New bacterial culture medium for production of mosquito pathogenic bacilli using agro-poultry industrial wastes

A cost-effective technology has been developed to utilise bioorganic wastes as culture media, to produce mosquitocidal biopesticides, Bacillus sphaericus and B. thuringiensis serovar israelensis. The mosquitocidal spore/crystal toxins produced from the experimental medium (chicken feather waste, CFW+paddy husk waste, PHW) was higher than that of the conventional medium (Nutrient Yeast Extract Salt Medium, NYSM). The bacterial toxins produced from different media (NYSM, CFW, PHW, CFW+PHW) were bioassayed against mosquito vectors and the toxic effect was found to be significant. The use of chicken feathers and paddy husk wastes as bacterial culture media is cost-effective and economical for the production of these mosquito pathogenic bacilli.     

Optimization of medium composition for the production of mosquitocidal toxins from Bacillus thuringiensis subsp. israelensis

Optimization of chicken feather (CF) based culture medium for the production of Bacillus thuringiensis subsp. israelensis (Bti) biomass in combination with the agro industrial by-product (coconut cake, CC) and manganese chloride (MnCl2) has been evaluated. The biomass yield of Bti spore/crystal toxin was highest (12.06 g/L) from the test medium (CF+CC+MnCl2) compared to the reference medium (Luria Bertani, LB). Toxicity assay with Bti produced from the test medium against mosquito vectors (Culex quinquefasciatus, Anopheles stephensi and Aedes aegypti) was also satisfactory and results were comparable with bacteria produced from LB. The results suggest that Bti can be produced to the maximum extent possible as a potential mosquitocidal activity as suggested by the test medium (CF+CC+MnCl2).     

Overproduction of a mosquitocidal chloramphenicol-resistant Lysinibacillus sphaericus mutant obtained through UV irradiation

A highly active mosquitocidal mutant UV-chloramphenicol-resistant mutant (UCR-146) of Lysinibacillus sphaericus 2362 was isolated by UV irradiation and selected through resistance to chloramphenicol which was inhibitory for growth of the parent strain. The growth of UCR-146 in NYSMCL at different concentrations of chloramphenicol (20–100 µg/ml) showed high mosquitocidal activity against Culex pipiens larvae with the optimum concentration of 35µg/ml. At this level, LC₅₀ of UCR-146 was decreased about five times from that of L. sphaericus 2362. Comparative efficiency of mosquitocidal activity of UCR-146 and L. sphaericus 2362 within 28 days of consecutive growth exhibited notable stability of both cultures through seven cycles of growth in their optimal media. UCR-146 was grown in industrial by-products media for production of the binary toxin under economic conditions. Offal medium at 2% showed the highest mosquitocidal activity of UCR-146 with LC₅₀ of 0.53 PPM which was 16.6, 13, 12.3 and 3.4 times less than that produced with L. sphaericus 2362 grown in NYSM, L. sphaericus 2362 grown in offal, UCR-146 grown in NYSM and UCR-146 grown in NYSMCL, respectively. Hence, the mosquitocidal activity of L. sphaericus 2362 increased several times through ultraviolet (UV) irradiation followed by chloramphenicol resistance selection and then growth in 2% offal medium. Finally, this procedure for selecting UV-chloramphenicol-resistant mutant and the medium used could potentially be a simple and cost effective approach for obtaining and producing a highly active mosquitocidal mutant.     

杀蚊苏云金芽孢杆菌及其晶体蛋白研究进展

自从发现苏云金芽孢杆菌Bacillusthuringiensis(Bt)具有杀蚊活性以来,目前已发现多种Bt亚种或血清型对蚊虫具有杀虫活性,同时也发现了一些新的杀蚊晶体蛋白。在对杀蚊晶体蛋白的分子结构进行研究的基础上,对其的作用机理有了一定的了解。近年来利用DNA重组技术显著提高杀蚊晶体蛋白的合成和将不同菌种的杀蚊晶体蛋白进行联合表达,为有效控制蚊虫危害展示广阔前景。     

Use of various coffee industry residues for the cultivation of Pleurotus ostreatus in solid state fermentation

Studies were carried out to evaluate the feasibility of using coffee industry residues, viz. coffee husk, coffee leaves and spent coffee ground as substrates in solid state fermentation (SSF) to cultivate edible mushrooms Pleurotus. Eight strains of Pleurotus ostreatus and two strains of Pleurotus sajor‐caju were screened on a medium prepared from aqueous extract of coffee husk and agar. Based on best mycelial growth (9.68 mm/day) and biomass production (43.4 mg/plate in 9 days at 24°C), the strain P. ostreatus LPB 09 was selected for detailed studies. SSF was carried out using these substrates under different moisture conditions (45–75%) and spawn rates (2.5–25%). In general, although a 25% spawn rate appeared superior, the 10% spawn rate was recommended for all the three substrates in view of the process economics, as there was not any significant difference in the increase with 10 to 15%. The ideal moisture content for mycelial growth was 60–65% for coffee husk and spent coffee ground, and 60–70% for coffee leaves. The biological efficiency (BE), which is defined as the ratio of the weight of fresh fruiting bodies to the weight of dry substrate, multiplied by 100, and which indicates the fructification ability of the fungus for utilizing the substrate, was best with coffee husk. With coffee husk as the substrate, the first fructification occurred after 20 days of inoculation, and the biological efficiency reached about 97% after 60 days. When coffee leaves were used as the substrate, no fructification was observed even upon prolonged cultivation. With spent ground as the substrate, the first fructification occurred 23 days after inoculation and the biological efficiency reached about 90% in 50 days. There was a significant decrease in the caffeine and tannin contents (61 and 79%, respectively) of coffee husk after 60 days. It was remarkable to observe that caffeine was adsorbed onto the fruiting body (0.157%), indicating that it was not completely degraded by the fungal culture. However, no tannins were found in the fruiting body, indicating that the fungal strain was capable of degrading them. The results showed the feasibility of using coffee husk and spent coffee ground as substrates without any pre‐treatment for the cultivation of edible fungi in SSF, and provided one of the first steps towards an economical utilization of these otherwise unutilized or poorly utilized residues.     

Characterization of a new highly mosquitocidal isolate of Bacillus thuringiensis – An alternative to Bti?

The mosquito is a very important vector involved in the worldwide transmission of disease-causing viruses and parasites. Controlling the mosquito population remains one of the best means for preventing the serious infectious diseases of malaria, yellow fever, dengue, filariasis and so on and there has been an increasing interest in developing biopesticides as a useful substitute to chemical insecticides. As a result, Bacillus thuringiensis subsp. israelensis (Bti) has been extensively used due to its specificity and high toxicity to a variety of mosquito larvae. However it is prudent to seek alternatives to Bti with alternative spectra of mosquitocidal activity or that are able to overcome any resistance that might develop against Bti. The Bt S2160-1 strain was isolated from soil samples collected from Southern China and found to have a comparable mosquitocidal activity to Bti. However there were significant differences in terms of their plasmid profiles, crystal proteins produced and cry gene complement. A PCR-restriction fragment length polymorphism identification system was developed and used in order to identify novel cry-type genes and four such genes (cry30Ea, cry30Ga, cry50Ba and cry54Ba) were identified in Bt S2160-1. In conclusion, Bt S2160-1 has been identified as a potential alternative to Bti, which could be used for the control of mosquito populations in order to reduce the incidence of mosquito-borne diseases.     

Effects and mechanisms of Bacillus thuringiensis crystal toxins for mosquito larvae

Bacillus thuringiensis is a Gram‐positive aerobic bacterium that produces insecticidal crystalline inclusions during sporulation phases of the mother cell. The virulence factor, known as parasporal crystals, is composed of Cry and Cyt toxins. Most Cry toxins display a common 3‐domain topology. Cry toxins exert intoxication through toxin activation, receptor binding and pore formation in a suitable larval gut environment. The mosquitocidal toxins of Bt subsp. israelensis (Bti) were found to be highly active against mosquito larvae and are widely used for vector control. Bt subsp. jegathesan is another strain which possesses high potency against broad range of mosquito larvae. The present review summarizes characterized receptors for Cry toxins in mosquito larvae, and will also discuss the diversity and effects of 3‐D mosquitocidal Cry toxin and the ongoing research for Cry toxin mechanisms generated from investigations of lepidopteran and dipteran larvae.     

Pilot-scale production of mosquitocidal toxins by Bacillus thuringiensis and Lysinibacillus sphaericus under solid-state fermentation

Mosquitocidal toxins of Bacillus thuringiensis israelensis (Bti) and Lysinibacillus sphaericus 14N1 (Ls14N1) were produced under solid-state fermentation using agro-industrial wastes. Sugar beet pulp–sesame meal (1:1) and wheat germ meal–linen meal (1:1) at 9% were the efficient substrate mixtures for the growth and toxin production of Bti and Ls14N1, respectively. Bti was more active after the addition of beef extract (0.2%) or yeast extract (0.5%) to the medium. On the other hand, the addition of yeast extract (0.2%) or NYSM salts (2%) significantly enhanced the toxicity produced by Ls14N1. The optimum conditions for the maximum toxicity of Bti were at pH 7–8, 20–30% moisture, 4–10% inoculum and 7 days incubation. For Ls14N1, the best conditions were pH 6.5–7.5, 20–30% moisture, 4–10% inoculum and 5 days incubation. It was found that the best thickness of carrier-substrates in the plate (15?cm in diameter) for the maximum mosquitocidal activity was about 0.5?cm for Bti and 0.5–1?cm for Ls14N1. Pilot-scale production in aluminium trays applying the above conditions showed a decrement of toxicity of fermented cultures and some plates were contaminated. These problems were dissolved by reducing the moisture content to 15%, increasing inoculum to 10% and manual agitation of trays every-day.     

A novel approach for the production of natural aroma compounds using agro-industrial residue

Experiments were carried out to study the production of aroma compounds from coffee husk by a fungal culture of C. fimbriata in solid state fermentation. Hot water treated coffee husk seemed to be useful substrate for aroma production in comparison to whole coffee husk or its water extract. Raw data were integrated in order to calculate the total volatiles (TV) accumulated during the fermentation using the Gompertz model. Glucose addition between 20-35% increased significantly the yields of aroma compounds. Supplementation of leucine further improved the TV production significantly (about 58%), specially the esters. When soybean oil was added, TV production was similar to the control, showing that the fungus was not able to use soybean oil for its primary metabolism, nor it acted as precursor for the synthesis of methyl ketones as reported for other fungi. Addition of saline solution drastically decreased the volatile production. Under optimized conditions, a total of 13 compounds were produced which included alcohols (2), aldehyde (1), ketones (2) and esters (8). Ethyl acetate was the prominent compound, followed by ethanol.     

Evaluation of mosquitocidal activity of Annona squamosa leaves against filarial vector mosquito,Culex quinquefasciatus Say

Methanolic extract of leaves of A. squamosa was tested for mosquitocidal effect against C. quinquefasciatus. A liquid mosquito insecticide formulation was prepared with the extract (1, 3 and 5 %w/w) using deodorized kerosene as solvent and investigated for its knock-down and 24 hr mortality. The extract formulation produced dose dependent activity, exhibited significantly shorter knock down KD50 and KD90 values and produced significant mortality. The results suggest the potential mosquitocidal effect of A. squamosa on C. quinquefasciatus.     

Mtx toxins synergize Bacillus sphaericus and Cry11Aa against susceptible and insecticide-resistant Culex quinquefasciatus larvae

Two mosquitocidal toxins (Mtx) of Bacillus sphaericus, which are produced during vegetative growth, were investigated for their potential to increase toxicity and reduce the expression of insecticide resistance through their interactions with other mosquitocidal proteins. Mtx-1 and Mtx-2 were fused with glutathione S-transferase and produced in Escherichia coli, after which lyophilized powders of these fusions were assayed against Culex quinquefasciatus larvae. Both Mtx proteins showed a high level of activity against susceptible C. quinquefasciatus mosquitoes, with 50% lethal concentrations (LC(50)) of Mtx-1 and Mtx-2 of 0.246 and 4.13 microg/ml, respectively. The LC(50)s were 0.406 to 0.430 microg/ml when Mtx-1 or Mtx-2 was mixed with B. sphaericus, and synergy improved activity and reduced resistance levels. When the proteins were combined with a recombinant Bacillus thuringiensis strain that produces Cry11Aa, the mixtures were highly active against Cry11A-resistant larvae and resistance was also reduced. The mixture of two Mtx toxins and B. sphaericus was 10 times more active against susceptible mosquitoes than B. sphaericus alone, demonstrating the influence of relatively low concentrations of these toxins. These results show that, similar to Cyt toxins from B. thuringiensis subsp. israelensis, Mtx toxins can increase the toxicity of other mosquitocidal proteins and may be useful for both increasing the activity of commercial bacterial larvicides and managing potential resistance to these substances among mosquito populations.     

Coffee husk: an inexpensive substrate for production of citric acid by Aspergillus niger in a solid-state fermentation system

Aspergillus niger CFTRI 30 produced 1.3 g citric acid/10 g dry coffee husk in 72 h solid-state fermentation when the substrate was moistened with 0.075 M NaOH solution. Production was increased by 17% by adding a mixture of iron, copper and zinc to the medium but enrichment of the moist solid medium with (NH₄)₂SO₄, sucrose or any of four enzymes did not improve production. The production of about 1.5 g citric acid/10 g dry coffee husk at a conversion of 82% (based on sugar consumed) under standardized conditions demonstrates the commercial potential of using the husk in this way.The authors are with the Department of Microbiology and Bioengineering, Central Food Technological Research Institute, Mysore-570 013, India;     

Comparison of Bacillus thuringiensis subsp. israelensis CryIVA and CryIVB cloned toxins reveals synergism in vivo

When the gene for the mosquitocidal protein CryIVA was expressed in two strains of Bacillus thuringiensis (Bt) cured of their resident delta-endotoxin genes, the protein accumulated as large inclusions. The inclusions produced in the Bt subsp. kurstaki recipient strain were twice as soluble at alkaline pH as the inclusions produced in Bt subsp. israelensis. Solubilized protoxins were activated by treatment with mosquito gut extracts or trypsin for varying lengths of time and tested for in vitro cytotoxicity on cell lines of three genera of mosquito. CryIVA treated with any of the mosquito gut extracts for 6 h showed significant toxicity against Anopheles gambiae cells and slight activity on Culex quinquefasciatus cells. For CryIVB, the only significant cytotoxicity observed was against Aedes aegypti cells after treatment with Aedes gut extract. In in vivo bioassays, both CryIVA, purified from either of the Bt recipient strains, and CryIVB inclusions were similarly toxic to A. aegypti and A. gambiae larvae but CryIVA was 25-fold more toxic to C. quinquefasciatus. Synergism in vivo between the two toxins was revealed when results from assaying single toxins and mixtures were compared. Mixtures of CryIVA and CryIVB proved to be 5-fold more toxic to Culex than either toxin used singly and showed a reduced but similar synergism when tested against Aedes and Anopheles larvae. The synergism was not duplicated in vitro using cell lines from these three insects.     

Mosquitocidal toxins, genes and bacteria: the hit squad

Certain entomopathogenic species of bacilli and Clostridium produce one or more toxins that kill mosquito larvae even at concentrations in the picomolar range. Altogether, 19 distinct genes are known that encode mosquitocidal toxins, which vary in their potency, species specificity and mode of action. Unlike chemical insecticides, mosquitocidal bacilli used as larvicides are safe for animals and the environment, and do not affect non-pest insects. Mosquitocidal bacteria are effective to varying degrees against Culex, Anopheles and Aedes mosquito larvae, but their rapid sedimentation from the larval feeding zone, UV-light sensitivity and narrow host range have hampered their development. New genetic engineering approaches are being investigated that could overcome these limitations and allow stable expression of broad host range combinations of toxins in UV-resistant, buoyant recombinant bacteria, as discussed here by Alan Porter.     

Utilization of oil extracted from spent coffee grounds for sustainable production of polyhydroxyalkanoates

Spent coffee grounds (SCG), an important waste product of the coffee industry, contain approximately 15 wt% of coffee oil. The aim of this work was to investigate the utilization of oil extracted from SCG as a substrate for the production of poly(3-hydroxybutyrate) (PHB) by Cupriavidus necator H16. When compared to other waste/inexpensive oils, the utilization of coffee oil resulted in the highest biomass as well as PHB yields. Since the correlation of PHB yields and the acid value of oil indicated a positive effect of the presence of free fatty acids in oil on PHB production (correlation coefficient R ²?=?0.9058), superior properties of coffee oil can be probably attributed to the high content of free fatty acids which can be simply utilized by the bacteria culture. Employing the fed-batch mode of cultivation, the PHB yields, the PHB content in biomass, the volumetric productivity, and the Y _P/S yield coefficient reached 49.4 g/l, 89.1 wt%, 1.33 g/(l h), and 0.82 g per g of oil, respectively. SCG are annually produced worldwide in extensive amounts and are disposed as solid waste. Hence, the utilization of coffee oil extracted from SCG is likely to improve significantly the economic aspects of PHB production. Moreover, since oil extraction decreased the calorific value of SCG by only about 9 % (from 19.61 to 17.86 MJ/kg), residual SCG after oil extraction can be used as fuel to at least partially cover heat and energy demands of fermentation, which should even improve the economic feasibility of the process.     

Production, statistical optimization and application of endoglucanase from Rhizopus stolonifer utilizing coffee husk

Coffee cherry husk (CH) is one of the major by-products obtained from coffee processing industry and accounts to 43 ± 5.9 % of cellulose. Screening of fungal organism for cellulase production was carried out and the potential organism was identified as Rhizopus stolonifer by internal transcribed spacer’s (ITS)—5.8S rDNA analysis. A systematic study with response surface methodology (RSM) based on CCRD was used to study the interactions among the variables such as pH (3–7), moisture (40–80 %) and progression duration (72–168 h) of the fermentation process to maximize the enzyme production. Under the optimized cultivation condition, R. stolonifer synthesized 22,109 U/gds. Model validations at optimum operating conditions showed excellent agreement between the experimental results and the predicted responses with a confidence level of 95 %. Endoglucanase thus produced was utilized for ethanol production by simultaneous saccharification and fermentation and maximum of 65.5 g/L of ethanol was obtained. This fungal cellulase has also reported to be efficient detergent additives and promising for commercial use. The present study demonstrates coffee husk as a significant bioprocess substrate. Statistical optimization with major parameters for cellulase production can be highly applicable for industrial scale. Furthermore, value addition to coffee husk with sustainable waste management leading to environment conservation can be achieved.     

Biological detoxification of coffee husk by filamentous fungi using a solid state fermentation system

Studies were carried out on detoxification of coffee husk in solid state fermentation using three different strains of Rhizopus, Phanerochaete, and Aspergillus sp. Fungal strains were selected by their ability to grow on a coffee husk extract-agar medium. Using R. arrizus LPB-79, the best results on the degradation of caffeine (87%) and tannins (65%) were obtained with pH 6.0 and moisture 60% in 6 days. When P. chrysosporium BK was used, maximum degradation of caffeine and tannins were 70.8 and 45%, respectively, with coffee husk having 65% moisture and pH 5.5 in 14 days. The Aspergillus strain, isolated from the coffee husk, showed best biomass formation on coffee husk extract-agar medium. Optimization assays were conducted using factorial design, and surface response experiments with Aspergillus sp. The best detoxification rates achieved were 92% for caffeine and 65% for tannins. The results showed good prospects of using these fungal strains, in particular Aspergillus sp., for the detoxification of coffee husk.     

Biotechnological management of coffee pulp — isolation,screening, characterization,selection of caffeine-degrading fungi and natural microflora present in coffee pulp and husk

A total of 272 strains of filamentous fungi were isolated from soil, leaves of coffee plants and coffee cherries collected in coffee-growing areas of Mexico on three semi-synthetic culture media containing coffee extract, coffee extract with sucrose and coffee pulp extract. The isolated strains were purified by conventional techniques and identified by microscopic examination. Strains were selected on the basis of their caffeine-degrading ability in well-defined liquid medium containing caffeine. Most of the isolated microorganisms belong to Aspergillus, Penicillium, Trichoderma, Fusarium, and Humicola genera. Five strains belonging to Aspergillus species and two strains belonging to Penicillium species had the ability to degrade almost 100% of the caffeine in liquid medium. A comparative study on the evaluation of natural microflora present in coffee pulp and coffee husk revealed the presence of a wide variety of microorganisms. The percentage distribution of fungi, bacteria and yeast was almost similar in all the samples, except in coffee husk where the fungal population was slightly higher than in the other two samples. The yeast population was predominant when the coffee pulp was lyophilized immediately after pulping. However, there was a wide diversity in the microbial population with respect to selective media containing functional nutritional groups like cellulose, starch and pectin.     

Mosquitocidal toxins of bacilli and their genetic manipulation for effective biological control of mosquitoes.

The identification, cloning, and characterization of protein toxins from various species of bacilli have demonstrated the existence of mosquitocidal toxins with different structures, mechanisms of action, and host ranges. A start has been made in understanding the polypeptide determinants of toxicity and insecticidal activity, and the purification of toxins from recombinant organisms may lead to the elucidation of their X-ray crystal structures and the cloning of brush border membrane receptors. The results of cloning mosquitocidal toxins in heterologous microorganisms show the potential of expanding the range of susceptible mosquito species by combining several toxins of different host specificity in one cell. Toxins have been expressed in new microorganisms with the potential for increasing potency by persisting at the larval feeding zone. The powerful tools of bacterial genetics are being applied to engineer genetically stable, persistent toxin expression and expand the insecticidal host ranges of Bacillus sphaericus and Bacillus thuringiensis strains. These techniques, together with modern formulation technology, should eventually lead to the construction of mosquitocidal microorganisms which are effective enough to have a real impact on mosquito-borne diseases.     

Mtx Toxins Synergize Bacillus sphaericus and Cry11Aa against Susceptible and Insecticide-Resistant Culex quinquefasciatus Larvae

Two mosquitocidal toxins (Mtx) of Bacillus sphaericus, which are produced during vegetative growth, were investigated for their potential to increase toxicity and reduce the expression of insecticide resistance through their interactions with other mosquitocidal proteins. Mtx-1 and Mtx-2 were fused with glutathione S-transferase and produced in Escherichia coli, after which lyophilized powders of these fusions were assayed against Culex quinquefasciatus larvae. Both Mtx proteins showed a high level of activity against susceptible C. quinquefasciatus mosquitoes, with 50% lethal concentrations (LC₅₀) of Mtx-1 and Mtx-2 of 0.246 and 4.13 μg/ml, respectively. The LC₅₀s were 0.406 to 0.430 μg/ml when Mtx-1 or Mtx-2 was mixed with B. sphaericus, and synergy improved activity and reduced resistance levels. When the proteins were combined with a recombinant Bacillus thuringiensis strain that produces Cry11Aa, the mixtures were highly active against Cry11A-resistant larvae and resistance was also reduced. The mixture of two Mtx toxins and B. sphaericus was 10 times more active against susceptible mosquitoes than B. sphaericus alone, demonstrating the influence of relatively low concentrations of these toxins. These results show that, similar to Cyt toxins from B. thuringiensis subsp. israelensis, Mtx toxins can increase the toxicity of other mosquitocidal proteins and may be useful for both increasing the activity of commercial bacterial larvicides and managing potential resistance to these substances among mosquito populations.