CA2193378A1 - Process for cultivating bacillus thuringiensis biopesticides in wastewater treatment sludges - Google Patents

Abstract

Disclosed herein is a process for preparing a Bacillus thuringiensis bioinsecticide, 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 .delta.-endotoxin proteins in the form of parasporal crystals; (c) recovering the sludge(s) containing the resulting bio-insecticide.

Description

~woss/3s36s PCT/CA95/00387 TI~T~ OF THE ~ h V ~n ~ lUN

PROCESS FOR CULTIVATING ~A~TTTT~S T~ ~TNGI~N~IS
BIOPESTICIDES IN WASTEWATER ~T~A~M~NT SLUDGES
R~r~nND OF ~u~ Ih v ~h ~ l~h . FTT~'T,n QF THE INVENTION
The present invention relates to a novel process for the production of Baçillus thurinqiensis (all serotypes and strains) biopesticides. More specifically, the novel process uses sludges generated by wastewater LL~ai ~ plants as a growth substrate.

~. DISCUSSION OF T~ PRIOR ART

CHENICAL INSECTICIDES
rh~;cAl insecticides have traditionally been used to control various insects which adversely affect agriculture and forestry or that constitute disease vectors. Although rh~m;rAl insecticides have generally been effirArir~ their production costs are high and they present envi,, t~l c~nr~n~. 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, orgAn~rh~crorus ~ ds and carbamates act by disrupting or inhibiting the nervous sy6tem functions of insect6. This may also represent a risk to all living organisms. Furth ~, some insects have become resistant to rh~;cAl insecticides. rh~;r~l pesticides can also AC: lAte in the environment and become a soil or water contaminant.

wo95l3s36s 2 1 9 3 3 7 8 PCT/CA9S/00387 BIO-INSECTICIDES
The use of ~n; ~athogenic (microorganisms path~g~n;~ 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 ~- AniFm of pathogenicity.

RArTT.T.TT.S THUR I N(; I ~:N.~ I 6 (hereinafter identified in shortened form as RT): RArTT~RTAT~ INSECTICIDE
BT represents a major class of microbes used for insect biocontrol. Most ~ strains produce several different insecticidal ~-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 b~n~fici~l insects. In addition, food products treated with this insecticide are safe for human or animal cu.-~u~tion. The BT insecticide is also biodegradable and will not a~ lAte in the environment or cause pollution problems. Accordingly, increasing attention is directed to ~ as a viable alternative to chemical pesticides.
Between the 35 or so ~ serotypes that have so far been identified, approximately 3 are currently used as microbial insecticides. These will now be briefly ~iC~llCC~ in se~uence.
RT seL~Ly~e 3a3b (R'lrstAki 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).
~ serotype 1~ fIsraelensis variety) is known as a specific pathogen to the larvae of certain D~p~era ~WO9~135365 ~1 ~337B PCT/CA95100387 (mosquitoes and black flies). Serotype 14 is also used to fight the vector of some tropical (Onchocercosis, Filariosis. etc.) or for the sanitation of public areas.
BT serotype Tenebrion;~ is a pathogen to the larvae of certain kind of coleopters, in particular to the Colorado potato beetle.

PRIOR ~RT pR~r~ s EOR PRODUCING ~
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, l~pI~du~Lion and spore formation: water, a carbon source for biosynthesis and energy, a nitrogen source, mineral elements and other optimal growing factors. Nost 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 ~ is conducted by batch liquid f~ L~tion process or yed fermentation in which the cultures grow dispersed by air in liquid media at controlled pH and t~ ~ ~LUL~. 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 30 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.

WOss/3~36s PCTIC~95100387 KNOWN GROWTH NEDIA:
The type of media used for the growth, sporulation and ~-endotoxin production of ~ can influence pro~nc~i~n 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 E~ growth media ingredients.
For example the following ingredients have been suggested: cheese whey, corn steep liguor, 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 Pnh~nr;ng additives such as nitrogen sources, protein sources usually in the form of leguminous seeds, such as peanuts, chick pea5, lima beans, horse beans, kidney beans and soya beans, mineral salts such as CaCO3, NaCl, K2HP4, MgSO4, CaCl2, FeSO4 and CuS04 and small amounts of some amino acids. Although these pr ~osed growth media use by-products of agro-industrial operations, their availability and acquisition costs may often be prohibitive since a number of other Pc~n ic~lly attractive products can be made from them, for example:
proteins, organic solids, ethanol. Fur~hP ~, the use of synthetic media and the use of additives add to the cost and complexity of the media.
Chilcott and Pillai (see Nircen Journal, 1, pages 327-332, 1985) have investigated the use of waste products of the coconut oil industrial processes as a BT

21 9337~
~WO 9513536S PCTrCA95rO0387 , .
growth medium ingredient. The ingredient is usually coconut ~n~cpPrm extract which is prepared by boiling finely ground ~n~sp~rm in distilled water for 2 minutes.
The ~n~cr~rm is then extracted by filtration through ~ 5 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 ~ lo~o~ m glutamate production. For example, a medium could be ~ of 4 to 7%/vol of hydrolyzed liquor (HDL) by-product from a ~ium glutamate factory, supplemented with 0.05% K2HPO4.
However, the availability of this by-product (hydrolyzed liquor by-product from -- ~inm glutamate production) is not reliable for the industrial production of ~3T.
Obeta and Okafor have for their part pluposed the use of cow blood as a ~ growth media ingredient (see Applied and Env. Nicrob, 47, pages 863-867, 1984). For example, they have proposed a media comprising lOg/l of cow blood, 0.02g/l MnCl.4H20; 0.05g/l MgSO4.7H20 and l.Og/l CaC03 combining it with different types of legume seeds in an agueous base.
Mummigatti et al. have plu~06ed the use of ~h~ d greengram powder, defatted soybean powder soluble starch and cane sugar molasses as ~ 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 ~ bio-insecticides. Among the woss/3s36s PCT/cAssloo387 drawbacks, it i5 noted that in many cases the media must be submitted to complicated and expensive ~LL~a~ Ls before they are adequate for use. These P~L~a; LS
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 growth and sporulation (substances with certzin kind of amino acids, high concentrations of caLbohydL~tes, etc). Moreover, many of the proposed ingredients do not contain all the n~c~ ry elements for ~~ growth, sporulation and ~-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 thLuuyh~uL 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 PYpl~in~d in detail hereinbelow, the growth media of the present invention are b~cic~lly devoid of nutrients and _ osed 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 inPYp~n~ive when compared to prior art growth media.

There is therefore a need for an alternative ~ growth medium which will overcome the above-mentioned drawbacks of the prior art.

~YO 95135365 2 1 9 3 3 7 8 PCT/CA95/003~

-STTMM~v OF THE INVENTION
In response to the above-mentioned drawbacks, it is an object of the present invention to present an ~ 5 economically efficient and novel process for sustaining growth, sporulation and insecticidal toxin production of all serotype6 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 nP~PsS~ry nutrients for adequate ~T growth without the requirement of additives;
- has plentiful local availability so as to m;n;m;7e purchase or tran~olLation costs.

Surprisingly, it has been found that wastewater LLea, ~ sludges constitute a well suited medium for the efficient growth, sporulation and ~-endotoxin synthe6is for the production of ~ bio-insecticides. Accordingly, the invention provides a process for preparing a Bacillus thllrinaien5i5 bio-insecticide, the process comprising the steps of:
(a) inoculating a given quantity of wastewater treatment plant sludge(s) with Bacillus thurinqiensis bacteria;
(b) aerobically cultivating the bacteria in the sludge(s) for a period of time sufficient for producing the bio-;nCPct;~;~o resulting from the sporulation of the bacteria and synthesis of insecticidal ~-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 ~ nt of the invention, the process further comprises the hydrolyzing of the wastewater W095/3536~ 2 1 9 3 3 7 8 PC~/CA9S/00387 ~

treatment sludges prior to inoculation with the ~acillnc thurinqiensis bacteria.
In a preferred embodiment of the invention, the inventive process is specifically directed to the production of ~ of the Rllr5t~k; 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 ; ~;r~' 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.
~ydrolysis as a pretreatment step generally increases spore production and ~-endotoxin is not time conCllm;ng 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.

R~TrR L~:,o~ ON OF T~lE FIGIJRE:~3 FIGs. 1 to 7 show graphical ~epLes~.L~ions 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 r~yLese~lL~tions of the viable BT spores and cell count in m; 11; onC over a time scale in days for the previously hydrolyzed wastewater sludges from various sources in Canada.

~WO 9513S365 PCTICA95/00387 DE~TTT~ JE~_n~
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, phncphnrU5, mineral salts, a pH near neutrality and other elements that render them a well suited medium for the production of ~. Wastewater treatment sludges are of course cheaply and plentifully available. Noreover, 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 , liPC well with the sludge use as fertilizer on agricultural land.
To 3 LL~te the use of wastewater sludges as a growth medium, various experiments were conducted using sludges from different sources. The experiments are L~s~--Lative in that they involve the growth, sporulation and ~-endotoxin production of BT of the Kllr5t~ki variety. It will be understood by those skilled in the art that the experiments are for illustrative purposes and that all other ~ varieties could be similarly cultivated.

~-r~ot~ri~;on of primary nnd 5~ ' ry wastewater sludges, t~rm;nology ~nd d~finitions:
It should be observed tlat Pr;r~rv sludqes are produced during the primary treatment of wastewater (the process consists of removal of sucpPndPd materials by sP~ir Lation of by some other te~hni~lPc)~ Sec~n~ry sludaes are produced during the cP~n~ry tL~a L of wastewater (activated sludge process or some other microbiological process configuration: involves conversion of wastewater by microorganisms). Primary _ _ _ _ _ _ _ _ , ..... . . . .. _ . .

W095/35365 PCTICA95l0~387 sludges contain s~cp~n~d solids present in the raw wastewater. Secondary sludges contain rhP~;r~l or biological solids produced during the LL~ nt process.

T M ~ Ar.
The experiments were conducted to illustrate and d L,~te the feasibility of using wastewater treatment sludges in the process of the present invention.
1. ~ STRAIN AND INOCULUM.
Culture tubes containing 5ml of nutrient broth prepared with 30g/1 of tryptic soy broth and 3g/1 of yeast extract were sterilized at 121-C during 20 min.
These tubes were inoculated with a loopful of BT Kurst~k HD-l grown on tryptic soy agar.
The culture was incubated during 18 hours at 30-C
in a shaking water bath. The dilution te~hni~P 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 SLUD~
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 ~-endotoxin production of ~. 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)~5 2.4) Secondary sludge from Ste-Claire, Ste-Claire (Sec) ~ 0 9513S365 2 1 9 3 3 7 8 PCT/CA95~003~7 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) ~5 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.

Wo 9s/3s36s ~ l ~ 3 3 ~ PCr/~A95/00387 CONCENTRATION OF SOLIDS

5SAMPLE mg/L
TS VS SS VSS
Valcartier (PS) 20430 16340 18610 15876 Black Lake (Sec) 5100 ~ 2500 2490 1820 1 o Beauceville (Sec) 43220 21620 37850 19060 Ste. Claire (Sec) 24230 14 160 22520 13760 Black Lake (ADS) 25240 12770 22830 12060 Sludge 1 (PP Sec) 20530 16180 15790 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 sludc~e les 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 LL~j L of organic matter can be used to obtain these _ _ _ _ _ _ . .. . .... . . . .

~W095/35365 2 1 ~ 3 3 ~ ~ PcT/cAgsroo387 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 ~eLL~ai 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 lN H2SO4. After acid addition, the sludge samples were sterilized at 121-C
during 20 min., cooled, pH adjusted to 7 with lN 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 s~ron~ry 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--NDOTOXIN PRODUCTION: ~h~lhl~ATION

3.1 Reference standard ~;um (RSM~: soybean flol~r 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.

WosS/35365 2 1 9 3 3 7 8 PCT/CA95/00387 ~

COMPOSITION OF STANDARD REF~RFNCE S~ANDARD MFDIUM (RSM) (G/L) SOYBEA~
DExTRo!cF
MAIZE STAR(~T 5 K2HPO4 1.0 KH2PO4 1.0 MgSO47H2O 03 FeSO47H2O 0.02 ~nSO47~2Q 0.02 CaC03 1.0 3 . 2 ~ x~r:~ I M~ T. PROCEDURE
~Z~l Reference Standard M~;ll~
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 ~topped, 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 ~ d with 2ml of sterilised distilled water.

~WO 95135365 2 1 9 3 3 7 8 PCTICA95/00387 The viable spore and cell count o~ this concentrate and its spore-crystal complex conce~,LL~tion were ~otorm;nPd by dilution technique.

3.2.2 SLUDGE M~nIUM
One milliliter of the sllcponcion 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 ~otorm;nod daily by dilution torhni~o 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-sllcpon~o~ with two ml of distilled sterilised water. This ~ul.cu-,LL~te was used for the bioassays. The viable spore and cell count and the ~O~ y~Lal complex cu.,c~l.L~ion of each concentrate were detomm;nod by the spore dilution terhni~lo.

4. ES~CTMATION OF SPORES AND ~ - hl~UU~OXlN PROD~CTION
4.1 M;croscoPic analvsis To follow the progress of the sporulation process, each sludge sample under incubation was observed daily under a mi~,uscuue. Preparations for microscopic observations were made by placing a drop of the sludge under incubation on a mi~,us~u~e slide with a sterilized loop; the observations were made with an immersion oil objective and phase contrast dark field method (x 1200).

2~ 93378 4.1.2 Coloration There are basic coloration methods for microscopic identification of spores, cry6tals and cells of BT. One method consists in the use of malachite green and the other is by using Buffalo black. Both terhn;quD~ were used to follow sporulation of BT in the nutrient broth and in the soybean solution.
4.2 EVATJTA~IO~ OF BT GROWTH
~ 1 Viable sT~ore and cell count The dilution t~rhni~l~ used to ~t~rminP 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.
Sllhse~l~ntly, 0.lml 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 det~rmin~ with a colony counter (see FIGs 1 to 14 for viable spore and cell counts).
~2~2 Viable s~ore Count of the s~ore-crYstal com~lex 10ml of distilled water and 0.02ml of 1~ Tween 80TM
solutions were placed in a test tube followed by sterilization at 121-C for 15 minutes. Test tubes were then cooled to room t~ , ~LuLe and 1 ml of RT 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 ~et~rmino~
following the dilution t~hni~l~ described above.
Duplicate samples were also used for viable spore count.

5. Potencv (Bioassav) _ To evaluate the potency of the BT grown sludge, bioassays were conducted against 3rd instar larvae of the ~W 095/35365 17 PCT/CA95/00337 spruce budworm (Choristoneura fumiferan~, an insect pest that causes great damages to the coniferous forests of North America. Standard laboratory larvae of this insect were raised at room t~ , ~L~L~ on artificial diet contained in plastic cups and used at the 3rd stage.
Table 3, below, presents the characteristics of the diet.

CEUiRACTERlS~CS OFARllFICLUL DIET
Distilled water ............................... 176 mL
Casein (vitamin free) ......................... 28 g Potassium hydroxide 4M ......................... 4 mL
~phacel ......................................... 4 g Salt mixture Wesson ............................. 8 g Wheat embryo .................................... 24 g Cho~e chloride ................................. 0.8 g Vitamin solution ................................ 8 mL
Ascorbic acid .................................. 3.2 g F~ '''JJ~ 40% .................................. 0.4 mL
Sucrose ......................................... 28 g Raw linseed oil ................................ O~i %
Agar ............................................ 20 g Distilled water ................................ 500 mL

Vitamin solution Distilled water ................................ 100 mL
Niacine ........................................ 100 mg Calcium I ' .................................... 100 mg 3 5 Riboflavine .................................... 50 mg Thiamine hrJ.u.,lllulidc ....................... 25 mg Pyridoxine hJilu~,hluliJ~....................... 25 mg Folic acid ..................................... 25 mg Biotine ......................................... 2 mg Vitamin B 12 ................................... 0.2 mg W095/35365 2 ~ 9 3 3 7 8 PCT/CA95/00387 ~

The assay procedure for each kind of BT grown sludge is described in Table 4, below.

BIOASSAYSPROTOCQL

No. RSM Sludge Control Sludge Foray of with(dist.H2O) control lalvae BT
lllL
l~L
2~L
3~L
1~L
2~L
3~L
I~L
2~L
3~1 I~L
Number of tubes60 60 50 60 20 Fifty larvae were placed in fifty bioassay tubes (one larvae each tube) containing the artifir~ diet mentioned before added with 1 ~1 of distilled sterilised water; these larvae served as control.
Control bioassays using 1, 2 and 3 ~1 of each fresh, sterilised and not inoculated sludge were also conducted using same larvae (20x3=60 tubes).
1, 2 and 3 ~1 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 ~ro 95/35365 2 1 q 3 3 7 8 PCT/CA95100387 of 1,2 and 3 l~l into 20 tubes (20x3=60 tubes).
Commercial biopesticide Foray 43 B~ was also included in the bioassays using a dosage of 1 ~1 added to 20 tubes having a larvae in each tube.
- 5 This sequence was repeated for each centrifuged BT
grown sludge. The 250 tubes ne~cs~rY 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/~l" each sample was compared with the known potency of commercial preparation Foray 48B~. Its spore concentration is 64x109 spores/ml and its potency is 12.7x109 IU/L
(12.7xlO~/~l). The high biological activity of this in~cti~ is because it i5 a purified and concentrated preparation obtained from soybean f~ tion.
The biological activity of the ~01~ ~Ly~al complex of this commercial biopesticide is expressed in activity units, for a specific insect test species, related to an international standard preparation. The 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 ~-endotoxin production 6.1 Microscopic analysis In the case of BT growa sludges coloration t~hni~ for spore identification were not used because the high content of organic and inorganic material contained in the sludges made it ~;ff;cl~lt to dist;n~l;~h spores from crystals and cells.
However, daily analysis of the inoculated sludges shows differences in sporulation process between them.
The results are:

woss/3s36s ~ 9 3 3 7 ~ ~CT/CA95l00387 ~

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 f~ L~tion.
Beauceville (Sec) - Sporulation process was normal beginning at the second day taking 11 days for total sporulation.
Ste Claire (Sec) - Sporulation process was normal 10 b~g;nn;ng 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 b~g;nn;ng at third day. Total sporulation was achieved after 11 days.
PP(NBR) 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 sesond 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.

337~8 ~ro 9s/3536s 21 PCT/CA95/00387 PP (HMBR) - Sporulation began second day: total sporulation was completed after nine days.
As shown, hydrolysis helped to ~;m;n;ch 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 ~-endotoxin production of BT var. Kurstaki. Results in Table 5, below, show that before concentration viable spore and cell count and viable spore count Ispore-crystal complex) in sludges at the end of sporulation period ranged from 0 spores and cells/ml for the sludge from Valcartier (PS) to 1.4x107 spores and cells/ml for Beauceville (Sec). The highest spore-crystal complex concentration co~Le~nd6 also to the same sludge (1.3x107 spores/ml) of Beauceville (Sec).

WO 95/353652 1 9 3 3 7 8 PCT/CA95/00387 ~

CONCENTRATION OF VIABLE SPORES IN
UNHYDROLYZED BT GROWN SLUDGES
(non-centrifuged) Sample Viable spores andSpore crystal complex cells count Spore and cell/mL Spore/mL
Valcartier (PS) O O
Black Lake (Sec) 1.3 x 106 1.0 x 106 Beauceville (Sec) 1.4 x 107 1.3x 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) 1 x 107 1.5 x 106 RSM 2.4 x 108 2x 108 Note: RSM ~u~ ,uuul~ to the reference standard medium prepared with 2 5 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 oss/3s365 2 1 9 3 3 7 8 PCT/CA95/00387 Tlae viable spore counts after ~u.lc_..L,~tion of unhydrolyzed ~ grown sludges by centrifugation is shown - in Table 6.

VIABLE SPORES COUNT IN
UNHYDROLYZED BT GROWN CENTRIFUGED SLUDGES

Sample Viable spores andSpore crystal complex cells count Spores and cells/mL Spores/mL
Valcartier (PS) O O
Black Lake (Sec) 1.85x 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 (SecADS) 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 1o8 Note: RSM ~,u~ ,u--.i~ 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 cu--c~l-LL~Led sludges ranged from 1.5x106 spores/ml for the sludge from Black Lake (Sec ADS) to 2.3x107 spores/ml Beauceville (Sec).
5pore crystal complex concentrations varied from 1.3xlO~
spores/ml for Black Lake (ADS) to 2x107 spores/ml for Beauceville (Sec).

W095/35365 2 1 q 3 3 7 8 PCT/CA95/00387 ~

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.

VIABLE SPORES COUNl' I~
HYDROLYZED ~T 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 1x107 Black Lake (HSec) 1.4 x 107 1.2 x 107 Beauceville (HSec) 2x 107 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 108 Note: RSM ~,-r~ to the reference standard medium prepared with soybean flour. Sludges 1 and 2 are the same as in Table 5 35 In this case, the values of viable spores and cells count vary from l.7xlO6 spores/ml for Black Lake (~ADS~ to ~ ~ogs/3s36s 2 ~ 9 3 3 7 8 PCT/CA9~00387 2xlO7 spores/ml for Beauceville (HSec). The spore-crystal complex concentration5 vary from l.6x106 ~ spores/ml for Black Lake (HADS) to 1.8x107 spores/ml for Beauceville (HSec).
After centrifugation of hydrolyzed Bt grown sludges, the viable spore and cell count varies from 2.8xlO6 spores/ml for Black Lake (~ADS) to 2.7x107 spores/ml for Beauceville (HSec). The spore-crystal complex concentrations vary from 2.5xlO6 spores/ml for Black Lalce (HADS) to 2.4x107 spores/ml for Beauceville (H
Sec). See Table 8, below.

VIABLE SPORE COUNT IN
HYDROLYZED BT GROWN CENTRIFUGED SLUDGES

Sample Viable spores ;mdSpore crystal complex cells count Spores and cells/mL Spores/mL
Valcartier (HPS) 1 6x107 1.2x107 Black Lake (HSec) 2.2x107 2x107 Beauceville (HSec) 2.7x107 2.4x107 Ste. Claire (HSec) 25x107 2x107 Black Lake (HADS) 2.8x106 2.5x106 Sludge I
(PP HSec) 2.2x107 4 2xl06 Sludge 2 (PP HMBR) 2x107 1.6x107 RSM 3.2x108 3x1o8 Note: RSM ~..~- ' to the reference standard medium prepared with soybean flour. Sludges 1 and 2 are the same as in Table 5 WO95/3S365 2 1 ~ 3 3 7 ~ PCT/CA9C/00387 ~

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.

MORTALITY LARVAE PERCENTAGES

UNHYDBOLYZED BT GROWN SLUDGES
DAY IlL RSM 2.2 23 2.4 25 2 6 2.7 3 10 lD 5 0 10 0 0 Note: RSM ~UII~ Jlld:~ to the reference standard medium prepared with ~oybean flour.

~ro 95135365 PCT/CA95/00387 MORTALITY LARVAE PERCENTAGES

HYDROLYZED BT GROWN SLUDGES
DAY ~L RSM 2.8 2.9 2.10 2.11 2.12 2.13 2.14 2 0 Note: RSM ~,u~ u~ 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 WO9~/35365 2 1 9 3 3 7 ~ PCT/CA95/00387 ~

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 day5. 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) ~Rperi~lly after 6 days.
Biological activities of unhydrolyzed sludges expressed in international units/~l (IU/~l) are presented in Table lO, below.

TABLElO
BIOLOGICAL POTENCY OF UNHYDROLYZED BT GRO~N SLUDGES

Sample Potency IU/IlL 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 I
(PP Sec) 0.878 Sludge 2 (PP MBR) 1.288 Note~ L = ;"l~ t;.. :1 units/llL
35 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.

~ro 95/35365 2 1 9 3 3 7 8 PCTICA95/00387 Between the sludges used without prior hydrolysis, sludge Black Lake (Sec) gave the highest biological potency (3260 IU/~l) followed by sludges Beauceville (Sec) (3000 IU/~l) and Black Lake (Sec ADS) (3020 IU/~l). The lowest ~ 5 potency was obtained with PP(Sec) (878 IU/~l).
Hydrolysis had, in general, a favorable impact on biological potency of the sludges. Black Lake (HSec) gave a potency of 4090 IU/~l, followed by Black Lake (HADS) (3600 IU/~l) and PP(HMBR) (3500 IU/~l). Table ll, below.

TABLEl1 BIOLOGICAL POTENCY OF HYDROLYZED BT GROWN SLUDGES

Sample Potency L x 103 Soybean (reference media) 3.811 2 0 Valcartier (HPS) 3 000 Black Lake (HSec) 4 090 Beauceville (HSec) 3200 Ste. Claire (HSec) 3 000 Black Lake (HADS) 3.600 Sludge 1 (PP E~Sec) 3.220 Sludge 2 (PP HMBR) 3500 Note~ L = inlPrr7~inn7l units/;lL
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.

., _ . .. . .. _ . . . .. _ .. _ . . . . _ _ .. .. . . . _ _ _ _ _ W095/35365 PCT1CAgS/0038 These results show that hydrolyzed and non-hydrolyzed sludges, with the exception of non-hydrolyzed primary sludges, are capable of sustaining growth, sporulation and ~-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 (19)

What is claimed:

1. Cancelled

2. Cancelled

3. A process for preparing a sludge containing Bacillus thuringiensis bio-insecticide having effective insecticidal potency, said process using as the growth medium the sedimented sludge generated by municipal or industrial wastewater purification treatment plants said process comprising the steps of:
(a) hydrolyzing said municipal or industrial wastewater treatment plant sedimented sludge;
(b) inoculating the resulting sludge with Bacillus thuringiensis bacteria;
(c) aerobically cultivating said bacteria in said sludge for a period of time sufficient for producing said bio-insecticide resulting from the sporulation of said bacteria through synthesis of insecticidal ~-endotoxin proteins in the form of parasporal crystals:
(d) recovering said sludge containing said resulting bio-insecticide.

4. The process of claim 3 wherein said hydrolysis step (a) comprises thesequential steps of:

- lowering the pH of said municipal or industrial wastewater treatment plant sedimented sludge to about 2 by adding an acid;
- raising the pH of the resulting sludge to about 7 by adding a neutralising 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 3 wherein said Bacillus Thuringiensis bacteria consists of Bacillus thuringiensis Kurstaki.

7. Cancelled.

8. Cancelled.

9. Cancelled.

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

11. 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. Cancelled.

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

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

16. The process of claim 3 wherein said sludge(s) consist of secondary sludge(s).

17. Cancelled.

18. Cancelled.

19. The process of claim 3 wherein said growth medium is municipal wastewater sedimented sludge.

The use of the sedimented sludge generated by municipal or industrial wastewater purification treatment plants as a growth medium for cultivating Bacillus thuringiensis bio-insecticide