WO1995035365A1

WO1995035365A1 - Process for cultivating bacillus thuringiensis biopesticides in wastewater treatment sludges

Info

Publication number: WO1995035365A1
Application number: PCT/CA1995/000387
Authority: WO
Inventors: Rajeshwar D. Tyagi; Jose R. Valero; Maria De Lourdes Tirado
Original assignee: Institut National De La Recherche Scientifique
Priority date: 1994-06-22
Filing date: 1995-06-22
Publication date: 1995-12-28
Also published as: CA2193378A1; AU2782495A

Abstract

Disclosed herein is a process for preparing a Bacillus thuringiensis bio-insecticide, the process comprising the steps of: (a) inoculating wastewater treatment plant sludge(s) with Bacillus thuringiensis bacteria; (b) aerobically cultivating the bacteria in the sludge(s) for a period of time sufficient for producing the bio-insecticide resulting from the sporulation of the bacteria through synthesis of insecticidal δ-endotoxin proteins in the form of parasporal crystals; (c) recovering the sludge(s) containing the resulting bio-insecticide.

Description

TITLE OF THE INVENTION

PROCESS FOR CULTIVATING BACILLUS THURINGIENSIS BIOPESTICIDES IN WASTEWATER TREATMENT SLUDGES

BACKGROUND OF THE INVENTION

1. FIELD OF THE INVENTION

The present invention relates to a novel process for the production of Bacillus thuringiensis (all serotypes and strains) biopesticides. More specifically, the novel process uses sludges generated by wastewater treatment plants as a growth substrate.

2. DISCUSSION OF THE PRIOR ART

CHEMICAL INSECTICIDES

Chemical insecticides have traditionally been used to control various insects which adversely affect agriculture and forestry or that constitute disease vectors. Although chemical insecticides have generally been efficacious, their production costs are high and they present environmental concerns. For example, because of their mode of action, they can cause many ecological problems by destroying harmful and harmless and even useful insects. For example, chlorinated hydrocarbons, pyrethroids, organophosporus compounds and carbamates act by disrupting or inhibiting the nervous system functions of insects. This may also represent a risk to all living organisms. Furthermore, some insects have become resistant to chemical insecticides. Chemical pesticides can also accumulate in the environment and become a soil or water contaminant. BIO-INSECTICIDES

The use of entomopathogenic (microorganisms pathogenic to insect pests) microorganisms as biological insecticides have resulted in a valuable option to chemical insecticides. Various groups of microorganisms are considered useful as entomopathogenic agents. These groups include a range of bacteria, viruses, protozoa and fungi; each species can vary in its mode of insect infection, site of replication and mechanism of pathogenicity.

BACILLUS THURINGIENSIS (hereinafter identified in shortened form as BT) : BACTERIAL INSECTICIDE

BT represents a major class of microbes used for insect biocontrol. Most BT strains produce several different insecticidal δ-endotoxin proteins in the form of parasporal crystals. It has been shown that BT strains can be very specific in their lethal activity against different insect pests while being harmless to mammals, birds or beneficial insects. In addition, food products treated with this insecticide are safe for human or animal consumption. The BT insecticide is also biodegradable and will not accumulate in the environment or cause pollution problems. Accordingly, increasing attention is directed to BT as a viable alternative to chemical pesticides.

Between the 35 or so BT serotypes that have so far been identified, approximately 3 are currently used as microbial insecticides. These will now be briefly discussed in sequence.

BT serotype 3a3b (Kurstaki variety) which is known as a specific pathogen to the larvae of the Lepidoptera. BT 3a3b is currently used in agriculture and forestry (protection of plants and cereals) . BT serotype 14 (Israelensis variety) is known as a specific pathogen to the larvae of certain Diptera (mosquitoes and black flies) . Serotype 14 is also used to fight the vector of some tropical diseases (Onchocercosis. Filariosis. etc.) or for the sanitation of public areas. BT serotype Tenebrionis is a pathogen to the larvae of certain kind of coleopters, in particular to the Colorado potato beetle.

PRIOR ART PROCESSES FOR PRODUCING BT One of the keys to successful commercialization of BT insecticides is the development of an adequate culture medium. When cultured in appropriate nutrient broth, vegetative cells sporulate and lyse, releasing spores and parasporal crystals into the medium. Like other microorganisms BT needs the following ingredients for growth, reproduction and spore formation: water, a carbon source for biosynthesis and energy, a nitrogen source, mineral elements and other optimal growing factors. Most strains of BT grow best at 30°C under vigorous aeration and a pH level between 6.8 and 7.2.

Current industrial production of BT is conducted by batch liquid fermentation process or submerged fermentation in which the cultures grow dispersed by air in liquid media at controlled pH and temperature. Such processes are expensive in terms of initial capital investment and operation. The typical production scheme begins with the inoculation of a 15 L vessel with a seed culture. This culture serves to inoculate larger vessels arriving to tank volumes of 30,000 to 100,000 L. After harvest, the product is concentrated and either dried or stabilized as a liquid suspension using different preservatives such as sorbitol, sodium benzoate, xylol, etc. , to avoid further growth and germination of the spores. KNOWN GROWTH MEDIA:

The type of media used for the growth, sporulation and 5-endotoxin production of BT can influence production cost of the bio-insecticide. Consequently, various attempts have been made to evolve efficacious yet cheaply available media. For example, the following have been proposed: AGRO-INDUSTRIAL BY-PRODUCTS:

It is known in the prior art to use various agro- industrial by-products as BT growth media ingredients. For example the following ingredients have been suggested: cheese whey, corn steep liquor, sorter liquor, cottonseed meal, wheat bran, extracts of potatoes, carrots and sweet potatoes, cassava starch, maize, cowpea liquor, fodder yeast, fish meal, cotton seed meal, horse beans, wheat bran, citrus peels and seeds of dates, (see Salama et al. in Entomophaga, 28, pages 151-160, 1983,; in J. of Invert Pathology, 41, pages 8-19, 1983) . These ingredients are generally added to synthetic media comprising water, glucose, yeast extract and a plethora of growth enhancing additives such as nitrogen sources, protein sources usually in the form of leguminous seeds, such as peanuts, chick peas, lima beans, horse beans, kidney beans and soya beans, mineral salts such as CaC0₃, NaCl, K_jHP^., MgS0₄, CaCl₂, FeS0₄ and CuS0₄ and small amounts of some amino acids. Although these proposed growth media use by-products of agro-industrial operations, their availability and acquisition costs may often be prohibitive since a number of other economically attractive products can be made from them, for example: proteins, organic solids, ethanol. Furthermore, the use of synthetic media and the use of additives add to the cost and complexity of the media.

Chilcott and Pillai (see Mircen Journal, 1, pages 327-332, 1985) have investigated the use of waste products of the coconut oil industrial processes as a BT growth medium ingredient. The ingredient is usually coconut endosperm extract which is prepared by boiling finely ground endosperm in distilled water for 2 minutes. The endosperm is then extracted by filtration through several layers of muslin. Although satisfactory results are obtained, the required pretreatment and the non¬ availability of coconuts in many parts of the world represent drawbacks for its use in commercial production of BT. Dharmsthiti et al. (see J. of Invert. Pathology, 46, pages 231-238, 1985) have proposed the use of by¬ products of monosodium glutamate production. For example, a medium could be composed of 4 to 7%/vol of hydrolyzed liquor (HDL) by-product from a monosodium glutamate factory, supplemented with 0.05% K_jHPO^. However, the availability of this by-product (hydrolyzed liquor by-product from monosodium glutamate production) is not reliable for the industrial production of BT.

Obeta and Okafor have for their part proposed the use of cow blood as a BT growth media ingredient (see Applied and Env. Microb, 47, pages 863-867, 1984). For example, they have proposed a media comprising lOg/1 of cow blood, 0.02g/l MnC1.4H₂0; 0.05g/l MgS0₄.7H₂0 and l.Og/1 CaC0₃ combining it with different types of legume seeds in an aqueous base.

Mummigatti et al. have proposed the use of dehusked greengram powder, defatted soybean powder soluble starch and cane sugar molasses as BT growth media ingredients (see J. of Invert. Pathology 55, pages 147- 151, 1990) . However these ingredients must generally be subjected to pretreatment such as defatting prior to their use. These requirements limit their use in commercial production.

In summary, the known BT growth media carry various drawbacks which has limited their use for the commercial production of BT bio-insecticides. Among the drawbacks, it is noted that in many cases the media must be submitted to complicated and expensive pretreatments before they are adequate for use. These pretreatments can include heating, defatting, long drying times, steeping protein precipitation and concentration. Some of the proposed media ingredients cannot be used directly and must be diluted or will cause inhibitory effects on BT growth and sporulation (substances with certain kind of a ino acids, high concentrations of carbohydrates, etc) . Moreover, many of the proposed ingredients do not contain all the necessary elements for BT growth, sporulation and δ-endotoxin production. The use of additives will of course cause a rise in BT production costs. Additionally, some of the proposed ingredients are not cheaply and widely available throughout the world.

The common point in prior growth media is that they are nutrient-rich broths capable of sustaining the growth of most varieties of bacteria. These broths often comprise additives to optimize growth rates. In contrast, and as will be explained in detail hereinbelow, the growth media of the present invention are basically devoid of nutrients and composed mainly of bacteria protoplasm (live and dead bacteria cell mass) . It has been found, surprisingly, that BT is able to grow in these specific growth media which are plentiful and inexpensive when compared to prior art growth media.

There is therefore a need for an alternative BT growth medium which will overcome the above-mentioned drawbacks of the prior art. SUMMARY OF THE INVENTION

In response to the above-mentioned drawbacks, it is an object of the present invention to present an economically efficient and novel process for sustaining growth, sporulation and insecticidal toxin production of all serotypes and all strains of BT. Further objects of the process of the present invention are to provide the use of a novel nutrient medium which: - requires minimal pretreatment; contains practically all of the necessary nutrients for adequate BT growth without the requirement of additives; has plentiful local availability so as to minimize purchase or transportation costs.

Surprisingly, it has been found that wastewater treatment sludges constitute a well suited medium for the efficient growth, sporulation and δ-endotoxin synthesis for the production of BT bio-insecticides. Accordingly, the invention provides a process for preparing a Bacillus thuringiensis bio-insecticide, the process comprising the steps of:

(a) inoculating a given quantity of wastewater treatment plant sludge(s) with Bacillus thuringiensis bacteria;

(b) aerobically cultivating the bacteria in the sludge(s) for a period of time sufficient for producing the bio- insecticide resulting from the sporulation of the bacteria and synthesis of insecticidal fS-endotoxin proteins in the form of parasporal crystals; (c) recovering the sludge(s) containing the resulting bio- insecticide, with the proviso that the said sludge(s) is(are) not non-hydrolyzed primary sludge(s) .

In one embodiment of the invention, the process further comprises the hydrolyzing of the wastewater treatment sludges prior to inoculation with the Bacillus thuringiensis bacteria.

In a preferred embodiment of the invention, the inventive process is specifically directed to the production of BT of the Kurstaki variety. Nevertheless, the process is not limited to and can be used for all serotypes and strains.

The process of the present invention can use the plentiful and widely available wastewater treatment sludges of various wastewater treatment plants such as municipal wastewater treatment plants and industrial wastewater treatment plants such as pulp and paper or food and beverages industries or any other similar biological sludge. It has been observed that nutrient additives are generally not required to support the growth and sporulation of BT in wastewater sludges. Hydrolysis as a pretreatment step generally increases BT spore production and <S-endotoxin is not time consuming or expensive. BT grown in sludge has essentially the same insecticidal potency as that obtained in any standard medium, for example soybean flour.

Other features and advantages of the invention will become apparent to those of ordinary skill in the art upon review of the following detailed description, claims, and drawings.

BRIEF DESCRIPTION OF THE FIGURES

FIGs. 1 to 7 show graphical representations of the viable

BT spore and cell count in millions over a time scale in days for different non-hydrolyzed wastewater sludges from various sources in Canada.

FIGs. 8 to 13 show graphical representations of the viable BT spores and cell count in millions over a time scale in days for the previously hydrolyzed wastewater sludges from various sources in Canada. DETAILED DESCRIPTION

Surprisingly, it has been found that wastewater sludges constitute a well suited medium for the production of BT and bio-insecticides therefrom. The sludges generated by wastewater treatment plants generally contain organic matter, protein, nitrogen, phosphorus, mineral salts, a pH near neutrality and other elements that render them a well suited medium for the production of BT. Wastewater treatment sludges are of course cheaply and plentifully available. Moreover, sludges having therein BT grown bio-insecticides can be, in most instances, directly applied to agricultural land and/or disposed of in forested areas for the control of defoliating agricultural and/or forest insects. This process also complies well with the sludge use as fertilizer on agricultural land.

To demonstrate the use of wastewater sludges as a BT growth medium, various experiments were conducted using sludges from different sources. The experiments are representative in that they involve the growth, sporulation and 5-endotoxin production of BT of the Kurstaki variety. It will be understood by those skilled in the art that the experiments are for illustrative purposes and that all other BT varieties could be similarly cultivated.

Characterization of primary and secondary wastewater sludges, terminology and definitions:

It should be observed that primary sludges are produced during the primary treatment of wastewater (the process consists of removal of suspended materials by sedimentation of by some other techniques) . Secondary sludges are produced during the secondary treatment of wastewater (activated sludge process or some other microbiological process configuration: involves conversion of wastewater by microorganisms) . Primary sludges contain suspended solids present in the raw wastewater. Secondary sludges contain chemical or biological solids produced during the treatment process.

EXPERIMENTAL

The experiments were conducted to illustrate and demonstrate the feasibility of using wastewater treatment sludges in the process of the present invention. 1. BT STRAIN AND INOCULUM.

Culture tubes containing 5ml of nutrient broth prepared with 30g/l of tryptic soy broth and 3g/l of yeast extract were sterilized at 121°C during 20 min.

These tubes were inoculated with a loopful of BT Kurstaki HD-1 grown on tryptic soy agar.

The culture was incubated during 18 hours at 30^βC in a shaking water bath. The dilution technique was used to estimate the viable spore count of the nutrient broth. From this preparation, a suspension containing 1000 spores/ml was prepared by diluting in sterilized physiological solution (0.9% NaCl) . This suspension was kept at 4°C until it was used as an inoculum for reference standard medium and sludge samples.

2. TESTED SLUDGES

Seven sludges from different wastewater treatment plants (municipal and industrial) in the Province of Quebec (Canada) were tested to determine their ability to sustain growth, sporulation and rS-endotoxin production of BT. The sludges were:

2.1) Primary sludge of Valcartier, Valcartier (PS)

2.2) Secondary sludge from Black Lake, Black Lake (Sec)

2.3) Secondary sludge from Beauceville, Beauceville (Sec) 2.4) Secondary sludge from Ste-Claire, Ste-Claire (Sec) 2.5) Secondary aerobically digested sludge from Black Lake, Black Lake Sec (ADS)

2.6) Sludge from activated sludge process of a paper mill plant, (PPSec) 2.7) Sludge from the membrane reactor of a paper mill plant, (PPMBR)

It is to be understood that primary sludges refer to the sludges produced in the primary treatment of wastewaters. Secondary sludges are obtained by further treatment of the supernatant of the primary treatment.

The samples were collected in sterile polypropylene bottles, shipped cold to the laboratory and kept at 4°C until used in the experiments. The physical characteristics of the sludges are presented in Table 1 below.

TABLE 1

CONCENTRATION OF SOLIDS

SAMPLE mg/L

TS VS SS VSS

Valcartier (PS) 20430 16340 18610 15876

Black Lake (Sec) 5100 2500 2490 1820

Beauceville (Sec) 43220 21620 37850 19060

Ste. Claire (Sec) 24230 14160 22520 13760

Black Lake (ADS) 25240 12770 22830 12060

Sludge 1 (PP Sec) 20530 16180 15990 15800

Sludge 2 (PP MBR) 32180 22600 28190 22080

Note: Sludge 1 is from the activated sludge treatment at the pulp and paper mill.

Sludge 2 comes from the membrane reactor of the same pulp and paper mill.

TS - Total solids

VS - Volatile solids

SS - Suspended solids

VSS - Volatile suspended solids

50ml Erlenmeyer flasks containing 25ml of each sludge sample were sterilized at 121°C during 20 min. and used as culture media for BT.

Pretreatment of sludge samples

It is known in the prior art that the presence of certain amino acids may stimulate the growth and sporulation of BT. It is further known that hydrolysis treatment of organic matter can be used to obtain these amino acids from the organic matter (see Muratov et al. in Biotekhnologiya, 5, pages 592-595, 1987) . To study also the influence of hydrolysis as a simple pretreatment over spore production 25ml of each of the sludges mentioned above were added to a 50ml Erlenmeyer flask and their pH was adjusted to 2 with IN H₂S0₄. After acid addition, the sludge samples were sterilized at 121°C during 20 min. , cooled, pH adjusted to 7 with IN NaOH and sterilized again at 121°C during 20 min. and cooled. These samples were then used as culture media of BT.

The pretreated sludges were:

2.8) Hydrolyzed primary sludge of Valcartier, Valcartier (HPS)

2.9) Hydrolyzed secondary sludge of Black Lake, Black Lake (HSec)

2.10) Hydrolyzed secondary sludge of Beauceville, Beauceville (HSec)

2.11) Hydrolyzed secondary sludge of Ste-Claire, Ste- Claire (HSec) 2.12) Hydrolyzed aerobically digested sludge of Black Lake, Black Lake (HADS)

2.13) Hydrolyzed secondary sludge from activated sludge process of a pulp and paper mill plant (PPHSec)

2.14) Hydrolyzed sludge of membrane reactor of a paper mill plant, (PP HMBR)

3. SPORE AND S-ENDOTOXIN PRODUCTION: FERMENTATION MEDIUM

3.1 Reference standard medium (RSM^ : soybean flour solution.

A soybean solution was used as reference standard medium to compare results obtained with the hydrolyzed and non-hydrolyzed sludge samples. The composition of this medium is shown in Table 2 below. TABLE 2

COMPOSITION OF STANDARD REFERENCE STANDARD MEDIUM (RSM)

(G/L)

SOYBEAN 15

DEXTROSE 5

MAIZE STARCH 5

K₂HPO₄ 1.0 KH₂PO₄ 1.0

MgSO₄.7H₂0 0.3

FeSO₄.7H₂0 0.02

ZnSO₄.7H₂0 0.02

CaCO₃ 1.0

3.2 EXPERIMENTAL PROCEDURE 3.2.1 Reference Standard Medium

25ml of this solution were transferred to 50 ml Erlenmeyer flask followed by sterilization at 121°C for 20 minutes. After cooling at room temperature the soybean solution was inoculated with one ml of the 1000 spores/ml suspension prepared as explained before. This permitted to have an initial concentration of 40 spores/ml in the standard reference media. The flask was incubated in a temperature control shaking water bath at 30°C and 100 oscillations/minute (osc/min) until total lysis of cells and liberation of spores was achieved. This process was followed with daily viable spore counts and microscopic analysis.

When the sporulation was completed, incubation was stopped, the standard reference media (3 ml) was transferred to a polycarbonate tube and centrifuged in a superspeed automatic refrigerated centrifuge at 4500 rpm during 20 min. Supernatant was discarded and the pellets were re-suspended with 2ml of sterilised distilled water. The viable spore and cell count of this concentrate and its spore-crystal complex concentration were determined by dilution technique.

3.2.2 SLUDGE MEDIUM

One milliliter of the suspension containing 1000 spores/ml was added as an inoculum to 25ml of sterilized hydrolyzed and non hydrolyzed sludges contained in 50ml

Erlenmeyer flasks. The flasks were incubated in a shaking water bath at 30°C and 100 osc/min to assure mixing and oxygen transfer. The viable spore and cell count was determined daily by dilution technique and the process was followed daily also by microscopic analyses.

Once the sporulation was completed the incubation was terminated. 3ml of inoculated sludge was centrifuged at 4500 rpm during 20 min in a superspeed automatic refrigerated centrifuge.

The supernatants were discarded and the pellets were re-suspended with two ml of distilled sterilised water. This concentrate was used for the bioassays. The viable spore and cell count and the spore-crystal complex concentration of each concentrate were determined by the spore dilution technique.

4. ESTIMATION OF SPORES AND 5-END0T0XIN PRODUCTION 4.1 Microscopic analysis

To follow the progress of the sporulation process, each sludge sample under incubation was observed daily under a microscope. Preparations for microscopic observations were made by placing a drop of the sludge under incubation on a microscope slide with a sterilized loop; the observations were made with an immersion oil objective and phase contrast dark field method (x 1200) . 4.1.2 Coloration

There are basic coloration methods for microscopic identification of spores, crystals and cells of BT. One method consists in the use of malachite green and the other is by using Buffalo black. Both techniques were used to follow sporulation of BT in the nutrient broth and in the soybean solution. 4.2 EVALUATION OF BT GROWTH

4.2.1 Viable spore and cell count The dilution technique used to determine the viable spore and cell count was as follows: 0.5ml of the sample (nutrient broth, soybean or sludges) was added to culture tubes containing 4.5ml of previously sterilised physiological solution. Appropriate dilutions were made with each sample. Subsequently, 0.1ml of each diluted sample was aseptically spread on Petri dishes containing sterilised tryptic soy agar. Duplicate samples were used for each dilution. Petri dishes were incubated at 30°C during 18 hours. Viable spore and cell count was determined with a colony counter (see FIGs 1 to 14 for viable spore and cell counts) .

4.2.2 Viable Spore Count of the spore-crystal complex 10ml of distilled water and 0.02ml of 1% Tween 80™ solutions were placed in a test tube followed by sterilization at 121°C for 15 minutes. Test tubes were then cooled to room temperature and 1 ml of BT grown sludge was added. The solution thus obtained was heat treated at 65°C for 15 minutes aseptically. The viable spore count (spore-crystal complex) was then determined following the dilution technique described above. Duplicate samples were also used for viable spore count.

5. Potency (Bioassav) To evaluate the potency of the BT grown sludge, bioassays were conducted against 3rd instar larvae of the spruce budworm (Choristoneura fumiferana) , an insect pest that causes great damages to the coniferous forests of North America. Standard laboratory larvae of this insect were raised at room temperature on artificial diet contained in plastic cups and used at the 3rd stage. Table 3, below, presents the characteristics of the diet.

TABLE 3

CHARACTERISTICS OF ARTIFICIAL DIET

Distilled water 176 mL

Casein (vitamin free) 28 g Potassium hydroxide 4M 4 mL

Alphacel 4 g

Salt mixture Wesson 8 g

Wheat embryo 24 g

Choline chloride 0.8 g Vitamin solution 8 mL

Ascorbic acid 3.2 g

Formaldehyde 40% 0.4 mL

Sucrose 28 g

Raw linseed oil 0.5 % Agar 20 g

Distilled water 500 mL

Vitamin solution

Distilled water 100 mL

Niacine 100 mg

Calcium pantothenate 100 mg Riboflavine 50 mg

Thiamine hydrochloride 25 mg

Pyridoxine hydrochloride 25 mg

Folic acid 25 mg

Biotine 2 mg Vitamin B 12 0.2 mg The assay procedure for each kind of BT grown sludge is described in Table 4, below.

TABLE4

BIOASSAYS PROTOCOL

No. RSM Sludge Control Sludge Foray of with (dist.H₂O) control larvae BT

50 lμL

20 lμL

20 2μL

20 3μL

20 IμL

20 2μL

20 3μL

20 lμL

20 2μL

20 3μl

20 lμL

Number of tubes 60 60 50 60 20

Fifty larvae were placed in fifty bioassay tubes (one larvae each tube) containing the artificial diet mentioned before added with 1 μl of distilled sterilised water; these larvae served as control.

Control bioassays using 1, 2 and 3 μl of each fresh, sterilised and not inoculated sludge were also conducted using same larvae (20x3=60 tubes) .

1, 2 and 3 μl of centrifuged BT grown sludge were spread in 20 tubes for each dosage (20x3= 60 tubes) having one larvae in each tube. The reference standard medium (soybean flour solution) was also added in doses of 1,2 and 3 μl into 20 tubes (20x3=60 tubes). Commercial biopesticide Foray 48 B™ was also included in the bioassays using a dosage of 1 μl added to 20 tubes having a larvae in each tube. This sequence was repeated for each centrifuged BT grown sludge. The 250 tubes necessary to test potency of BT grown sludges were left at room temperature. Larval mortality was recorded on a daily basis. 5.2 Use of Foray 48B™ To determine the biological potency of BT grown sludges in terms of "international units/μl" each sample was compared with the known potency of commercial preparation Foray 48B™. Its spore concentration is 64xl0⁹ spores/ml and its potency is 12.7x10° IU/L (12.7xl0³/ _L) . The high biological activity of this insecticide is because it is a purified and concentrated preparation obtained from soybean fermentation.

The biological activity of the spore-crystal complex of this commercial biopesticide is expressed in activity units, for a specific insect test species, related to an international standard preparation. The BT preparation E-61 of the Pasteur Institute in Paris was arbitrarily chosen as primary standard, and was assigned a specific activity of 1000 IU/mg.

RESULTS

6. Sporulation and δ-endotoxin production

6.1 Microscopic analysis

In the case of BT grown sludges coloration techniques for spore identification were not used because the high content of organic and inorganic material contained in the sludges made it difficult to distinguish spores from crystals and cells.

However, daily analysis of the inoculated sludges shows differences in sporulation process between them. The results are: NON-HYDROLYZED SLUDGES:

Valcartier (PS) - No spore production was detected.

Black Lake (Sec) - The analysis showed that sporulation process was normal, beginning at the second day. Total sporulation was completed after 10 days of fermentation.

Beauceville (Sec) - Sporulation process was normal beginning at the second day taking 11 days for total sporulation.

Ste Claire (Sec) - Sporulation process was normal beginning at the fourth day. Total sporulation was obtained after 10 days.

Black Lake (Sec ADS) - Sporulation process was normal beginning at the fourth day. Total sporulation was obtained after 10 days. PP (Sec) . - Sporulation process was normal beginning at third day. Total sporulation was achieved after 11 days.

PP(MBR)

HYDROLYZED SLUDGES: Valcartier (HPS) - Hydrolysis process favors sporulation.

The primary sludge as such was not able to sustain growth and sporulation. Sporulation began the third day and was completed after 10 days.

Black Lake (HSec) - Sporulation began the second day, number of spores was higher than in the same sludge without hydrolysis. Total sporulation was completed after 8 days.

Beauceville (HSec) - Sporulation began at second day.

Total sporulation was completed after 10 days. Ste Claire (HSec) - Sporulation began at third day.

Total sporulation was achieved after 9 days.

Black Lake (HADS) - Sporulation began at third day, number of spores increase and total sporulation was completed after 8 days. PP (HSec) - Sporulation began at second day; total sporulation was completed after nine days. PP (HMBR) - Sporulation began second day; total sporulation was completed after nine days.

As shown, hydrolysis helped to diminish time required to complete sporulation in all BT grown sludges.

6.2 Viable spore counts 6.2.1 Non-hydrolized sludges

All the sludges, except that of Valcartier (PS) , support growth, sporulation and δ-endotoxin production of BT var. Kurstaki. Results in Table 5, below, show that before concentration viable spore and cell count and viable spore count (spore-crystal complex) in sludges at the end of sporulation period ranged from 0 spores and cells/ml for the sludge from Valcartier (PS) to 1.4xl0⁷ spores and cells/ml for Beauceville (Sec) . The highest spore-crystal complex concentration corresponds also to the same sludge (1.3xl0⁷ spores/ml) of Beauceville (Sec).

TABLE 5

CONCENTRATION OF VIABLE SPORES IN UNHYDROLYZED BT GROWN SLUDGES

(non-centrifuged)

Sample Viable spores and Spore crystal complex cells count

Spore and cell/mL Spore/mL

Valcartier (PS) 0 0

Black Lake (Sec) 1.3 x 10⁶ 1.0 x 10⁶

Beauceville (Sec) 1.4 x 10⁷ 1.3 x 10⁷

Ste. Claire (Sec) 1.2 x 10⁷ 1.0 x 10⁷

Black Lake (Sec ADS) 1.1 x 10⁶ 1.0 x 10⁶

Sludge 1 (PP Sec) 1.0 x 10⁷ 3.1 x 10⁶

Sludge 2 (PP MBR) l x lO⁷ 1.5 x 10⁶

RSM 2.4 x 10⁸ 2 x 10⁸

Note: RSM corresponds to the reference standard medium prepared with soybean flour

Sludge 1: sludge from activated sludge treatment at the pulp and paper mill Sludge 2: sludge from the membrane reactor of the pulp and paper mill

The viable spore counts after concentration of unhydrolyzed BT grown sludges by centrifugation is shown in Table 6.

TABLE 6

VIABLE SPORES COUNT IN UNHYDROLYZED BT GROWN CENTRIFUGED SLUDGES

Sample Viable spores and Spore crystal complex cells count

Spores and cells/mL Spores/mL

Valcartier (PS) 0 0

Black Lake (Sec) 1.85 x 10⁶ 1.5 x 10⁶

Beauceville (Sec) 2.3 x 10⁷ 2.0 x 10⁷

Ste. Claire (Sec) 1.8 x 10⁷ 1.6 x 10⁷

Black Lake (Sec ADS) 1.5 x 10⁶ 1.3 x 10⁶

Sludge 1 (PP Sec) 1.4 x 10⁷ 4.1 x 10⁶

Sludge 2 (PP MBR) 1.3 x 10⁷ 1.83 x 10⁶

RSM 3.2 x 10⁸ 3 x 10⁸

Note: RSM corresponds to the reference standard medium prepared with soybean flour. Sludges 1 and 2 are the same as in Table 5

Viable spores and cells counts in concentrated sludges ranged from 1.5xl0⁶ spores/ml for the sludge from Black Lake (Sec ADS) to 2.3xl0⁷ spores/ml Beauceville (Sec). Spore crystal complex concentrations varied from 1.3xl0⁶ spores/ml for Black Lake (ADS) to 2xl0⁷ spores/ml for Beauceville (Sec) . 6.2.2 Hydrolyzed sludges

As has been observed by the microscopic analysis results, hydrolysis increased growth and sporulation of BT in sludges. The viable spore counts before concentration by centrifugation are shown in Table 7, below.

TABLE 7

VIABLE SPORES COUNT IN

HYDROLYZED BT GROWN SLUDGES

(non-centrifuged)

Sample Viable spores and Spore crystal complex cells count

Spores and cells/mL Spores/mL

Valcartier (HPS) 1.3 x 10⁷ l x 10⁷

Black Lake (HSec) 1.4 x 10⁷ 1.2 x 10⁷

Beauceville (HSec) 2 x l0⁷ 1.8 x 10⁷

Ste. Claire (HSec) 1.5 x 10⁷ 1.1 x 10⁷

Black Lake (HADS) 1.7 x 10⁶ 1.6 x 10⁶

Sludge 1 (PP HSec) 1.6 x 10⁷ 3.2 x 10⁶

Sludge 2 (PP HMBR) 1.2 x 10⁷ 9.9 x 10⁶

RSM 2.4 x 10⁸ 2 x l0⁸

In this case, the values of viable spores and cells count vary from 1.7xl0⁶ spores/ml for Black Lake (HADS) to 2xl0⁷ spores/ml for Beauceville (HSec) . The spore- crystal complex concentrations vary from 1.6x10° spores/ml for Black Lake (HADS) to 1.8xl0⁷ spores/ml for Beauceville (HSec) .

After centrifugation of hydrolyzed Bt grown sludges, the viable spore and cell count varies from 2.8x10° spores/ml for Black Lake (HADS) to 2.7xl0⁷ spores/ml for Beauceville (HSec) . The spore-crystal complex concentrations vary from 2.5x10° spores/ml for Black Lake (HADS) to 2.4xl0⁷ spores/ml for Beauceville (H Sec). See Table 8, below.

TABLE 8

VIABLE SPORE COUNT IN HYDROLYZED BT GROWN CENTRIFUGED SLUDGES

Sample Viable spores and Spore crystal complex cells count

Spores and cells/mL Spores/mL

Valcartier (HPS) 1.6xl0⁷ 1.2xl0⁷

Black Lake (HSec) 2.2xl0⁷ 2xl0⁷

Beauceville (HSec) 2.7 x 10⁷ 2.4xl0⁷

Ste. Claire (HSec) 2.5xl0⁷ 2xl0⁷

Black Lake (HADS) 2.8xl0⁶ 2.5xl0⁶

Sludge 1 (PP HSec) 2.2xl0⁷ 4.2xl0⁶

Sludge 2 (PP HMBR) 2xl0⁷ 1.6xl0⁷

RSM 3.2xl0⁸ 3xl0⁸

Note: RSM corresponds to the reference standard medium prepared with soybean flour. Sludges 1 and 2 are the same as in Table 5 The results show that hydrolysis can be used as a simple pretreatment to increase spore production.

7. Potency

Mortality percentages after 3, 6, 9 and 12 days for all the sludges are presented in Tables 9 A and 9B below.

TABLE 9A

MORTALITY LARVAE PERCENTAGES

UNHYDROLYZED BT GROWN SLUDGES

DAY μL RSM 2.2 2.3 2.4 2.5 2.6 2.7

1 5 5 5 0 5 0 0

3 2 5 5 5 0 5 0 0

3 10 10 5 0 10 0 0

1 15 15 10 5 10 0 0

6 2 20 15 15 5 15 0 0

3 25 20 20 5 20 5 10

1 20 20 15 5 15 5 5

9 2 30 20 20 5 20 5 15

3 40 25 25 20 25 15 20

1 35 20 20 10 20 5 5

12 2 40 30 30 25 25 15 15

3 50 35 35 30 30 20 20

Note: RSM corresponds to the reference standard medium prepared with soybean flour. TABLE 9B

MORTALITY LARVAE PERCENTAGES

HYDROLYZED BT GROWN SLUDGES

DAY μL RSM 2.8 2.9 2.10 2.11 2.12 2.13 2.14

1 5 0 5 5 0 5 0 0

3 2 5 0 10 10 5 10 0 0

3 10 0 10 10 5 10 0 0

1 15 10 10 10 5 10 10 10

6 2 20 10 15 20 10 15 15 10

3 25 15 15 25 15 20 20 10

1 20 20 30 20 25 20 25 30

9 2 30 25 35 30 35 25 25 35

3 40 35 60 35 35 35 25 60

1 35 25 45 30 35 25 25 35

12 2 40 35 50 35 40 35 35 45

3 50 40 70 45 45 45 45 65 Note: RSM corresponds to the reference standard medium prepared with soybean flour.

The results can be summarized as follows: 7.1 Reference standard media (RSM)

Mortality percentages obtained with this substrate were utilised to compare the mortality percentages obtained with the sludges.

7.2 Sludge samples Mortality percentages of larvae for Ste Claire (Sec), PP(Sec) , PP(MBR) and Valcartier (HPS) was lower than RSM before 6 days but after mortality began to increase. It is important to note that hydrolysis is a good pretreatment for primary sludges because prior to hydrolysis BT growth was low and no bioassays were conducted.

Black Lake (Sec) and (HSec) gave the more acceptable percentages of mortality in comparison with those of RSM; Black Lake (HSec) gave higher mortality percentages than RSM after 6 days. The potency of

Beauceville (HSec) can be comparable to that of RSM.

Hydrolysis increased the potency of sludges as can be seen in sludges of Ste Claire (HSec) , PP(HSec) and PP(HMBR) especially after 6 days.

Biological activities of unhydrolyzed sludges expressed in international units/μl (IU/μl) are presented in Table 10, below.

TABLE 10

BIOLOGICAL POTENCY OF UNHYDROLYZED BT GROWN SLUDGES

Sample Potency IU/μL x 10³

Soybean (reference media) 3.811

Black Lake (Sec) 3.260

Beauceville (Sec) 3.000

Ste. Claire (Sec) 1.295

Black Lake (Sec ADS) 3.020

Sludge 1 (PP Sec) 0.878

Sludge 2 (PP MBR) 1.288

Note: IU/ μL = international units/ μL Sludge 1: sludge from activated sludge treatment of a pulp and paper mill.

Sludge 2: sludge from the membrane reactor of a pulp and paper mill. Between the sludges used without prior hydrolysis, sludge Black Lake (Sec) gave the highest biological potency (3260 IU/μl) followed by sludges Beauceville (Sec) (3000 IU/μl) and Black Lake (Sec ADS) (3020 IU/μl) . The lowest potency was obtained with PP(Sec) (878 IU/μl) .

Hydrolysis had, in general, a favorable impact on biological potency of the sludges. Black Lake (HSec) gave a potency of 4090 IU/μl, followed by Black Lake

(HADS) (3600 IU/μl) and PP(HMBR) (3500 IU/μl). Table 11, below.

TABLE 11 BIOLOGICALPOTENCYOFHYDROLYZED BTGROWNSLUDGES

Sample Potency IU/μL x 10³

Soybean (reference media) 3.811

Valcartier (HPS) 3.000

Black Lake (HSec) 4.090

Beauceville (HSec) 3.200

Ste. Claire (HSec) 3.000

Black Lake (HADS) 3.600

Sludge 1 (PP HSec) 3.220

Sludge 2 (PP HMBR) 3.500

Note: IU/ μL = international units/ μL Sludge 1: sludge from activated sludge treatment of a pulp and paper mill plant. Sludge 2: sludge from the membrane reactor of a pulp and paper mill plant. These results show that hydrolyzed and non- hydrolyzed sludges, with the exception of non-hydrolyzed primary sludges, are capable of sustaining growth, sporulation and 6-endotoxin production of BT and can be successfully used as an alternate media for its production.

Although the invention has been described above with respect with one specific form, it will be evident to a person skilled in the art that it may be modified and refined in various ways. It is therefore wished to have it understood that the present invention should not be limited in scope, except by the terms of the following claims.

Claims

What is claimed:

1. A process for preparing a Bacillus thuringiensis bio-insecticide, said process comprising the steps of:

(a) inoculating wastewater treatment plant sludge(s) with Bacillus thuringiensis bacteria;

(b) aerobically cultivating said bacteria in said sludge(s) for a period of time sufficient for producing said bio-insecticide resulting from the sporulation of said bacteria through synthesis of insecticidal 5- endotoxin proteins in the form of parasporal crystals;

(c) recovering said sludge(s) containing said resulting bio-insecticide.

2. The process of claim 1 wherein said process comprises the additional step (d) of recovering said parasporal crystals from said sludge(s).

3. A process for preparing a Bacillus thuringiensis bio-insecticide, said process comprising the steps of:

(a) hydrolyzing wastewater treatment plant sludge(s), said sludge(s) being selected from the group consisting of primary and secondary sludges; (b) inoculating said quantity of hydrolyzed wastewater treatment plant sludge(s) with Bacillus thuringiensis bacteria;

(c) aerobically cultivating said bacteria in said sludge(s) for a period of time sufficient for producing said bio-insecticide resulting from the sporulation of said bacteria through synthesis of insecticidal δ- endotoxin proteins in the form of parasporal crystals;

(d) recovering said sludge(s) containing said resulting bio-insecticide.

4. The process of claim 3 wherein said hydrolysis step (a) comprises each of the following two consecutive steps of: - lowering the pH of said sludge(s) to about 2 by adding an acid;

- raising the pH of said sludge(s) to about 7 by adding a neutralizing base.

5. The process of claim 4 wherein said acid is sulfuric acid and said neutralizing base is sodium hydroxide or lime.

6. The process of claim 1 wherein said Bacillus Thuringiensis bacteria consists of Bacillus thuringiensis Kurstaki.

7. The process of claim 2 wherein said Bacillus Thuringiensis bacteria consists of Bacillus thuringiensis Kurstaki.

8. The process of claim 3 wherein said Bacillus Thuringiensis bacteria consists of Bacillus thuringiensis Kurstaki.

9. The process of claim 1 wherein step (b) is conducted at about 30°C for about 10 days.

10. The process of claim 3 wherein step (c) is conducted at about 30°C for about 10 days.

ιι. The process of claim 9 wherein step (b) is conducted under agitation.

12. The process of claim 10 wherein step (b) is conducted under agitation.

13. The process of claim 1 comprising, prior to step (a) of claim 1, the additional initial step of sterilizing said wastewater treatment plant sludge(s) .

14. The process of claim 3 comprising, prior to step (a) of claim 1, the additional initial step of sterilizing said wastewater treatment plant sludge(s) .

15. The process of claim 4 comprising, prior to step (a) of claim 1, the step of heat sterilizing said sludge(s) after adding said acid and prior to adding said neutralizing base.

16. The process of claim 1 wherein said sludge(s) consist of secondary sludge(s) .