperature, and starch source and concentration. A growth condition with starch concentration approximately 20 g/l at pH 6.0 and 30°C was favourable for both starch saccharification and lactic acid fermentation, resulting in lactic acid yield of 0.87–0.97 g/g starch associated with 1.5–2.0 g/l fungal biomass produced in 36 h fermentation. R. arrhizus 3,6017 had a higher capacity to produce lactic acid, while R. oryzae 2,062 produced more fungal biomass under similar conditions.     

Potential of a granulovirus isolate to control <Emphasis Type="Italic">Phthorimaea operculella</Emphasis> (Lepidoptera: Gelechiidae)

In this study a Brazilian granulovirus strain, PhopGV, isolated from the potato tuber moth (PTM) Phthorimaea operculella, was investigated regarding its potential for biological control and in vivo production. The relationship between mortality of P. operculella larvae and virus concentration was determined at different temperatures on potato tubers and susceptibility of P. operculella to PhopGV was also determined on potato leaves. Virulence of PhopGV to P. operculella was not affected by temperatures from 18 to 30°C. The median lethal concentration (LC₅₀) of larvae fed on potato foliage treated with PhopGV was not higher than that verified with larvae fed on treated tubers. Optimal conditions for production of virus-infected larvae were obtained by using the virus suspensions of 41 × 10⁵, 6.3 × 10⁵ and 62 × 10⁵ OBs ml⁻¹ at 18, 24 and 30°C, which resulted in 32.0, 31.4 and 34.8% of infected larvae collected, respectively. The maximum percentage of infected larvae recovered from tubers was not affected by temperature. However, time for production of virus-infected larvae was longer at 18°C and shorter at 30°C. Persistence of PhopGV was determined on stored tubers and we observed that the virus remained effective for at least two months, causing up to 84.2% mortality of P. operculella at 1 × 10⁷ OBs ml⁻¹. The pathogen was also highly virulent to tomato pinworm, Tuta absoluta, inflicting high percentage of mortality, delaying larval growth and inhibiting pupation. This Brazilian PhopGV strain has potential to control PTM larvae on potato tubers at a broad range of temperature and can be produced in vivo using virus-treated tubers.