Comparacion_aguaresidual_biopesticida

8/3/2019 Comparacion_aguaresidual_biopesticida

1/8

A Comparison of sludge solids and solids-enriched starch

industry wastewater as potential raw materials for the

production of Bacillus thuringiensis based biopesticides

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

* INRS-ETE, Universit du Qubec, 490, Rue de la Couronne, Qubec (Qubec), 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 in

wastewater or wastewater sludge. In order to enhance growth rate and to achieve higher product concentration it is

essential that optimum solids concentration be used in a biological process to produce value added products. In this

study, different solids concentration of starch industry wastewater (SIW) and that of activated sludge were used to

compare the production of Bacillus thuringiensis var. kurstaki (Btk) based biopesticides. The SIW with solids

concentration of 15 g/l was settled and the settled solids were mixed with the supernatant in different ratio to obtain

a range of solids concentration between 15 to 66g/L. These different solids concentration were used as growth media

to produce Btk biopesticides. Growth, spore production and entomotoxicity potential were verified against spruce

budworm larvae (Choristoneura fumiferana) using fermented broth at the end of the experiment. The results showed

a higher entomotoxicity at solids concentration of 30 g/L which was higher than the entomotoxicity at similar solids

concentration in wastewater activated sludge. Based on these results, the optimal value of sludge as well as SIW

solids concentration was determined that could be used for eventual biopesticide production.

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

INTRODUCTION

Management of wastewater/wastewater sludge through bioconversion into value added products such as

biopesticides showed the advantages in waste management as well as in potential production of biopesticides

for 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 when

using 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 yields

of 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 minerals

sources, Table 1), and most of the nutrients required for growth of industrial microorganisms are embedded

in solids contained in wastewater or wastewater sludge. In order to enhance growth rate and to achieve

higher entomotoxicity or product yield, it is essential that optimum solids concentration should be used. In case

of sludge, different types of sludge (secondary sludge, mixed sludge and dewatered sludge) at different sludge

solids concentrations were used to produce Bacillus thuringiensisvar kurstakiHD-1 (Btk) biopesticides and

the 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, in

case of starch industry wastewater (SIW), the optimal solids concentration for biopesticides production has

not been yet investigated.

1101


2/8

Therefore, the purpose of this research was: (1) investigate the effect of different solids concentrations of

SIW in the growth and entomotoxicity production of Btk in shake flask experiments; (2) Verify the optimal

solids concentration, determined in shake flask, in terms of entomotoxicity by conducting experiments in

15-L computer-controlled fermentor under controlled parameters (pH, temperature, dissolved oxygen) and

compare the results obtained using optimal solids concentration of wastewater sludge.

MATERIAL AND METHODS

Bacterial strain and inoculum preparation

Bacillus thuringiensisvar. kurstakiHD-1 (Btk) was used for the production of biopesticides. The inoculum was

prepared in two-stage process according to Vidyarthi et al. (2002).

Biopesticides production media

Secondary sludge: JQS, CUQS and BLS were obtained from Jonquiere, Communaut Urbain de Quebec (CUQ)

and Black Lake wastewater treatment plants, respectively. Mixed sludge (primary + secondary sludge) was

obtained from CUQ. Dewatered sludge (JQC) was procured from Jonquiere wastewater treatment plant. Starch

Industry wastewater (SIW) was obtained from ADM-Ogilvie (Candiac, Qubec, Canada). The characteristics of

sludge and SIW (Table 1) were determined according to the Standard Methods (1998).

Shake flask experiments

The starch industry wastewater (SIW) with solids concentration of 15 g/l was settled and the settled solids were

mixed with the supernatant in different ratio in order to obtain a range of solids concentrations between 15 to

66g/L. Different solids concentrations of SIW were used as growth media to produce Btk biopesticides; pH of

the media was adjusted to 7.0 followed by sterilisation at 121oC for 30 min. The sterilised media were

inoculated with 2% (v/v) Btk cells suspension and incubated in a rotary shaker (220 revolution per minute or

rpm) at 30oC for 60h. Samples were drawn at 48h and 60h of fermentation. The total cell count, spore count

and entomotoxicity in the samples were determined.

15L-computer-controlled fermentor experiment

Fermentation 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 automatic

set-point control and integration of all parameters via PLC. Fermentor was filled with starch industry

wastewater (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 cooled

to 30 oC, it was then inoculated (2% v/v inoculum) aseptically with pre-culture of Btk harvested in exponential

phase (8-12 h age). In order to keep the dissolved oxygen (DO) above 25% saturation, air flow rate and agitation

rates were varied between 0.2-0.3 vvm and 300500 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 pH

sensor (Mettler-Toledo, USA), respectively. Samples were collected periodically to monitor the changes in total

cell 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). The

appropriately diluted samples (0.1mL) were plated on TSA plates and incubated at 30 C for 16-24 h to form

fully developed colonies. For spore count (SC), the appropriately diluted samples were heated in an oil bath at80C for 10 min and then chilled in ice for 5 min. The TC and SC were performed by counting colonies grown on

nutrient agar medium. For all counts, the average of at least three replicate plates was used for each tested

dilution. For enumeration, 30300 colonies were enumerated per plate. The results were expressed as colony

forming units per mL (CFU/ml). The standard deviation for TC and SC was 7 and 8%, respectively.

1102


3/8

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. The

bioassays were conducted using the diet incorporation method (Beegle, 1990). In this technique, 1.5 ml of

each 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 production

medium) 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 were

incubated at ambient temperature for 1 week and the mortality of the larvae was counted. If mortality in control

vials (three sets of control mentioned above) was higher than 10%, the bioassay was repeated. Tx of sample

preparations was obtained by comparing the final mortality (percentage) of spruce budworm larvae with that

of a standard commercial product (Foray 76B, Abbott Laboratories, Chicago, IL) and expressed as relative

SBU/ml. Foray 76 B contained spores and crystals of Btvar. kurstakiat a potency of 20.1 x 109 International

Unit (IU/l) measured against cabbage looper (Trichoplusia ni). On comparison of Tx of Bt-fermented sludge

samples, it was found that SBU reported in this study was 2025% higher than IU. The standard deviation forTx measurement was 8%.

RESULTS AND DISCUSSION

Shake flask experiment using SIW/sludge as raw materials

Effect of different solids concentrations of SIW on the growth and entomotoxicity of Btk

Table 2 presented the total cell count, spore count and entomotoxicity of the samples withdrawn at the end of

fermentation (60h) of the shake flask experiments using different solids concentrations of SIW. Increase in solids

concentration of SIW (from 15 to 43 g/L) caused an increase in the total cell count, however, further increase in

solids 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 higher

solids concentrations (from 43 to 66 g/L). It was possible that at higher solids concentration, the oxygen transfer

in the media was low causing deleterious effects on growth and sporulation of Btk cells. The entomotoxicity of

fermented broth increased as solids concentrations increased from 15 g/L to 30 g/L and attained the highest value

at 30 g/L. At solids concentration of 35 g/L, the entomotoxicity was not much different than at 30 g/L solids

concentration. However, at higher solids concentrations (from 43 to 66 g/L), the entomotoxicity decreased

considerably. It is interesting to note that even the spore count was higher at solids concentration of 35 g/L as

compared to that of the optimal solids concentration (30 g/L), the entomtoxicity was less.

1103


4/8

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 entomotoxicity

of the fermented broth (Sachdeva et al., 2000; Vidyarthi et al., 2002). It has also been reported that a high spore

count is not sufficient to ensure a good entomotoxicity (Avignone-Rossa and Mignone 1993). Moreover, it should be

noted here that in this study, there was not much difference between the entomotoxicity at 48h (14.2 x 109 SBU/L)

andat60h(14.3x109 SBU/L) of the fermentation process at solids concentration of 30 g/L. Therefore, the fermentor

experiment of Btk using the optimal solids concentration of SIW at 30g/L was carried out for a 48h-period.

1104


5/8

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 when

grown in different sludge with different total solids concentrations. In case of secondary sludge (JQS), it was

found that the total cell count increased with increasing solids concentration indicating increased availability of

nutrients at higher sludge solids concentration. The spore count, however, increased as the solids concentration

was 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 entomotoxicity

were obtained at the solids concentration of 26 g/L. Higher sludge solids concentrations also caused a decrease

in the entomotoxicity value (Lachhab et al., 2001). The same results were observed when using dewatered sludge

(JQC) as raw material, 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 was

more mobile due to lower viscosity and so, better aeration (oxygen transfer) than in the higher solids

concentrations. -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 solids

concentration, all the cells may not be in the same phase: some are sporulating while others are releasing the

mature spore into the medium and even some spores are germinating, i.e. there is a desynchronization of the

sporulation 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-fermentation

in the 15L-fermentor using SIW at 15 g/L (as control) and 30 g/L (as optimal value) solids concentration are

presented in the Table 3. The results obtained from the other experiments using the optimal total solids

1105


6/8

concentration of 26 g/L (Lachhab et al., 2001) as well as the other experiments using the optimal suspended

solids 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 as

raw 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 other

sludge types. Especially, BLS had the same total solids as that of SIW (30 g/L), however, the entomotoxicity in

this 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 ICPs

produced per spore. For example, Dulmage (1971); Salama et al. (1983) found that different media could change

the relative entomotoxicity against several target insects or even change the entomotoxicity of products obtained

from the same Bacillus thuringiensisstrain. Moreover, it should also be noted that the best conditions for spore

production 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 in

other cases, however, the entomotoxicity obtained was the highest (Table 3). Our results were also in accordance

with the results from other authors in respect that the high final spore counts are not proportional to high

entomotoxicities 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 because

landfilling is too expensive due to non-availability of landfill sites. Incineration produces air pollution and

green house gases, and ocean dumping is banned (Meyer et al., 2001). However, in this case, wastewater

sludge can be used for biopesticides production and it can be the most abundantly available raw material for

Bt 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 require

expensive 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 as

raw materials, as compared to other cheap raw materials used to produce Btk based biopesticides, do not

require addition or fortification of nutrients for growth, sporulation and entomotoxicity production by Bacillus

thuringiensis. Therefore, sludge and SIW are eventually the potential industrial raw materials for Btk-

biopesticides production.

1106


7/8

CONCLUSION

Following conclusions were drawn from the foregoing study

1. The optimal total solids concentrations required, as raw materials, for Btk- biopesticides production were

30g/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 to

other sludge types. However, SIW at solids concentration of 30 g/L gave higher entomotoxicity (17.5 x 10

9

SBU/L) than the BLS secondary sludge.

3. Fermentations of Bacillus thuringiensisvar kurstakiHD-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 costs

by handling lesser volumes of broths.

ACKNOWLEDGEMENTS

The authors are sincerely thankful to the Natural Sciences and Engineering Research Council of Canada (Grants

A4984, STP235071, Canada Research Chair) for financial support. The views or opinions expressed in this article

are those of the authors and should not be construed as opinions of the U.S. Environmental Protection Agency.

REFERENCES

Annachhatre A. P. and Amatya P. (2000). UASB treatment of tapioca starch wastewater. Journal of

Environmental Engineering, 126(12), 1149-1152.

Arcas J., Yantorno O. M. and Ertola R. J. (1987). Effect of high concentration of nutrients on Bacillus

thuringiensiscultures. Biotechnology Letters, 9(2), 105-110.

Avignone-Rossa C., Arcas J., Mignone C. (1992). Bacillus thuringiensis, sporulation and -endotoxin

production in oxygen limited and nonlimited cultures. World Journal of Microbiology and Biotechnology, 8(3),

301-304.

Avignone-Rossa C. and Mignone C. (1993). -endotoxin activity and spore production in batch and fed-

batch cultures of Bacillus thuringiensis. Biotechnology Letters, 15(3), 295-300.

Beegle C. C. (1990). Bioassay methods for quantification of Bacillus thuringiensis -endotoxin, Analytical

Chemistry of Bacillus thuringiensis. In: Analytical Chemistry of Bacillus thuringiensis, L. A. Hickle and W. L.

Fitch (eds.), USA: American Chemical Society, ISBN 0841218153, pp. 1421.

Brar S. K., Verma M., Tyagi R. D., Valro J. R. and Surampalli R. Y. (2005). Starch industry wastewater based

stable Bacillus thuringiensisliquid formulations. Journal of Economic Entomology, 98(6), 1890 - 1898.

Dulmage H. T. (1971). Production of -endotoxin by eighteen isolates of Bacillus thuringiensis, serotype 3, in

3 fermentation media. Journal of Invertebrate Pathology, 18(3), 353-358.

Farrera R. R., Prez-Guevara F. and de la Torre M. (1998). Carbon:nitrogen ratio interacts with initial

concentration of total solids on insecticidal crystal protein and spore production in Bacillus thuringiensis

HD-73. Applied Microbiology and Biotechnology, 49(6), 758-765.

Lachhab K., Tyagi R. D. and Valro J. R. (2001). Production of Bacillus thuringiensisbiopesticides using

wastewater sludge as a raw material: effect of inoculum and sludge solids concentration. Process

Biochemistry, 37(2), 197-208.

Meyer V. F., Redente E. F., Barbarick K. A. and Brobst R. (2001). Ecoystem restoration. Biosolids applications

affect runoff water quality following forest fire. Journal of Environmental Quality, 30(5), 1528-1532.

Morris O. N, Converse V., Kanagaratnam P. and Davies J. S. (1996). Effect of cultural conditions on spore-

crystal yield and toxicity of Bacillus thuringiensissubsp. aizawai(HD 133). Journal of Invertebrate

Pathology, 67(2), 129-136.

1107


8/8

Sachdeva V., Tyagi R. D. and Valro J. R. (2000). Production of biopesticides as a novel method of

wastewater sludge utilization/disposal. Water Science and Technology, 42(9), 211-216.

Salama, H. S., Foda M. S., Dulmage H. T. and El-Sharaby A. (1983). Novel fermentation media for production

of delta-endotoxins from Bacillus thuringiensis. Journal of Invertebrate Pathology, 41(1), 8-19.

Scherrer P., Luthy P. and Trumpi B. (1973). Production of -Endotoxin by Bacillus thuringiensisas a function

of glucose concentrations. Applied Microbiology, 25(4), 644-646.Standard Methods for the Examination of Water and Wastewater (1998). 20th edn, American Public Health

Association/American Water Works Association/Water Environment Federation, Washington DC, USA.

Vidyarthi A. S., Tyagi R. D., Valero J. R. and Surampalli R. Y. (2002). Studies on the production of B.

thuringiensisbased biopesticides using wastewater sludge as a raw material. Water Research, 36(19),

4850-4860.

Yezza A., Tyagi R. D., Valero J. R., Surampalli R. Y. and Smith J. (2004). Scale-up of biopesticide production

process using wastewater sludge as a raw material. Journal Industrial Microbiology and Biotechnology,

31(12), 545-552.

Yezza A., Tyagi R.D., Valero J. R. and Surampalli R. Y. (2006). Bioconversion of industrial wastewater and

wastewater sludge into Bacillus thuringiensisbased biopesticides in pilot fermenter. Bioresource

Technology, 97(15), 1850-1857.

Zouari N. and Jaoua S. (1999). The effect of complex carbon and nitrogen, salt, Tween-80 and acetate on

delta-endotoxin production by a Bacillus thuringiensissubsp kurstaki. Journal of Industrial Microbiology and

Biotechnology, 23(6), 497-502.

1108

Comparacion_aguaresidual_biopesticida

Documents

Transcript of Comparacion_aguaresidual_biopesticida