Industrial wastewaters and dewatered sludge: rich nutrient source for production and formulation of biocontrol agent, <Emphasis Type="Italic">Trichoderma viride</Emphasis>

Axenic cultivation of biocontrol fungus Trichoderma viride was conducted on a synthetic medium and different wastewaters and wastewater sludges in shake flasks to search for a suitable raw material resulting in higher biocontrol activity. Soluble starch based synthetic medium, dewatered municipal sludge, cheese industry wastewater sludge, pre-treated and untreated pulp and paper industry wastewater and slaughter house wastewater (SHW) were tested for T. viride conidia and protease enzyme production. The maximum conidia production followed the order, soluble starch medium (>10⁹ c.f.u./mL), untreated pulp and paper industry wastewater (4.9 × 10⁷ c.f.u./mL) > cheese industry wastewater (1.88 × 10⁷ c.f.u./mL) ≈ SHW (1.63 × 10⁷ c.f.u./mL) > dewatered municipal sludge (3.5 × 10⁶ c.f.u./mL) > pre-treated pulp and paper industry wastewater (1.55 × 10⁶ c.f.u./mL). The protease activity of T. viride was particularly higher in slaughterhouse wastewater (2.14 IU/mL) and dewatered municipal sludge (1.94 IU/mL). The entomotoxicity of soluble starch based synthetic medium was lower (≈6090 SBU/μL) in contrast to other raw materials. The entomotoxicity inversely decreased with carbon to nitrogen ratio in the growth medium and the conidia concentration and protease activity also contributed to the entomotoxicity. The residual c.f.u./g formulation of T. viride conidia were up to approximately, 90% after 1 month at 4 ± 1 °C and about 70% after 6 months at 25 ± 1 °C. Thus, production of T. viride conidia would help in marketability of low cost biopesticide from the sludge and safe reduction of pollution load.     

Starch industry wastewater as a substrate for antagonist, Trichoderma viride production

Starch industry wastewater was investigated to assess and improve its potential as a raw material for the conidia production of biocontrol fungi, Trichoderma viride. The wastewater was tested with and without supplements of glucose, soluble starch, meat peptone and probable conidiation inducer chemicals in shake flask culture. Addition of complex carbon source (soluble starch, 1% and 2% w/v) produced maximum conidia ( approximately 3.02 and 4.2 x 10(10)CFU/mL, respectively). On the other hand, glucose addition as a simpler carbon source was either ineffective or, reduced conidia production (from 1.6 x 10(8) in control to 3.0 x 10(7)CFU/mL in 5% w/v glucose supplement). Supplement of nitrogen source showed a small increase of conidia concentration. Propionic, maleic and humic acids, EDTA, pyridine, glycerol and CaCO(3) were examined as probable conidiation inducers and showed effect only on initial rate of conidiation with no increase in final conidia concentration. Intra and extracellular ATP correlation with spore production showed dependence on growth media used and conidia concentration at the end of fermentation. Addition of carbon and nitrogen sources showed an increase in protease activity (from 0.4985 to 2.43 IU/mL) and entomotoxicity (from 10448 to 12335 spruce budworm unit (SBU)/microL). Entomotoxicity was improved by 11% in fermenter over shake flask when starch industry wastewater was supplemented with meat peptone.     

Acid and alkaline treatments for enhancing the growth of rhizobia in sludge

Wastewater sludges have been proposed as an effective media for the production of rhizobia. The effect of total suspended solid (TSS) concentrations and pretreatments of sludge on the growth of Sinorhizobium meliloti were investigated. Acid (pH 2.0-6.0 obtained with H2SO4) and alkaline (50-200 mequiv.wt./L of NaOH) treatments were applied to enhance the biodegradability of primary (0.325%-3.2% TSS obtained by dilution of original sample) and secondary (0.2%-0.4% TSS obtained by concentration of original sample) sludges. In primary sludge without pretreatment, the highest cell count (11.10 x 10(9) cfu/mL) was obtained with 1.3% TSS. However, a maximum cell count of 13.00 x 10(9) cfu/mL was reached using an acid treatment of pH 2.0 and a 0.325% TSS concentration. Moreover, the alkaline treatment with 100 mequiv.wt./L of NaOH and 0.65% TSS increased the cell yield to 21.00 x 10(9) cfu/mL. For secondary sludge without pretreatment, no enhancement of growth was observed while increasing TSS concentration. This may be due to the increase of inhibitory substances, such as heavy metals, and of the Ca and Mg concentrations. As in primary sludge, some acid and alkaline treatments of secondary sludge tend to improve the cell count of S. meliloti. However, the highest value of 9.80 x 10(9) cfu/mL obtained with 0.4% TSS at pH 2.0 was lower than that obtained with primary sludge. It was also observed that S. meliloti grown in treated sludges maintained its capacity to nodulate alfalfa.     

Batch and fed-batch fermentation of Bacillus thuringiensis using starch industry wastewater as fermentation substrate

Bacillus thuringiensis var. kurstaki biopesticide was produced in batch and fed-batch fermentation modes using starch industry wastewater as sole substrate. Fed-batch fermentation with two intermittent feeds (at 10 and 20 h) during the fermentation of 72 h gave the maximum delta-endotoxin concentration (1,672.6 mg/L) and entomotoxicity (Tx) (18.5 × 10⁶ SBU/mL) in fermented broth which were significantly higher than maximum delta-endotoxin concentration (511.0 mg/L) and Tx (15.8 × 10⁶ SBU/mL) obtained in batch process. However, fed-batch fermentation with three intermittent feeds (at 10, 20 and 34 h) of the fermentation resulted in the formation of asporogenous variant (Spo−) from 36 h to the end of fermentation (72 h) which resulted in a significant decrease in spore and delta-endotoxin concentration and finally the Tx value. Tx of suspended pellets (27.4 × 10⁶ SBU/mL) obtained in fed-batch fermentation with two feeds was the highest value as compared to other cases.     

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 (μ_max) augmented from 0.17 to 0.22 h⁻¹ and entomotoxicity (Tx) increased by 29.7%. Meanwhile, volumetric mass transfer coefficient (k_La) 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 (D₅₀) 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.     

Improvement in post-treatment of digested swine wastewater

The performance of sequencing batch reactor (SBR) during post-treatment of digested effluent of swine wastewater was investigated. While operating SBR to treat the digested effluent directly, the performance was very poor with COD removal rate about 10%, and NH4+-N removal rate nearly 50%, with a scarce removal of total phosphorus. The performance apparently improved after adding raw swine wastewater or alkali to digested effluent. Although similar results for NH4+-N removal were achieved adopting both measures, the addition of raw wastewater proved superior in removing total nitrogen and total phosphorus. The addition of raw wastewater obtained effluent COD around 300 mg/L which was lower than that after alkali addition i.e. around 550 mg/L. Judged from the investment, oxygen demand, sludge yield, biogas production and running cost, the traditional combined anaerobic-SBR process is unfeasible to treat swine wastewater, while the combined anaerobic-SBR process with addition of raw swine wastewater can be a suitable biotechnology.     

Scale-up of biopesticide production processes using wastewater sludge as a raw material

Studies were conducted on the production of Bacillus thuringiensis (Bt)-based biopesticides to ascertain the performance of the process in shake flasks, and in two geometrically similar fermentors (15 and 150 l) utilizing wastewater sludge as a raw material. The results showed that it was possible to achieve better oxygen transfer in the larger capacity fermentor. Viable cell counts increased by 38–55% in the bioreactor compared to shake flasks. As for spore counts, an increase of 25% was observed when changing from shake flask to fermentor experiments. Spore counts were unchanged in bench (15 l) and pilot scale (5.3–5.5 e⁺⁰⁸ cfu/ml; 150 l). An improvement of 30% in the entomotoxicity potential was obtained at pilot scale. Protease activity increased by two to four times at bench and pilot scale, respectively, compared to the maximum activity obtained in shake flasks. The maximum protease activity (4.1 IU/ml) was obtained in pilot scale due to better oxygen transfer. The Bt fermentation process using sludge as raw material was successfully scaled up and resulted in high productivity for toxin protein yield and a high protease activity.     

Production of polyhydroxyalkanoates by <Emphasis Type="Italic">Azotobacter vinelandii</Emphasis> UWD using swine wastewater: Effect of supplementing glucose,yeast extract,and inorganic salts

This study examined the effect of adding glucose, yeast extract, and inorganic salts to swine wastewater (SWW) in a batch culture on the production of a biodegradable plastic, polyhydroxyalkanoate (PHA). A bacterial strain, Azotobacter vinelandii UWD, was used to produce PHA without limiting the non-carbon nutrients. The addition of glucose (30 g/L) to the SWW medium increased the level of cell growth (4.4∼7.0 times) and PHA production (3.8∼8.5 times) depending upon the dilution of SWW. A 50% dilution of SWW was found to be optimal considering the dry cell weight (9.40 g/L), PHA content (58 wt%), and hydroxyvalerate (HV) mol fraction in the PHA (4.3 mol%). A 75% SWW medium was more advantageous for producing PHA with a higher HV fraction (7.1 mol%) at the expense of losing 22% of PHA production. The undiluted SWW medium produced less than one third of the PHA compared with the 50% SWW medium, but the HV fraction was the highest (10.8 mol%). Regarding the effect of the glucose concentration, at 20 g/L glucose, the dry cell weight and level of PHA production increased to 9.34 g/L (0.63 g PHA/g dry cell weight) and 5.90 g/L, respectively. At 50 g/L glucose, there was no significant increase in PHA production. For the glucose-supplemented (30 g/L) 50% SWW medium, the addition of a nitrogen source (1 g/L of yeast extract) did not increase the level of cell growth or PHA production because the C:N ratio (23:1) was already close to the optimal value (22:1). Better aeration increased the productivity of PHA. External nitrogen supplements (1 g/L of yeast extract) and other essential mineral salts was not necessary for bacterial growth because they were contained in the SWW. These results suggest that SWW is an excellent feedstock for producing larger amounts of the value-added material, PHA, if it is combined with carbohydrate-rich organic waste.     

Neutral fat hydrolysis and long-chain fatty acid oxidation during anaerobic digestion of slaughterhouse wastewater

Neutral fat hydrolysis and long-chain fatty acid (LCFA) oxidation rates were determined during the digestion of slaughterhouse wastewater in anaerobic sequencing batch reactors operated at 25 degrees C. The experimental substrate consisted of filtered slaughterhouse wastewater supplemented with pork fat particles at various average initial sizes (D(in)) ranging from 60 to 450 microm. At the D(in) tested, there was no significant particle size effect on the first-order hydrolysis rate. The neutral fat hydrolysis rate averaged 0.63 +/- 0.07 d(-1). LCFA oxidation rate was modelled using a Monod-type equation. The maximum substrate utilization rate (kmax) and the half-saturation concentration (Ks) averaged 164 +/- 37 mg LCFA/L/d and 35 +/- 31 mg LCFA/L, respectively. Pork fat particle degradation was mainly controlled by LCFA oxidation rate and, to a lesser extent, by neutral fat hydrolysis rate. Hydrolysis pretreatment of fat-containing wastewaters and sludges should not substantially accelerate their anaerobic treatment. At a D(in) of 450 microm, fat particles were found to inhibit methane production during the initial 20 h of digestion. Inhibition of methane production in the early phase of digestion was the only significant effect of fat particle size on anaerobic digestion of pork slaughterhouse wastewater. Soluble COD could not be used to determine the rate of lipid hydrolysis due to LCFA adsorption on the biomass.     

Influence of pH Control Agents on Entomotoxicity Potency of Bacillus thuringiensis using Different Raw Materials

Summary In this investigation, ammonium hydroxide and acetic acid were used as pH control agents during Bacillus thuringiensis (Bt) fermentation in a pilot scale fermentor (150-l) employing two secondary wastewater sludges from two different wastewater treatment plants (CUQS and JQS) and semi-synthetic soybean meal medium as raw materials. Regardless of the cultivation medium, a substantial increase in total cell count, spore count, protease activity and entomotoxicity was achieved when the pH of the culture was controlled using NH₄OH/CH₃COOH. At harvest, total cell count increased by almost 17%, 33% and 25%; protease activity was enhanced by 12%, 33% and 53% and maximal spore count augmented by almost 28%, 48% and 33% in CUQS, JQS and soybean medium, respectively. Entomotoxicity potency was improved by 22%, 21% and 14% in CUQS, JQS and soybean medium, respectively compared to results obtained with NaOH/H₂SO₄ as pH control agents. A higher entomotoxicity was also observed using sludge compared to the soybean medium. This improvement of the Bt process performance was a consequence of the addition of rapidly utilizable carbon and nitrogen source through pH control, which stimulated endotoxin production in the crystal and enhanced sporulation.     

Study of microbial community structures in UASB sludge treating municipal wastewater by denaturing gradient gel electrophoresis of 16S rDNA

The structures of microbial communities in lab-scale upflow anaerobic sludge blanket (UASB) reactors for treating municipal wastewater with different ratios of COD soluble/COD total were studied using denaturing gradient gel electrophoresis (DGGE) of 16S rRNA genes.The microbial structure of the inoculum sludge obtained from a full-scale UASB reactor of treating potato processing wastewater was compared with the structures of sludges collected from three lab-scale UASB reactors after eight months feeding with raw municipal wastewater, with CEPS (chemically enhanced primary sedimentation) pretreated municipal wastewater, and with a synthetic municipal sewage, respectively. Computer-aided numerical analysis of the DGGE fingerprints showed that the bacterial community underwent major changes. The sludges for treating raw and CEPS pretreated wastewater had very similar bacterial and archaeal communities (82%and 96% similarity) but were different from that for treating the synthetic sewage. Hence, despite similar % COD in the particulate form in the synthetic and the real wastewater, the two wastewaters were selected for different microbial communities. Prominent DGGE bands of Bacteria and Archaea were purified and sequenced. The 16S rRNA gene sequences of the dominant archaeal bands found in the inoculum, and UASB sludge fed with raw sewage, CEPS pretreated wastewater, and synthetic sewage were closely associated with Methanosaeta concilii. In the UASB sludge fed with synthetic sewage, another dominant band associated with an uncultured archaeon 39-2 was found together with M. concilii.     

Kinetics of anaerobic treatment of slaughterhouse wastewater in batch and upflow anaerobic sludge blanket reactor

The kinetics of anaerobic treatment of slaughterhouse wastewater in batch and upflow anaerobic sludge blanket (UASB) reactors was investigated. Different concentrations of organic matter in slaughterhouse wastewater did not change the first order kinetics of the reaction. In batch digesters, methane and nitrogen production stopped after 30-40, 20-30 h, respectively, and in UASB reactors it was terminated after 30-40 days. The constant of velocity was 3.93 and 0.23 h(-1) respectively, for methane and nitrogen production. The yield coefficient, Yp was 343 and 349 ml CH4 per g of chemical oxygen demand at standard temperature and pressure conditions for batch reactors and UASB reactor, respectively.     

Wastewater Sludge Pre-treatment for Enhancing Entomotoxicity Produced by Bacillus thuringiensis var. kurstaki

Summary Wastewater sludge is a complex raw material for fermentation and requires pre-treatment in order to transform less biodegradable compounds into more easily degradable ones. In this study, sludge was treated by thermo-alkaline and oxidative pre-treatment methods and subjected to Bacillus thuringiensis fermentation in bench scale fermentors under controlled conditions. Thermo-alkaline treatment was found to be an effective process in order to enhance the entomotoxicity tested against spruce budworm. The total cell and spore counts were improved by 40 and 46%, respectively as compared to that using the untreated sludge. The final entomotoxicity potency increased from 12.3 × 10⁹ SBU/l of the raw sludge to 16.6 × 10⁹ SBU/l of the thermo-alkaline pre-treated sludge. The improvement of the process performance was attributed to a better oxygen transfer due to decrease in media viscosity and an improvement of nutrient availability due to the sludge solubilization and biodegradability.     

Study of microbial community structures in UASB sludge treating municipal wastewater by denaturing gradient gel electrophoresis of 16S rDNA

The structures of microbial communities in lab-scale upflow anaerobic sludge blanket (UASB) reactors for treating municipal wastewater with different ratios of COD soluble/ COD total were studied using denaturing gradient gel electrophoresis (DGGE) of 16S rRNA genes. The microbial structure of the inoculum sludge obtained from a full-scale UASB reactor of treating potato processing wastewater was compared with the structures of sludges collected from three lab-scale UASB reactors after eight months feeding with raw municipal wastewater, with CEPS (chemically enhanced primary sedimentation) pretreated municipal wastewater, and with a synthetic municipal sewage, respectively. Computer-aided numerical analysis of the DGGE fingerprints showed that the bacterial community underwent major changes. The sludges for treating raw and CEPS pretreated wastewater had very similar bacterial and archaeal communities (82% and 96% similarity) but were different from that for treating the synthetic sewage. Hence, despite     

Enumeration of Aeromonas hydrophila from domestic wastewater treatment plants and surface waters

Influents, effluents and sludges from sewage purification plants and surface water samples were examined quantitatively for Aeromonas hydrophila on the mA medium of Rippey and Cabelli. Between 10(4) and 10(6)/ml A. hydrophila were found in domestic wastewaters. On the average 99.975% were removed by activated sludge and 98.25% by trickling filters. Only 20.9% of A. hydrophila end up in the primary sludge, which contained up to 10(7)/g dry sludge. After 3 months, anaerobically (methane) fermented and partially dried sludge from trickling filters contained more than 10(6) A. hydrophila/g dry sludge. Surface water receiving raw sewage contained several hundreds of A. hydrophila/ml, comparable with the numbers found in effluent waters, while surface receiving no municipal wastewater and destined for the preparation of drinking water contained only small and negligible numbers. It was concluded that A. hydrophila was omnipresent in surface water.     

Effect of nitrogen and phosphorus concentration on their removal kinetic in treated urban wastewater by Chlorella vulgaris

This study evaluates the feasibility of removing nutrients by the microalgae Chlorella vulgaris, using urban wastewater as culture medium, namely the effluent subjected to secondary biological treatment in a wastewater treatment plant (WWTP). For this, laboratory experiments were performed in batch cultures to study the effect of initial nitrogen and phosphorus concentrations on growth and reduction of nutrient performance of C. vulgaris. The microalga was cultivated in enriched wastewater containing different phosphorus (1.3-143.5 mg x L(-1) P.PO4(3-)), ammonium (5.8-226.8 mg x L(-1) N-NH4+) and nitrate (1.5-198.3 mg x L(-1) N-NO3-) concentrations. The nutrient removal and growth kinetics have been studied: maximum productivity of 0.95 g SS x L(-1) x day(-1), minimum yield factor for cells on substrate (Y) of 11.51 g cells x g nitrogen(-1) and 0.04 g cells x g phosphorus(-1) were observed. The results suggested that C. vulgaris has a high potential to reduce nutrients in secondary WWTP effluents.     

Agro-industrial waste materials and wastewater sludge for rhizobial inoculant production: a review

Inoculating legumes with commercial rhizobial inoculants is a common agriculture practice. Generally, inoculants are sold in liquid or in solid forms (mixed with carrier). The production of inoculants involves a step in which a high number of cells are produced, followed by the product formulation. This process is largely governed by the cost related to the medium used for rhizobial growth and by the availability of a carrier source (peat) for production of solid inoculant. Some industrial and agricultural by-products (e.g. cheese whey, malt sprouts) contain growth factors such as nitrogen and carbon, which can support growth of rhizobia. Other agro-industrial wastes (e.g. plant compost, filtermud, fly-ash) can be used as a carrier for rhizobial inoculant. More recently, wastewater sludge, a worldwide recyclable waste, has shown good potential for inoculant production as a growth medium and as a carrier (dehydrated sludge). Sludge usually contains nutrient elements at concentrations sufficient to sustain rhizobial growth and heavy metals are usually below the recommended level. In some cases, growth conditions can be optimized by a sludge pre-treatment or by the addition of nutrients. Inoculants produced in wastewater sludge are efficient for nodulation and nitrogen fixation with legumes as compared to standard inoculants. This new approach described in this review offers a safe environmental alternative for both waste treatment/disposal and inoculant production.     

Study of microbial community structures in UASB sludge treating municipal wastewater by denaturing gradient gel electrophoresis of 16S rDNA

The structures of microbial communities in lab-scale upflow anaerobic sludge blanket (UASB) reactors for treating municipal wastewater with different ratios of COD_soluble/ COD_total were studied using denaturing gradient gel electrophoresis (DGGE) of 16S rRNA genes. The microbial structure of the inoculum sludge obtained from a full-scale UASB reactor of treating potato processing wastewater was compared with the structures of sludges collected from three lab-scale UASB reactors after eight months feeding with raw municipal wastewater, with CEPS (chemically enhanced primary sedimentation) pretreated municipal wastewater, and with a synthetic municipal sewage, respectively. Computer-aided numerical analysis of the DGGE fingerprints showed that the bacterial community underwent major changes. The sludges for treating raw and CEPS pretreated wastewater had very similar bacterial and archaeal communities (82% and 96% similarity) but were different from that for treating the synthetic sewage. Hence, despite similar % COD in the particulate form in the synthetic and the real wastewater, the two wastewaters were selected for different microbial communities. Prominent DGGE bands of Bacteria and Archaea were purified and sequenced. The 16S rRNA gene sequences of the dominant archaeal bands found in the inoculum, and UASB sludge fed with raw sewage, CEPS pretreated wastewater, and synthetic sewage were closely associated withMethanosaeta concilii. In the UASB sludge fed with synthetic sewage, another dominant band associated with an uncultured archaeon 39-2 was found together withM. concilii.     

Biodiesel production by in situ transesterification of municipal primary and secondary sludges

The potential of using municipal wastewater sludges as a lipid feedstock for biodiesel production was investigated. Primary and secondary sludge samples obtained from a municipal wastewater treatment plant in Tuscaloosa, AL were freeze-dried and subjected to an acid-catalyzed insitu transesterification process. Experiments were conducted to determine the effects of temperature, sulfuric acid concentration, and mass ratio of methanol to sludge on the yield of fatty acid methyl esters (FAMEs). Results indicated a significant interactive effect between temperature, acid concentration, and methanol to sludge mass ratio on the FAME yield for the insitu transesterification of primary sludge, while the FAME yield for secondary sludge was significantly affected by the independent effects of the three factors investigated. The maximum FAME yields were obtained at 75 degrees C, 5% (v/v) H(2)SO(4), and 12:1 methanol to sludge mass ratio and were 14.5% and 2.5% for primary and secondary sludge, respectively. Gas chromatography (GC) analysis of the FAMEs revealed a similar fatty acid composition for both primary and secondary sludge. An economic analysis estimated the cost of $3.23/gallon for a neat biodiesel obtained from this process at an assumed yield of 10% FAMEs/dry weight of sludge.     

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 12.4 × 10⁹ (without chitin) to 14.4 × 10⁹ SBU/L and from 18.2 × 10⁹ (without chitin) to 25.1 × 10⁹ 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 (26.7 × 10⁹ SBU/L) at 36 h of fermentation with chitin supplementation of SIW. In control (without chitin), the highest value of entomotoxicity of suspended pellet (20.5 × 10⁹ 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.