J.R. Valéro

Studies on the production of B. thuringiensis based biopesticides using wastewater sludge as a raw material.

Growth and delta-endotoxin yield of Bacillus thuringiensis (Bt) subsp kurstaki in tryptic soy yeast extract (TSY) medium, soybean meal based commercial medium and wastewater sludge medium were studied. The viable spores (VS) count in sludge medium was comparable to that obtained in laboratory and commercial media. The entomotoxicity of the fermentation liquid (Bt grown sludge) against Choristoneura fumiferana was comparable to the concentrated commercial Bt formulation available in the market (Foray 48B). A higher entomotoxicity was observed in a sludge medium than in the TSY or soybean meal media. The secondary and mixed (mixture of primary and secondary) sludges from various wastewater treatment plants were also evaluated for spore formation and entomotoxicity yield. The VS count was higher in a mixed sludge compared to the secondary sludge at a similar sludge solids concentration. Both VS count and entomotoxicity yield was found to be a function of sludge solids concentration in the medium. The optimum value of solids concentration for Bt production was found to be 25 g (-1) (dry weight basis). Beyond this concentration, a drop in VS count and entomotoxicity yield was observed. A low C:N ratio in the secondary sludge and a high C:N ratio in the mixed sludge resulted in a higher entomotoxicity. The optimum value of C:N ratio in combined sludge for Bt production was found to be 7.9-9.9. Relationships between entomotoxicity and maximum specific growth as well as with specific sporulation rate were developed.

Extraction and Analysis of Polyphenols: Recent trends

In recent years, there has been an increasing interest in diets rich in fruits and vegetables and this is mostly due to their presumed role in the prevention of various degenerative diseases, such as cancer and cardiovascular diseases. This is mainly due to the presence of bioactive compounds, such as polyphenols, carotenoids, among others. Polyphenols are one of the main classes of secondary metabolites derived from plants offering several health benefits resulting in their use as functional foods. Prior to the use of these polyphenols in specific applications, such as food, pharmaceutical, and the cosmetic industries, they need to be extracted from the natural matrices, then analyzed and characterized. The development of an efficient procedure for the extraction, proper analysis, and characterization of phenolic compounds from different sources is a challenging task due to the structural diversity of phenolic compounds, a complex matrix, and their interaction with other cellular components. In this light, this review discusses different methods of extraction, analysis, and the structural characterization of polyphenolic compounds.

Concurrent degradation of dimethyl phthalate (DMP) during production of Bacillus thuringiensis based biopesticides

Bacillus thuringiensis var. kurstaki (Btk) predominantly used as a microbial insecticide was tested for concomitant degradation of dimethyl phthalate (DMP). Different concentrations of DMP, 100, 200, 500, 1000 and 2000 mg/l were initially spiked in tryptic soya broth medium. A control of tryptic soya broth without DMP was also included. The experiments were carried out for 48 h at 30 degrees C and 250 rpm and growth, enzyme and biocontrol assay was evaluated. DMP concentration until 500 mg/l did not affect Btk cell growth, however, for DMP concentration of 1000-2000 mg/l, there was a 10 and 100 times decrease in cell and spore concentration, respectively. The enzyme concentration reduced by a factor of 1.5, 2, and 2.3 times for protease, esterase and amylase, respectively, at 1000 and 2000 mg/l suggesting toxicity of DMP at higher concentrations. Interestingly, the entomotoxicity (insect toxicity, taken as measure of biocontrol) was higher at 1000 and 5000 mg/l with 12 and 13 billion spruce budworm units/l, as tested against spruce budworm larvae. After 48h of incubation, DMP concentration decreased to 1-3mg/l at initial concentrations ranging from 100 to 500 mg/l, and the removal was relatively lower with 89 and 87% at 1000 and 2000 mg/l of DMP, respectively. These results suggest that Btk produces panoply of enzyme systems which can assist in degradation of DMP.

Bio-processing of agro-byproducts to animal feed

Agricultural and food-industry residues constitute a major proportion (almost 30%) of worldwide agricultural production. These wastes mainly comprise lignocellulosic materials, fruit and vegetable wastes, sugar-industry wastes as well as animal and fisheries refuse and byproducts. Agro-residues are rich in many bioactive and nutraceutical compounds, such as polyphenolics, carotenoids and dietary fiber among others. Agro residues are a major valuable biomass and present potential solutions to problems of animal nutrition and the worldwide supply of protein and calories, if appropriate technologies can be used for their valorization by nutrient enrichment. Technologies available for protein enrichment of these wastes include solid substrate fermentation, ensiling, and high solid or slurry processes. Technologies to be developed for the reprocessing of these wastes need to take account of the peculiarities of individual wastes and the environment in which they are generated, reprocessed, and used. In particular, such technologies need to deliver products that are safe, not just for animal feed use, but also from the perspective of human feeding. This review focuses on the major current applications of solid-state fermentation in relation to the feed sector.

Screening of Different Adjuvants for Wastewater/Wastewater Sludge-Based Bacillus thuringiensis Formulations

Screening of different adjuvants, namely, suspending agents, phagostimulants, stickers, antimicrobial agents, and UV screens to develop aqueous biopesticidal suspensions of Bacillus thuringiensis (Bt) variety kurstaki HD-1 fermented broths, specifically, nonhydrolyzed sludge, hydrolyzed sludge, starch industry wastewater, and soya (commercial medium), were investigated. The selected suspending agents [20% (wt:vol)] included sorbitol, sodium monophosphate, and sodium metabisulfite with corresponding suspendibility of 74-92, 69-85, and 71-82%, respectively. Molasses [0.2% (wt:vol)] increased adherence by 84-90% for all fermented broths. The optimal phagostimulants [0.5% (wt:vol)], namely, soya and molasses, caused entomotoxicity increase of 3-13 and 7-13%, respectively. Sorbic and propionic acids showed high antimicrobial action [0.5% (wt:vol)], irrespective of fermentation medium. Sodium lignosulfonate, molasses, and Congo red, when used as UV screens [0.2% (wt:vol)], showed percent corresponding entomotoxicity losses of 3-5, 0.5-5 and 2-16, respectively. The Bt formulations, when exposed to UV radiation, showed higher half-lives (with and without UV screens) than the fermented broths or semisynthetic soya medium and commercial Bt formulation. UV screen-amended nonhydrolyzed, hydrolyzed, and starch industry wastewater formulations showed 1.3-1.5-fold higher half-lives than commercial Bt formulation. Thus, the recommended formulation comprises sorbitol, sodium monophosphate, sodium metabisulfite (suspending agents); molasses, soya flour (phagostimulants); molasses and skimmed milk powder (rainfasteners); sorbic and propionic acids (antimicrobial agents) and sodium lignosulfate; and molasses and Congo red (UV screens). These waste-based Bt formulations offer better UV resistance in comparison with commercial formulation.

Induced production of chitinase to enhance entomotoxicity of Bacillus thuringiensis employing starch industry wastewater as a substrate

Induced production of chitinase during bioconversion of starch industry wastewater (SIW) to Bacillus thuringiensis var. kurstaki HD-1 (Btk) based biopesticides was studied in shake flask as well as in computer-controlled fermentors. SIW was fortified with different concentrations (0%; 0.05%; 0.1%; 0.2%; 0.3% w/v) of colloidal chitin and its consequences were ascertained in terms of Btk growth (total cell count and viable spore count), chitinase, protease and amylase activities and entomotoxicity. At optimum concentration of 0.2% w/v colloidal chitin, the entomotoxicity of fermented broth and suspended pellet was enhanced from 12.4x10(9) (without chitin) to 14.4x10(9) SBU/L and from 18.2x10(9) (without chitin) to 25.1x10(9) SBU/L, respectively. Further, experiments were conducted for Btk growth in a computer-controlled 15 L bioreactor using SIW as a raw material with (0.2% w/v chitin, to induce chitinase) and without fortification of colloidal chitin. It was found that the total cell count, spore count, delta-endotoxin concentration (alkaline solubilised insecticidal crystal proteins), amylase and protease activities were reduced whereas the entomotoxicity and chitinase activity was increased with chitin fortification. The chitinase activity attained a maximum value at 24 h (15 mU/ml) and entomotoxicity of suspended pellet reached highest (26.7x10(9) SBU/L) at 36 h of fermentation with chitin supplementation of SIW. In control (without chitin), the highest value of entomotoxicity of suspended pellet (20.5x10(9) SBU/L) reached at 48 h of fermentation. A quantitative synergistic action of delta-endotoxin concentration, spore concentration and chitinase activity on the entomotoxicity against spruce budworm larvae was observed.

Entomotoxicity, protease and chitinase activity of Bacillus thuringiensis fermented wastewater sludge with a high solids content.

This study investigated the production of biopesticides, protease and chitinase activity by Bacillus thuringiensis grown in raw wastewater sludge at high solids concentration (30 g/L). The rheology of wastewater sludge was modified with addition of Tween-80 (0.2% v/v). This addition resulted in 1.6 and 1.3-fold increase in cell and spore count, respectively. The maximum specific growth rate (micro(max)) augmented from 0.17 to 0.22 h(-1) and entomotoxicity (Tx) increased by 29.7%. Meanwhile, volumetric mass transfer coefficient (k(L)a) showed marked variations during fermentation, and oxygen uptake rate (OUR) increased 2-fold. The proteolytic activity increased while chitinase decreased for Tween amended wastewater sludge, but the entomotoxicity increased. The specific entomotoxicity followed power law when plotted against spore concentration and the relation between Tx and protease activity was linear. The viscosity varied and volume percent of particles increased in Tween-80 amended wastewater sludge and particle size (D(50)) decreased at the end of fermentation. Thus, there was an increase in entomotoxicity at higher suspended solids (30 g/L) as Tween addition improved rheology (viscosity, particle size, surface tension); enhanced maximum growth rate and OUR.

Photostabilization of Bacillus thuringiensis fermented wastewater and wastewater sludge based biopesticides using additives

Photoprotection (against UV-A and UV-B radiations) of the active components of Bacillus thuringiensis var. kurstaki obtained from the fermentation of various culture media was investigated. The culture media comprised: starch industry wastewater; secondary wastewater sludge (non-hydrolyzed and hydrolyzed) and soya (used as a reference). Photoprotection was carried out by using various UV-protection additives, namely, para-aminobenzoic acid, lignosulfonic acid and molasses at different concentrations (0.1%, 0.15% and 0.2%, w/w). In the absence of UV-protection agents, secondary sludge demonstrated natural UV protection with half-lives ranging from 3.25 to 3.4 d. The half-life for soya and starch industry wastewater was 1.9 and 1.8 d, respectively. Para-amino-benzoic acid as a UV-protection agent at 0.20% (w/w) gave excellent UV-protection for soya and starch industry wastewater with half-lives being 5.9 and 7 d, respectively. Likewise, lignosulfonic acid at 0.20% (w/w) was an effective photostabilizer for hydrolyzed and non-hydrolyzed secondary sludge with half-lives of 7.25 and 8 d, respectively. Hence, when similar concentration of the UV-protection additives was used, photoprotection was higher for the alternative media than the conventional soya medium, validating the technical feasibility of using three additives.

Bacillus thuringiensis fermentation of wastewater and wastewater sludge--presence and characterization of chitinases.

Abstract This study investigated the presence of chitinases in Bacillus thuringiensis ssp kurstaki HD‐1(Bt) fermented broths of wastewater sludge (non‐hydrolyzed and hydrolyzed); starch industry wastewater and soyameal. Chitinase activity was absent in soyameal and present in others. Chitinase demonstrated peaks at pH 4.0 and temperatures 40 and 50°C with higher activity between pH 4–5 and 10–11. The chitinase band on SDS‐PAGE was found to be between 36 and 45 kDa for non‐hydrolyzed (NH) and hydrolyzed sludge (TH) and starch industry wastewater. The chitinase profile during fermentation showed peaks at 15 and 30 h for non‐hydrolyzed and hydrolyzed sludge and 15 and 24 h for starch industry wastewater. Chitinase retained 96–99 % activity after two weeks incubation at room temperature and pH 4. Bioassays with supplementation of Bt chitinases showed 1.2 fold increase in entomotoxicity of wastewater sludge and a small increase in starch industry wastewater. This study sheds light on production of Bt chitinases in alternative media which will have a long term effect on entomotoxicity of these formulations.

Starch industry wastewater for production of biopesticides--ramifications of solids concentrations.

Total solids (TS) concentrations ranging from 15 to 66 g L−1 of starch industry wastewater (SIW) were tested as raw material for the production of Bacillus thuringiensis var. kurstaki HD‐1 (Btk) biopesticide in shake flasks and a 15 L bench‐scale fermenter. Shake flask studies revealed a higher delta‐endotoxin concentration of Btk at 30 g L−1 TS concentration and 2.5% (v v−1) volume of pre‐culture. The fermenter experiments conducted using SIW at 30 g L−1 TS concentration under controlled conditions of temperature, pH and dissolved oxygen showed higher spore count, enzyme production (protease and amylase) and delta‐endotoxin concentration as compared with those of SIW at 15 g L−1 TS concentration. The entomotoxicity, at the end of fermentation, with SIW at 30 g L−1 solids concentration (17.8 × 109 SBU L−1, measured against spruce budworm) was considerably higher as compared with entomotoxicity at 15 g L−1 solids concentration (15.3 × 109 SBU L−1) and semi‐synthetic medium (11.7 × 109 SBU L−1). The pellet, comprising spores and delta‐endotoxin complex obtained after centrifugation and followed by resuspension (in supernatant) in one‐tenth of the original volume, of SIW at 30 g L−1 solids concentration media registered the highest potential for application (to protect forests against spruce budworm) than other media in term of entomotoxicity.