Note: Descriptions are shown in the official language in which they were submitted.
2 1 93378
~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.
21 93378
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
2 1 93378
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.
2 1 93378
~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
_ _ _ _ _ _ _ _ , ..... . . . .. _ . .
21 93378
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
12
TABLE 1
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 ~
14
TABLE2
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
W095/35365 PCT/CA95/00387
16
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
21 93378
~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.
TABLE3
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 ~
18
The assay procedure for each kind of BT grown
sludge is described in Table 4, below.
TABLE4
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 ~
TABLE 5
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.
- 5 TABLE6
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 ~
24
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 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.
TABLE8
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 ~
26
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.
TABLE9A
MORTALITY LARVAE PERCENTAGES
UNHYDBOLYZED BT GROWN SLUDGES
DAY IlL RSM 2.2 23 2.4 25 2 6 2.7
1 5 5 5 0 5 0 0
3 2 5 5 5 0 5 0 0
3 10 lD 5 0 10 0 0
0 0
6 2 20 15 15 5 15 0 0
3 25 20 20 5 20 5 10
9 2 30 20 20 5 20 5 15
3 40 25 25 20 25 15 20
2 5 12 2 40 30 30 25 25 15 15
3 50 35 35 30 30 20 20
Note: RSM ~UII~ Jlld:~ to the reference standard medium prepared with
~oybean flour.
21 93378
~ro 95135365 PCT/CA95/00387
27
TABLE 9B
MORTALITY LARVAE PERCENTAGES
HYDROLYZED BT GROWN SLUDGES
DAY ~L RSM 2.8 2.9 2.10 2.11 2.12 2.13 2.14
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
6 2 20 10 15 20 10 15 15 10
3 25 15 15 25 15 20 20 10
9 2 30 25 35 30 35 25 25 35
3 40 35 60 35 35 35 25 60
12 2 40 35 50 35 40 35 35 45
3 50 40 70 45 45 45 45 65
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 ~
28
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
29
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.
., _ . .. . .. _ . . . .. _ .. _ . . . . _ _ .. .. . . . _ _ _ _ _
21 93378
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.
