a comparison of sludge solids and solids-enriched … · a comparison of sludge solids and...

A Comparison of sludge solids and solids-enriched starchindustry wastewater as potential raw materials for theproduction of Bacillus thuringiensis based biopesticides

K. D. Vu*, R. D. Tyagi*1, R. Y. Surampalli**and J. R. Valéro*

* INRS-ETE, Université du Québec, 490, Rue de la Couronne, Québec (Québec), G1K 9A9, Canada

** US EPA, P.O. Box-17-2141, Kansas City, KS 66117, USA

1 Corresponding author: R.D. Tyagi, Email: [email protected]; Phone: (418) 654 2617; Fax: (418) 654 2600

Abstract: Most of the nutrients required for growth of industrial microorganisms are embedded in solids contained inwastewater or wastewater sludge. In order to enhance growth rate and to achieve higher product concentration it isessential that optimum solids concentration be used in a biological process to produce value added products. In thisstudy, different solids concentration of starch industry wastewater (SIW) and that of activated sludge were used tocompare the production of Bacillus thuringiensis var. kurstaki (Btk) based biopesticides. The SIW with solidsconcentration of 15 g/l was settled and the settled solids were mixed with the supernatant in different ratio to obtaina range of solids concentration between 15 to 66g/L. These different solids concentration were used as growth mediato produce Btk biopesticides. Growth, spore production and entomotoxicity potential were verified against sprucebudworm larvae (Choristoneura fumiferana) using fermented broth at the end of the experiment. The results showeda higher entomotoxicity at solids concentration of 30 g/L which was higher than the entomotoxicity at similar solidsconcentration in wastewater activated sludge. Based on these results, the optimal value of sludge as well as SIWsolids concentration was determined that could be used for eventual biopesticide production.

Keywords: Biopesticide production; entomotoxicity; solids concentration; starch industry wastewater; wastewater sludge

INTRODUCTIONManagement of wastewater/wastewater sludge through bioconversion into value added products such asbiopesticides showed the advantages in waste management as well as in potential production of biopesticidesfor protection of agricultural and forestry plants from insects pest (Sachdeva et al., 2000; Lachhab et al., 2001;Yezza et al., 2006). In case of Bt biopesticides production, the entomotoxicity (insect kill rate) was higher whenusing wastewater/wastewater sludge as compared to soya medium (Sachdeva et al., 2000; Brar et al., 2005).However, to obtain higher potency for economical production of biopesticides, many process parameters(inoculum volume, C: N ratio, etc…) have been optimised (Lachhab et al., 2001; Vidyarthi et al., 2002).

In many reports, media with high concentration of nutrients have been successfully used for attaining high yieldsof spore-crystal preparations (Arcas et al., 1987; Farrare et al., 1998; Zouari and Jaoua, 1999). On the other hand,Scherrer et al. (1973) reported that high concentration of nutrients inhibited sporulation and toxin production.

Wastewater/wastewater sludge is known to contain the nutrients (carbon and nitrogen and other mineralssources, Table 1), and most of the nutrients required for growth of industrial microorganisms are embeddedin solids contained in wastewater or wastewater sludge. In order to enhance growth rate and to achievehigher entomotoxicity or product yield, it is essential that optimum solids concentration should be used. In caseof sludge, different types of sludge (secondary sludge, mixed sludge and dewatered sludge) at different sludgesolids concentrations were used to produce Bacillus thuringiensis var kurstaki HD-1 (Btk) biopesticides andthe results established that entomotoxicity was highest using 25-26g/l sludge solids concentrationirrespectively of sludge type (Sachdeva et al., 2000; Lachhab et al., 2001; Vidyarthi et al., 2002). However, incase of starch industry wastewater (SIW), the optimal solids concentration for biopesticides production hasnot been yet investigated.

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Therefore, the purpose of this research was: (1) investigate the effect of different solids concentrations ofSIW in the growth and entomotoxicity production of Btk in shake flask experiments; (2) Verify the optimalsolids concentration, determined in shake flask, in terms of entomotoxicity by conducting experiments in15-L computer-controlled fermentor under controlled parameters (pH, temperature, dissolved oxygen) andcompare the results obtained using optimal solids concentration of wastewater sludge.

MATERIAL AND METHODS

Bacterial strain and inoculum preparationBacillus thuringiensis var. kurstaki HD-1 (Btk) was used for the production of biopesticides. The inoculum wasprepared in two-stage process according to Vidyarthi et al. (2002).

Biopesticides production mediaSecondary sludge: JQS, CUQS and BLS were obtained from Jonquiere, Communauté Urbain de Quebec (CUQ)and Black Lake wastewater treatment plants, respectively. Mixed sludge (primary + secondary sludge) wasobtained from CUQ. Dewatered sludge (JQC) was procured from Jonquiere wastewater treatment plant. StarchIndustry wastewater (SIW) was obtained from ADM-Ogilvie (Candiac, Québec, Canada). The characteristics ofsludge and SIW (Table 1) were determined according to the Standard Methods (1998).

Shake flask experimentsThe starch industry wastewater (SIW) with solids concentration of 15 g/l was settled and the settled solids weremixed with the supernatant in different ratio in order to obtain a range of solids concentrations between 15 to66g/L. Different solids concentrations of SIW were used as growth media to produce Btk biopesticides; pH ofthe media was adjusted to 7.0 followed by sterilisation at 121oC for 30 min. The sterilised media wereinoculated with 2% (v/v) Btk cells suspension and incubated in a rotary shaker (220 revolution per minute orrpm) at 30oC for 60h. Samples were drawn at 48h and 60h of fermentation. The total cell count, spore countand entomotoxicity in the samples were determined.

15L-computer-controlled fermentor experimentFermentation was carried out in a stirred tank 15 L bioreactor (working volume: 10 L, Biogenie, Que., Canada)equipped with accessories and programmable logic control (PLC) system for dissolved oxygen (DO), pH, anti-foam, impeller speed, aeration rate and temperature. The software (iFix 3.5, Intellution, USA) allowed automaticset-point control and integration of all parameters via PLC. Fermentor was filled with starch industrywastewater (10 L) and polypropylene glycol (PPG, Sigma-Canada) (0.1% v/v) solution (10 mL) as an anti-foam agent. The fermentor with medium was sterilised in situ at 121 °C for 30 min. When the fermentor cooledto 30 oC, it was then inoculated (2% v/v inoculum) aseptically with pre-culture of Btk harvested in exponentialphase (8-12 h age). In order to keep the dissolved oxygen (DO) above 25% saturation, air flow rate and agitationrates were varied between 0.2-0.3 vvm and 300–500 rpm, respectively. The temperature was maintained at30 oC by circulating water through the fermenter jacket. Fermentation pH was controlled automatically at 7 ±0.1 through computer-controlled peristaltic pumps by addition of pH control agents: NaOH 4M or H2SO4 3 M.Both DO and pH were continuously monitored by means of a polarographic dissolved oxygen probe and of a pHsensor (Mettler-Toledo, USA), respectively. Samples were collected periodically to monitor the changes in totalcell count (TC), spore count (SC) and entomotoxicity (Tx) as described in the following sections.

Estimation of total cell count (TC) and spore count (SC)To determine TC and SC, the samples were serially diluted with sterile saline solution (0.85% w/v NaCl). Theappropriately diluted samples (0.1mL) were plated on TSA plates and incubated at 30 °C for 16-24 h to formfully developed colonies. For spore count (SC), the appropriately diluted samples were heated in an oil bath at80°C for 10 min and then chilled in ice for 5 min. The TC and SC were performed by counting colonies grown onnutrient agar medium. For all counts, the average of at least three replicate plates was used for each testeddilution. For enumeration, 30–300 colonies were enumerated per plate. The results were expressed as colonyforming units per mL (CFU/ml). The standard deviation for TC and SC was 7 and 8%, respectively.

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Bioassay to determine the entomotoxicity (Tx)Tx was evaluated through bioassays using eastern spruce budworm larvae (Choristoneura fumiferana,Lepidoptera: Tortricidae) of second instar, provided by Natural Resources Canada (Sault Ste-Marie, Ontario).The larvae were raised on an artificial diet for 7 days to obtain the third and fourth instar (L3-L4) larvae. Thebioassays were conducted using the diet incorporation method (Beegle, 1990). In this technique, 1.5 ml ofeach appropriately diluted Btk sample collected during fermentation was incorporated into 30 ml of molten-agar-based diet (at 60 oC). The mixture was distributed in aliquots of 1ml in twenty 15 x 45 mm glass vials(VWR Canlab, Canada) with perforated plastic caps. Three sets of control (diet with sterilized productionmedium) were also included in the procedure to correct the mortality of larvae due to the SIW only. One larva(third-instar) of eastern spruce budworm was placed in each vial after the diet solidified. The vials wereincubated at ambient temperature for 1 week and the mortality of the larvae was counted. If mortality in controlvials (three sets of control mentioned above) was higher than 10%, the bioassay was repeated. Tx of samplepreparations was obtained by comparing the final mortality (percentage) of spruce budworm larvae with thatof a standard commercial product (Foray 76B, Abbott Laboratories, Chicago, IL) and expressed as relativeSBU/ml. Foray 76 B contained spores and crystals of Bt var. kurstaki at a potency of 20.1 x 109 InternationalUnit (IU/l) measured against cabbage looper (Trichoplusia ni). On comparison of Tx of Bt-fermented sludgesamples, it was found that SBU reported in this study was 20–25% higher than IU. The standard deviation forTx measurement was 8%.

RESULTS AND DISCUSSION

Shake flask experiment using SIW/sludge as raw materialsEffect of different solids concentrations of SIW on the growth and entomotoxicity of BtkTable 2 presented the total cell count, spore count and entomotoxicity of the samples withdrawn at the end offermentation (60h) of the shake flask experiments using different solids concentrations of SIW. Increase in solidsconcentration of SIW (from 15 to 43 g/L) caused an increase in the total cell count, however, further increase insolids concentration (from 48 to 66 g/L), the total cell count decreased considerably. In case of spore production,the spore count increased when using high solids concentrations (from 15 to 35 g/L) and decreased at highersolids concentrations (from 43 to 66 g/L). It was possible that at higher solids concentration, the oxygen transferin the media was low causing deleterious effects on growth and sporulation of Btk cells. The entomotoxicity offermented broth increased as solids concentrations increased from 15 g/L to 30 g/L and attained the highest valueat 30 g/L. At solids concentration of 35 g/L, the entomotoxicity was not much different than at 30 g/L solidsconcentration. However, at higher solids concentrations (from 43 to 66 g/L), the entomotoxicity decreasedconsiderably. It is interesting to note that even the spore count was higher at solids concentration of 35 g/L ascompared to that of the optimal solids concentration (30 g/L), the entomtoxicity was less.

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Table 1. Characteristics of SIW/sludge used as raw materials*

It is reported in some of the previous research reports that the spore count can not reflect the overall entomotoxicityof the fermented broth (Sachdeva et al., 2000; Vidyarthi et al., 2002). It has also been reported that a high sporecount is not sufficient to ensure a good entomotoxicity (Avignone-Rossa and Mignone 1993). Moreover, it should benoted here that in this study, there was not much difference between the entomotoxicity at 48h (14.2 x 109 SBU/L)and at 60h (14.3 x 109 SBU/L) of the fermentation process at solids concentration of 30 g/L. Therefore, the fermentorexperiment of Btk using the optimal solids concentration of SIW at 30g/L was carried out for a 48h-period.

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Table 2. The cell/spore counts and entomotoxicity of Btk in SIW/sludge at different total solids concentrations

Effect of different sludge types and different sludge solids concentrations on the growth and entomotoxicity of Btk

Table 2 gives a summary of the total cell count, spore count as well as the entomotoxicity obtained for Btk whengrown in different sludge with different total solids concentrations. In case of secondary sludge (JQS), it wasfound that the total cell count increased with increasing solids concentration indicating increased availability ofnutrients at higher sludge solids concentration. The spore count, however, increased as the solids concentrationwas increased from 25 to 35 g/L but decreased markedly as the TS contents increased to 40 g/L (Sachdeva et al.,2000). In case of the mixed sludge (CUQM), the highest cell count and spore count as well as the entomotoxicitywere obtained at the solids concentration of 26 g/L. Higher sludge solids concentrations also caused a decreasein the entomotoxicity value (Lachhab et al., 2001). The same results were observed when using dewatered sludge(JQC) as rawmaterial, the highest entomotoxicity was at the solids concentration of 25 g/L (Vidyarthi et al., 2002).So, irrespective of the sludge types, it seems that the Btk growing in sludge solids concentration of 25-26 g/L wasmore mobile due to lower viscosity and so, better aeration (oxygen transfer) than in the higher solidsconcentrations. δ-endotoxin production and sporulation have been reported to decrease under oxygen limitation(Avignone-Rossa and Mignone, 1992). Moreover, according to Farrera et al. (1998) at high total solidsconcentration, all the cells may not be in the same phase: some are sporulating while others are releasing themature spore into the medium and even some spores are germinating, i.e. there is a desynchronization of thesporulation process and this may also be the reason for the lower entomotoxicity at higher solids concentrations,respectively the sludge types or the SIW.

Fermentor experimentsThe total cell count, spore count and the entomotoxicity of fermented broth (at 48h) of the Btk-fermentationin the 15L-fermentor using SIW at 15 g/L (as control) and 30 g/L (as optimal value) solids concentration arepresented in the Table 3. The results obtained from the other experiments using the optimal total solids

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concentration of 26 g/L (Lachhab et al., 2001) as well as the other experiments using the optimal suspendedsolids 20-25 g/L (Yezza et al., 2004; 2006) were also compiled in Table 3.

Table 3. Bacillus thuringiensis fermentation in fermentor using wastewater and wastewater sludge asraw materials.

The results showed that using optimal solids concentration of SIW (30 g/L) gave the highest entomotoxicity(17.5 x 109 SBU/L) as compared to SIW at 15 g/L solids concentration (15.2 x 109 SBU/L). In all cases of sludge,the secondary sludge from BLS obtained the highest entomotoxicity (16 x 109 SBU/L) as compared to othersludge types. Especially, BLS had the same total solids as that of SIW (30 g/L), however, the entomotoxicity inthis case was less than SIW. It should be noted that media with different compositions (or different origin)resulted in changes in the insecticidal crystal proteins (ICPs) specific production, i.e. different amounts of ICPsproduced per spore. For example, Dulmage (1971); Salama et al. (1983) found that different media could changethe relative entomotoxicity against several target insects or even change the entomotoxicity of products obtainedfrom the same Bacillus thuringiensis strain. Moreover, it should also be noted that the best conditions for sporeproduction were different from those for insecticidal crystal proteins (ICPs) production; that was the reason whyin case of SIW at optimal solids concentration (30 g/L), the spore concentration obtained was less than that inother cases, however, the entomotoxicity obtained was the highest (Table 3). Our results were also in accordancewith the results from other authors in respect that the high final spore counts are not proportional to highentomotoxicities of the culture (Avignone-Rossa and Mignone 1993; Morris et al. 1996; Farrera et al., 1998).

Nowadays, land application of biosolids (wastewater sludge) has increased as a disposal practice becauselandfilling is too expensive due to non-availability of landfill sites. Incineration produces air pollution andgreen house gases, and ocean dumping is banned (Meyer et al., 2001). However, in this case, wastewatersludge can be used for biopesticides production and it can be the most abundantly available raw material forBt production. SIW is also the popular and abundant available source of wastewater and contains highconcentration of chemical oxygen demand (COD) that cause serious environment pollution and requireexpensive treatment (Annachhatre and Amatya, 2000). Use of SIW for the production of biopesticides is better,since the nutrients in SIW are converted into useful and eco-friendly products. In summary, SIW and sludge asraw materials, as compared to other cheap raw materials used to produce Btk based biopesticides, do notrequire addition or fortification of nutrients for growth, sporulation and entomotoxicity production by Bacillusthuringiensis. Therefore, sludge and SIW are eventually the potential industrial raw materials for Btk-biopesticides production.

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CONCLUSIONFollowing conclusions were drawn from the foregoing study

1. The optimal total solids concentrations required, as raw materials, for Btk- biopesticides production were30g/l and 25-26 g/l for starch industry wastewater and wastewater sludge, respectively.

2. Secondary wastewater sludge from BLS gave the highest entomotoxicity (16 x 109 SBU/L) as compared toother sludge types. However, SIW at solids concentration of 30 g/L gave higher entomotoxicity (17.5 x 109

SBU/L) than the BLS secondary sludge.3. Fermentations of Bacillus thuringiensis var kurstaki HD-1 at high and optimal solids concentration (30 g/L)of sludge and SIW are important, not only to obtain high entomotoxicity, but also to reduce operation costsby handling lesser volumes of broths.

ACKNOWLEDGEMENTSThe authors are sincerely thankful to the Natural Sciences and Engineering Research Council of Canada (GrantsA4984, STP235071, Canada Research Chair) for financial support. The views or opinions expressed in this articleare those of the authors and should not be construed as opinions of the U.S. Environmental Protection Agency.

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a comparison of sludge solids and solids-enriched … · a comparison of sludge solids and...

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