Chemosphere 67 (2007) 674683

www.elsevier.com/locate/chemosphere

Bacillus thuringiensis fermentation of hydrolyzed sludge Rheology

and formulation studies
Satinder K. Brar a, M. Verma a, R.D. Tyagi a,*
, J.R. Valero a, R.Y. Surampalli b

a
INRS-ETE, Universite du Quebec, 490, de la Couronne, Quebec, Canada G1K 9A9
b
US EPA, P.O. Box-17-2141, Kansas City, Kansas, KS 66117, USA

Received 17 June 2006; received in revised form 31 October 2006; accepted 1 November 2006
Available online 20 December 2006

Abstract

Rheology of Bacillus thuringiensis fermentation of hydrolyzed sludge was investigated in bench scale fermenter. Stable liquid formu-
lations were developed and optimized for two-year based studies comprising various physical/chemical (viscosity, particle size, corrosion
and suspendibility) and biological (microbial contamination, viable spores and entomotoxicity) parameters at dierent pHs and temper-
atures. The hydrolyzed sludge depicted non-Newtonian and pseudoplastic behaviour during fermentation with 90% to 96% condence of
ts into Casson, Power and IPC paste models. Higher values of consistency and ow index during exponential growth and stationary
phase, respectively, aected downstream processing. The power law was also followed by stable formulations. Sorbitol, sodium mono-
phosphate and sodium metabisulte (2.2:1:1) as suspending agents produced suspendibility ranging from 69% to 94%. The stable for-
mulation (FH-4) comprising sorbitol, sodium monophosphate and sodium metabisulte deteriorated at pHs 6, 6.5 and temperatures,
40 and 50 C, with no signs of corrosion and microbial contamination. The viscosity of FH-4 formulations decreased with shear rate
which could improve handling and consequent spraying.
 2006 Elsevier Ltd. All rights reserved.

Keywords: Bacillus thuringiensis; Biopesticide; Hydrolyzed Sludge; Liquid formulation; Rheology; Shelf-life

1. Introduction Vidyarthi et al., 2002; Yezza et al., 2004). Additionally,

Bacillus thuringiensis (Bt) based biopesticides are
conventionally used options for pest control in agriculture,
forestry and public health sectors (Burges, 1998). The con-
ventional production of Bt in semi-synthetic medium has
been recently replaced with non-conventional wastes like
wastewater/wastewater sludge (Lisansky et al., 1993;
Abbreviations: ANOVA, Analysis of variance; Bt, Bacillus thuringiensis;
EPS, Extra cellular polymeric substances; F, Fisher-Snedecor distribution
(continuous probability distribution); SBU, Spruce budworm units; SD,
Standard deviation; SS, Suspended solids (g l1); TC, Total cell count
(103 CFU m3); TH-30, Hydrolyzed sludge at solids concentration of
30 g l1; TS, Total solids (g l1); TVS, Total volatile solids (g l1); Tx,
Entomotoxicity (SBU ll1); VS, Viable spore count (103 CFU m3);
VSS, Volatile suspended solids (g l1).
*
Corresponding author. Tel.: +1 418 654 2617; fax: +1 418 654 2600.
E-mail address: tyagi@ete.inrs.ca (R.D. Tyagi).
in order to achieve higher entomotoxicity (biopesticidal
potential), hydrolysis (pre-treatment) of wastewater sludge
has been investigated to increase nutrient availability (Bar-
nabe et al., 2005).
Rheology, during fermentation, aects oxygen transfer
and hence entomotoxicity, likewise downstream processing
(centrifugation) for recovery of products. The fermented
non-hydrolyzed wastewater sludge depicted pseudoplastic
and thixotropic behaviour as established earlier (Brar
et al., 2005a). The rheology of fermented microbial broth
is a net eect of formation of dierent metabolites, increase
in biomass and degradation of growth medium compo-
nents causing mass and heat transfer problems resulting
in decreased productivity (Hwang et al., 2004). Changing
the fermentation medium from non-hydrolyzed to hydro-
lyzed sludge would also shift the rheological behaviour.
Further, these problems are compounded during scale-up

0045-6535/$ - see front matter  2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.chemosphere.2006.11.007
 S.K. Brar et al. / Chemosphere 67 (2007) 674683 675

Nomenclature

g10 10 rpm viscosity (103 kg m1 s1) R Rotational speed (rpm)

K Consistency index (103 kg m1 sn) D Shear rate (s1)
Km Consistency multiplier ns Shear sensitivity factor
N Flow behaviour index s Shear stress (101 kg m1 s2)
g Plastic viscosity and/or viscosity s0 Yield stress (101 kg m1 s2)
(103 kg m1 s1)
A Ratio of spindle radius and inner cup radius

due to medium complexity and generally lower agitation concentrated and homogenized sludge (Waring blender)
intensities. Thus, the rheological proles during Bt fermen- was hydrolyzed by direct steam injection in a custom made
tation of hydrolyzed sludge need to be explored to better hydrolyzer at 140 1 C for 30 min (Barnabe et al., 2005).
understand the fermentation process. A dilution factor of 1.67 (empirically obtained) due to
Despite the availability of alternative economical raw steam condensation was incorporated requiring an initial
materials producing higher nal entomotoxicity, the formu- SS of 50 g l1 to achieve a nal SS of 30 g l1. Hydrolyzed
lation cost still eludes the broader application of biopesti- sludge was henceforth, referred to as TH-30. The sludge
cides. Moreover, rheological considerations are important characteristics (APHA,AWWA,WPCF, 1998) are given in
for the selection of dierent adjuvants required to obtain Table 1.
requisite properties (higher shelf-life, suspendibility, rain-
fastness, UV resistance, higher eld ecacy, compatibility 2.1.2. Bacterial strain and fermentation
with pre-existing application equipment, and cost-eective- Bt var. kurstaki HD-1 (ATCC 33679) was used with cul-
ness) of nal Bt formulations (Burges, 1998). Formulation ture conditions, maintenance and inoculum production
feasibility studies have been carried out using Bt fermented described elsewhere (Vidyarthi et al., 2002).
non-hydrolyzed wastewater sludge (Brar et al., 2004).
Meanwhile, hydrolyzed sludge oered various advantages
higher entomotoxicity (Barnabe et al., 2005; Brar et al.,
Table 1
2005b); low viscosity (Brar et al., 2005b); possible lower
Composition of hydrolyzed wastewater sludge
production cost by incorporating sterilization in hydrolysis
Parameter (s) Concentration SE(mg kg1 of TS,
step (Barnabe et al., 2005); lower centrifugation time to
unless otherwise stated)
recover targeted entomotoxicity in the pellet (Brar et al.,
TS (g l1) 53.5 0.1
2006a); higher half-life (UV protection) enhancing ecacy
TVS (g l1) 28.4 0.1
during eld application (Brar et al., 2006b) and possibly SS (g l1) 44 0.23
lower particle size causing synergy with conventional appli- VSS (g l1) 2.3 0.4
cation equipments. At this stage, it becomes imperative to pH 8.5 0.1
investigate the rheology eects on development of stable Total carbon 418977 785
Total nitrogen 39989 2211
hydrolyzed sludge formulations.
Total phosphorus 15702 1734
Hence, the present study will focus on following objec- NH3-N 549 73.4
tives: (a) rheology of hydrolyzed sludge fermentation and NO 
2 -N, NO3 -N 21.9 2.30
formulation; (b) shelf-life stability (pH, temperature) of PO3
4 -P 8004 3335
dierent formulations by analyzing various physical/chem- S 5978 332.3
Al 13978 2522
icalbiological test parameters.
Ca 23697 336
Cd 0.3 0.1 (3)
2. Materials and methods Cr 71 23.6 (210)
Cu 300 161.5 (100)
Fe 8061.9 758.6
2.1. Upstream processing K 23.3 4.2
Pb 4.1 1.9 (150)
2.1.1. Sludge procurement, amendment and characterization Zn 395.8 100.2 (500)
The secondary wastewater sludge was obtained from Na 15287 1021
Communaute Urbaine du Quebec wastewater treatment Ni 5.1 2.2 (62)
As n.d. (13)
plant at Ste-Foy. The sludge was concentrated from
N.B.: The mg kg1 conversion corresponds to weight of metals per unit
approximately 1.65% (w v1) suspended solids (SS) by
weight of TS (dry basis).
gravity settling followed by centrifugation at 7650 g for n.d. not detected
15 min at 20 1 C. The sludge supernatant stored at Values in parentheses represent the levels recommended by Ministry of
4 1C was used to dilute the sludge as required. The Environment, Quebec, Canada (MENV, 2004).
676 S.K. Brar et al. / Chemosphere 67 (2007) 674683

Fermentation was carried out in a bioreactor at pH series of TH-30 formulations, designated as FH-1 through
7 0.1 and 30 1 C (10 l working volume, Biogenie FH-5 (Table 2) and were blended as slurry in a Waring
Inc., Quebec, Canada) equipped with accessories and pro- Blender.
grammable logic control (interfaced with iFix 3.5 software, Further, the slurry was diluted with supernatant of cen-
Intellution, GE Fanuc Inc., USA) for dissolved oxygen, trifuged broth to achieve nal Tx of 19.5 109 SBU l1.
pH, anti-foam, impeller speed, aeration rate and tempera- The pH stability studies were carried out at 4.0, 4.5, 5.0,
ture. Fermenter dimensions, wastewater charging and ster- 6.0 and 6.5 0.1 by adjusting the pH with 2 N NaOH
ilization procedure are described elsewhere (Brar et al., and/or H2SO4 and stored at 20 1 C for two years. Sim-
2005b). ilarly, temperature storage studies were carried out at 4, 10,
20, 30, 40 and 50 1C at pre-adjusted pH 5.0 0.1. Sam-
2.2. Downstream processing ples were drawn at regular intervals (0, 3, 7, 10, 15, 30, 60,
90, 120, 150, 180, 365, 545 and 730 d) to determine physical
2.2.1. Post-fermentation and pre-formulation step (viscosity, particle size, suspendibility and corrosion) and
The pH of TH-30 fermented broth was lowered in-situ biological (microbial contamination, viable spores and
from 7 0.1 to 4.5 0.1 (Brar et al., 2006b). The fermented entomotoxicity) parameters.
broth (entomotoxicity denoted as Tx, 19 109 SBU l1,
spruce budworm units/l) was centrifuged aseptically in order 2.3. Physical/chemical and biological analysis
to compare with industrial standard equivalent of 19.5
109 IU l1. The concentrated hydrolyzed sludge broth at Rheological properties of fermented hydrolyzed sludge
70 g l1 solids yielded an approximate entomotoxicity of were determined by using a rotational viscometer Brooke-
26.4 109 SBU l1. eld DV II PRO + (Brookeld Engineering Laboratories,
Inc., Stoughton, MA, USA) equipped with Rheocalc32
2.2.2. Storage stability tests software (for rheological models). Three dierent spindles,
The concentrated broth of known Tx was further namely, SC-34, L2 and ultra-low centipoise adapter were
amended with basic adjuvants/additives (Table 2). The used with a sample cup volume of 18 ml/50 ml (spindle
basic adjuvants served following purposes: propionic and dependent). Time dependent prole was studied at low
sorbic acid anti-microbial agents; glycerol humectant shear rate (7.34 s1) and viscosity at each sampling point
and anti-evaporant; Tween-80 and Triton X-100 surfac- was measured at 36.71 s1. The shear rate behaviour was
tants and wetting agents, respectively. Furthermore, the determined from 0.1 to 200 s1. All measurements were
basic formulations were supplemented with dierent con- done at 25 1C with a measurement time lag of 1 min
centrations of specic adjuvants (suspending agents, sorbi- between consecutive shear rates. Viscosity was referred to
tol, sodium monophosphate and sodium metabisulte, apparent viscosity, unless stated otherwise.
Burges, 1998). The amendments resulted in ve dierent Particle size analysis was carried out by LASER dirac-
tion method as reported earlier (Brar et al., 2005b). A stan-
Table 2 dard deviation of 8% and 10% was observed for viscosity
Formulation recipe chart (concentrations in% w v1, unless stated and particle size measurements, respectively.
otherwise) Suspendibility was determined by comparing ratio of
No. Basic additives/adjuvantsa Concentration supernatant turbidity and weight of settled solids after
1 Propionic acid 0.5 30 min settling by using the procedure established earlier
2 Sorbic acid 0.4 (Brar et al., 2006b). The standard deviation for suspendibil-
3 Glycerol 2 ity measurement was 5%.
4 Tween-80 0.2 Corrosion was tested by immersing iron and aluminum
5 Triton X-100 0.1
strips (10 5 cm2) overnight in various formulations at dif-
6 TH Broth (% v v1) 67.3
7 Supernatant To make up ferent pHs. The pH was measured by simple glass electrode
8 Dispersing/suspending agents Variation (Delta 320 Mettler Toledo, NJ, USA). Further, microbial
contamination was tested as per the protocol reported by
Specic adjuvantsb Lisansky et al. (1993) as fermentation was prone to
Formulations Sorbitol Sodium Sodium Final total contamination.
monophos- metabisulte solids Total cell (TC) and viable spore (VS) count was ana-
phate concentration
lyzed according to earlier methods (Vidyarthi et al.,
FH-1 21 0 0 14.3 2002). Three replicates were performed and data analyzed
FH-2 18 3 0 13.6
with one way analysis of variance (ANOVA) to test signif-
FH-3 15 5 0 14.1
FH-4 11 5 5 14.7 icant dierence at P = 0.05. The standard deviation for cell
FH-5 9 7 5 15.3 and spore count was 7.0% and 8.0%, respectively.
a
Basic adjuvants were added to all formulations. The bioassay was performed to determine entomotoxic-
b
Specic adjuvants were added to respective formulations to study their ity by using diet incorporation method as described in
eect during shelf storage. details in earlier studies (Brar et al., 2005b, 2006b; Yezza
 S.K. Brar et al. / Chemosphere 67 (2007) 674683 677

et al., 2004). The SBU reported in this study was 2530%
higher than international units with standard deviation of
810%.
3. Results and discussion
3.1. Rheology of hydrolyzed sludge (TH-30)
Fig. 1a and b illustrate rheogram and shear rate behav-
iour, respectively of TH-30 at dierent fermentation times
with various rheological models presented in Table 3.
The rheograms (Fig. 1a and b) all through fermentation
showed non-linear relationship (strong non-Newtonian
behaviour) and viscosity vs. shear rate curves showed
pseudoplastic (viscosity decreased with shear rate) behav-
iour. Interestingly, the shear stress increased from 0 to
15 h with higher values from 9 to 15 h possibly due to
the resistance oered by actively growing Bt cells. How-
ever, the shear stress decreased during stationary phase
(2148 h) probably due to cell lysis; release of spores and
other virulence factors (namely, enzymes, phospholipases,
proteases, chitinases; vegetative insecticidal proteins and
cytolytic proteins) in the fermented broth. Spores, crystal
proteins and the virulence factors synergize to contribute
to Tx (Hansen and Salamitou, 2000). The microbial
fermentation broths show diverse rheology changes from
initial Newtonian to pseudoplastic behaviour during expo-
nential phase (Hwang et al., 2004). The pseudoplasticity
will have a signicant eect on mass and heat transfer in
the fermenter as shear rates will be higher near the impeller
and low elsewhere.
Table 3 also showed that TH-30 during fermentation
followed various rheological models with varying degree
of condence of ts. The TH-30 sludge strongly followed
Bingham plastic model during the exponential phase (0
21 h, Table 3 and Fig. 2) whereas a poor t was observed
during stationary phase (3048 h). Bingham model is a sim-
ple rheological model that relates shear stress and shear

7
Shear stress (10 kg m s )
-1 -2

5
-1

0
0h
3h
40 6h
9h
Viscosity (10 kg m s )
-1 -1

12h
15h
18h
21h
-3

24h
30h
20 36h
48h
FH-4Formulation

0
0 50 100 150 200
-1
Shear rate (s )
Fig. 1. Rheograms of hydrolyzed sludge during fermentation and formulation: (a) shear stress prole and; (b) viscosity prole.
678 S.K. Brar et al. / Chemosphere 67 (2007) 674683

Table 3
Dierent rheological model ts of Bt fermented hydrolyzed sludge and stable formulation (FH-4)
Time (h)) 0 3 6 9 12 15 18 21 24 30 36 48 FH-4
Bingham law
Plastic viscosity (g, 103 kg m1 s1) 2.44 2.46 2.38 2.82 7.4 5.06 3.84 3.49 3.91 4.82 3.8 3.6
Yield stress (s0, 101 kg m1 s2) 0.17 0.13 0.15 0.14 0.25 0.21 0.18 0.15 0.15 0.09 0.07 0.07
Condence of t (%) 87.7 90.4 89.7 95.8 82.8 85.8 88 86.5 85.6 67.3 74.2 68.5
Casson law
Plastic viscosity (g, 103 kg m1 s1) 0.85 1.07 0.93 1.27 3.32 2.37 1.97 2 2.25 3.64 2.92 2.84 1.9
Yield stress (s0, 101 kg m1 s2) 0.13 0.09 0.11 0.09 0.15 0.13 0.1 0.07 0.07 0.03 0.02 0.02 0
Condence of t (%) 82.5 87 85.3 89.8 91.4 93.9 96.1 95.7 95.2 88.9 92.6 90.6 71.0
NCA/CMA Casson
Pastic viscosity (g, 103 kg m1 s1) 0.85 1.07 0.93 1.27 3.32 2.37 1.97 2 2.25 3.64 2.92 2.84 1.89
Yield stress (s0, 101 kg m1 s2) 0.12 0.08 0.1 0.08 0.14 0.12 0.09 0.06 0.07 0.02 0.02 0.02 0
Condence of t (%) 82.5 87 85.3 89.8 91.4 93.9 96.1 95.7 95.2 88.9 92.6 90.6 71.0
Power law
Consistency index (K, 103 kg m1 sn) 150.6 100 123.2 114.4 177.7 140.1 105.2 76.6 79.6 34.6 26 21.3 18.5
Flow behaviour index (n) 0.22 0.29 0.25 0.29 0.32 0.33 0.36 0.41 0.41 0.61 0.62 0.66 0.84
Condence of t (%) 73.6 79.5 76.9 78.6 96.9 94.8 91.9 92.5 92.3 96.8 95.2 96.3 93.6
IPC paste
Shear sensitivity factor (ns) 21.3 17 19 19.1 32.5 26.3 21.2 17.6 18.4 13 10.1 9.12 5.6
10 rpm viscosity (g10) 0.78 0.71 0.75 0.71 0.68 0.67 0.64 0.59 0.59 0.39 0.38 0.34 0.24
Condence of t (%) 73.6 79.5 76.9 78.6 96.9 94.8 91.9 92.5 92.3 96.8 95.2 96.3 91.6
VS (10-3 CFU m-3)

110 1e+10 400 0.8 9

100 1e+9
Consistency Index (K, 10-3 kg m-1 s-n)

0.7 8
90 1e+8 300

Viscosity (10-3 kg m-1 s-1)

7
0.6
,
D.O. (%)

80 1e+7
6
Flow Index (n)

70 1e+6 200 0.5

5
60 1e+5
TC

0.4
4
50 1e+4 100
0.3 3
40 1e+3

30 1e+2 0 0.2 2
0 6 12 18 24 30 36 42 48
Time (h)
Fig. 2. Power law behaviour of hydrolyzed sludge and its correlation with Bt growth (15 l fermenter).

rate and quanties yield stress and high-shear viscosity.
The Bingham law is represented mathematically by Eq. (1):
s so gD
The eects of yield stress on ow are somewhat similar
to those of pseudoplasticity but are much more pro-
nounced. For example, the Bingham plastic behaviour of
TH-30 until 24 h of fermentation suggested that higher
shear stress will be required to overcome the heterogeneity
of fermentation medium as reected in higher agitation
rates required (Brar et al., 2005b). The lower yield stress
of fermented broth during stationary phase may be
contributed by two factors- larger proportion of cells con-
verted to spores and hence less resistance (shear thinning);
and extracellular polymeric substances (EPS) possibly
1
formed during the exponential phase (cellular growth) were
broken due to weaker interactions during stationary phase,
which remains to be ascertained. In fact, Bt was identied
as a principal EPS producing bacterial strain in marine
environment (Kwon et al., 2002).
Meanwhile, Casson and NCACMA Casson (modied
Casson) model was followed with good condence of ts
(8296%) throughout the fermentation. Casson model quan-
ties yield stress and high shear viscosity, a model which
 S.K. Brar et al. / Chemosphere 67 (2007) 674683
incorporates features of the power law and the Bingham
plastic equations. The mathematical representation of
Casson law is given as:
p p p well represented by Eq. (4):
s so gD 2
Likewise, NCACMA Casson model has been derived
from the standard set forth by the National Confectioners
Association (NCA) and the Chocolate Manufacturers
Association (CMA). Although based on the original Cas-
son equation, this implementation has been tailored by
the NCA and CMA specically to applications involving
chocolate type behaviour. The NCACMA model is math-
ematically represented by Eq. (3):
p p p g KRn
1 a s 2 so 1 a gD 3
Considering Casson law relation, plastic viscosity
increased from 012 h and later decreased with again a sud-
den peak at 30 h due to unknown reasons. The decrease in
plastic viscosity and yield stress towards the end of fermen-
tation suggested that the fermented broth would require
lower shear stress to maintain ow. The yield stress aected
ow in a similar manner as pseudoplasticity with more pro-
nounced eects. This will aid in pump design for centrifu-
gation facilities where cell debris aggregates are identied
as being reversibly de-aggregated by low shear stresses
which might exist in the separation zone of the disc space
of a disc stack centrifuge (Maybury et al., 2000). Further-
more, when the fermented centrifuged broth is carried over
for formulations, the owability will be maintained and
dierent adjuvants can be amended homogeneously (as dis-
cussed later). Additionally, during eld application, the
owability could aid in the spray application to achieve
desired droplet spectrum and ecient spray which is a
function of viscosity.
3.2. Fermentation and power law behaviour
The Bt fermentation and power law behaviour proles
of hydrolyzed sludge are illustrated in Fig. 2. Power and
IPC paste (extension of power model) models were
followed with moderate ts in the rst 6 h of growth
(Table 3). However, power law was followed with good ts
(7996%) from 9 to 48 h of fermentation (Table 3). The
power law was used to dene Bt fermentation as it has been
very widely utilized to determine the rheology of several
microbial fermentation broths (Hwang et al., 2004). Power
law model is a useful rheological model that describes the
relationship between viscosity or shear stress and shear rate
Table 4
List of ANOVA factors for Bt fermentation of hydrolyzed sludge
Figure Mean (103 CFU m3)
9
2 1.03 10 (TC at 48 h)
2 7.68 108 (VS at 48 h)
df : degree of freedom, between groups.
df*: degree of freedom, within group.
679
over the range of shear rates where shear thinning occurs in
a non-Newtonian uid. It quanties overall viscosity range
and degree of deviation from Newtonian behaviour and is
s KDn 4
Akin to power law, IPC paste model is intended to cal-
culate the shear sensitivity factor and the 10 RPM viscosity
value of pastes. The law is primarily used in the solder
paste industry, thus the name IPC (Institute for Intercon-
necting and Packaging Electronic Circuits) and is mathe-
matically denoted as:
5
It was seen in Fig. 2 that the TC and VS concen-
tration increased during 024 h, from 2.4 104 to 1.1
106 CFU m3 and 0.9 to 5.8 105 CFU m3, respectively
(P 6 0.05). Likewise, the consistency index (K, measure
of thickness of the fermented broth) and the ow index
(n) also varied. The corresponding ANOVA factors for
TC and VS are also given in Table 4. During 03 h of fer-
mentation, viscosity decreased and later attained a hump at
12 h as seen in Fig. 2 in agreement with earlier studies (Brar
et al., 2005b). Likewise, K decreased from 0 to 3 h which
may be due to mechanical force (agitation) that led to oc
disruption and hence thinning of medium. Floc, in this
case, refers to the aggregates of biomass present in hydro-
lyzed sludge. However, K increased from 0 to 12 h with a
peak at 12 h (Fig. 2) which may be due to: (a) increase in
medium consistency with Bt cell growth; (b) addition of
anti-foam during active growth phase (012 h, note dis-
solved oxygen values, Fig. 2) causing a peak and (c)
increase in the phase volume (volume of suspended mate-
rial/volume of continuous phase) which has been reported
to increase K in fermented broths (Hwang et al., 2004).
Interestingly, K decreased towards the end of fermen-
tation and n increased continuously attaining constancy
between 30 and 36 h of fermentation. In fact, n followed
exponential prole (n = 0.2373 e0.0245t; R2 = 0.92, Fig. 2) so
that increase in fermentation period resulted in a broth with
higher owability. Likewise, a correlation between K and
n followed exponential prole (K = 468.5 e4.4584n;
R2 = 0.91) implying that increase in owability (broth thin-
ness) will decrease the consistency (broth thickening). The
correlation suggested ease of downstream processing
(centrifugation) which was also established in our previous
studies where the centrifugation time to achieve certain
degree of separation (77% entomotoxicity recovery) was
F df ; df* P
1.643 2;6 >0.05
0.879 2;6 >0.05
680 S.K. Brar et al. / Chemosphere 67 (2007) 674683

lower for hydrolyzed Bt fermented sludge when compared Table 5

to other fermented broths (Brar et al., 2006a). Eect of dierent specic additives/adjuvants on percent spore survival
and suspendibility of dierent formulations
As n tends to 1, the shear-thinning properties nor-
mally increase so as to reach Newtonian behaviour. In con- Percent spore survival
trast, TH-30 during fermentation exhibited a marked pH FH-1 FH-2 FH-3 FH-4 FH-5
shear-thinning response (n 6 0.6, Fig. 2). Furthermore, a 4 79a 79a 84b 87bd 77a
gradual increase in n from 0.2 to 0.6 (Fig. 2) also con- 4.5 71ac 77a 80b 88bd 80ab
rmed shear rate as a function of Bt growth (as discussed 5 84b 84b 90d 84b 86b
6 75a 81b 87bd 81b 88b
earlier, viscosity decreased with shear rate). Meanwhile, 6.5 66c 77a 82b 77a 86b
in concentrated microbial suspensions, the increase in vis-
Temperature (C)
cosity with cell concentration is less pronounced due to
4 90d 81a 82b 82b 80ab
increase in shear rate (Hwang et al., 2004). The viscosity 10 89b 94d 84b 85b 76a
of FH-4 stable formulation (discussed later) was found to 30 88b 92d 88bd 78a 77a
decrease with shear rate (Fig. 1b) showing relatively less 50 77a 88b 74ac 83b 61c
non-Newtonian behaviour (Fig. 1a). This is necessary for Percent suspendibility
practical applications because marked dierences in viscos-
pH FH-1 FH-2 FH-3 FH-4 FH-5
ity may be expected at dierent operative shear rates during
4 65 5.6a 51 5.6d 54 6.2b 69 6.3ac 68 7.1ac
pouring, mixing, pumping, and spraying which will conse-
4.5 54 7.4b 63 7.3a 64 5.3a 76 5.2c 69 6.4ac
quently aect the droplet size spectrum and hence perfor- 5 55 3.5b 65 4.7a 66 5.1a 82 4.3e 81 7.3e
mance as biopesticides. In fact, viscosity has also been 6 72 5.1ac 71 7.6ac 62 2.8a 85 3.7e 82 4.3e
found to aect synergy with spray equipment and eld 6.5 71 5.6ac 73 4.2ac 63 4.2a 90 6.4f 84 7.8e
application of Bt biopesticides (Burges, 1998). Temperature (C)
4 48 4.4d 49 6.4bd 54 3.4b 62 2.3a 45 6.3d
3.3. Optimization of TH-30 formulations 10 49 5.2bd 54 4.3b 56 6.5b 68 3.4c 59 5.9b
30 44 6.7d 57 4.5b 57 6.3b 86 4.6e 62 7.7a
50 64 3.9a 68 5.6ac 59 7.3b 80 5.3e 84 8.1e
Five dierent formulations of Bt fermented TH-30 were
developed (FH-1 to FH-5) and analyzed for various physical Values within a column followed by dierent letters are signicantly dif-
ferent at P 6 0.05.
(viscosity, particle size, suspendibility, corrosion) and bio- Mean SD, means (n = 3). Refer to Table 2 for formulation composition.
logical (Tx and VS) stability parameters at dierent pHs
and temperatures over a period of two years. Further, the
optimal formulation was selected by adopting a quantitative ow behaviour index was higher than 48 h fermented broth
judgement analysis approach. The quantitative judgement (Table 3). Hence, the pseudoplasticity of FH-4 formulation
referred to selection of formulations (optimal and stable) was lower and hence the decrease in viscosity with shear rate
which showed no signicant dierence (P > 0.05) in the was tending more towards Newtonian behaviour. This is
physical and biological parameters over two years of shelf desired for the owability of a formulation so as to achieve
storage. If any signicant change (P 6 0.05) occurred, the better droplet spectrum and thus, eld application. The FH-
formulations were rejected. However, the most important 4 formulation (Fig. 3ac) performed dierently during pH
criterion for selection of the optimal formulation was the and temperature storage studies for 730 d. Viscosity and
entomotoxicity. The viscosity, particle size and Tx proles particle size did not vary signicantly (P > 0.05) over the
of FH-1, FH-2, FH-3 and FH-5 at dierent pHs and temper- selected pH range except for a decrease in particle size
atures showed signicant decline after 30 d period (P 6 0.05, (10 lm units) and viscosity (102 kg m1 s1) after 90 d
results unreported). Moreover, percent suspendibility and storage at pHs 6 and 6.5 (Fig. 3a). Considering the isoelec-
spore survival (Table 5) decreased signicantly (P 6 0.05) tric point of bacteria from pH 24 (Tenney and Stumm,
over the storage period (730 d). Thus, FH-4 comprising 1965), it was clear that sludge particles would carry increas-
sorbitol, sodium monophosphate and sodium metabisulte ingly negative charges at pH 6 and 6.5. Thus, increased sim-
(2.2:1:1) was found to be the optimal with nominal varia- ilar charges in the oc structure caused repulsion and hence
tions (discussed later). expansion of oc matrix. Further, the oc may be acted
upon by enzymes, especially, proteases produced during
3.4. Stable liquid formulation (FH-4) Bt fermentation which were active at these pHs (Tyagi
et al., 2001). This would have caused degradation of oc
3.4.1. Rheology, physical and biological parameters matrix and hence inuenced the particle size which also
The rheology of stable FH-4 formulation was investi- showed a similar declining prole after 90 d storage
gated by studying dierent mathematical models as seen (Fig. 3a). At lower pH, these issues may not arise and hence
in Table 3. The results suggested that with further increase most of the formulations are developed at these pHs main-
in solids concentration of the formulation, the viscosity will tained by buers (Burges, 1998).
increase as per exponents of the power law and IPC paste The eect of temperature on viscosity and particle size
equations. Further, the consistency index was lower and proles (viscosity; 57 103 to 22 103 kg m1 s1 and
 S.K. Brar et al. / Chemosphere 67 (2007) 674683 681

70 40
35

Viscosity (10 kg m s )
-1
60

-1
30

Particle size (m)

a a 25
50 aa aa a a aa aa a aa a a aa a aa a a aa aa aa a a a a a aa aa a aa
a aa a
-3
a b
b bb b b bb 20
b
40 15
cc c c
c c 10
30 d c c
d d d d d 5
20 0
0 3 7 10 15 30 60 90 120 150 180 365 545 730
Time (d)
4 4.5 5 6 6.5
4(P) 4.5(P) 5(P) 6(P) 6.5(P)

60 35
Viscosity (10 kg m s )

30
-1

Particle size (m)

50 a
-1

aa a a a
a a1a aa aa a 25
aa b a aa a aa b aa a
a a a aa
a b b b ba b aa
b b b bb b b a a a 20
b b a
-3

40 b b b
b b
b 15
c c c
10
30 d dd
dd dd
5

20 0
0 3 7 10 15 30 60 90 120 150 180 365 545 730
Time (d)
4 C 10 C 30 C 40 C 50 C
4 C( P) 10 C( P) 30 C( P) 40 C( P) 50 C( P)

20500
20000
19500
ba aa
Tx (SBU l )

19000
-1

18500
a a cd d ee e ee e e ee ee e e
e e
18000
a
17500 e g
a
ee f g
g
17000 h
g g
16500 g

16000
7 10 15 30 60 90 120 150 180 365 545 730
Time (d)
4 5 6.5 4 C 30 C 50 C

Fig. 3. Stability proles of TH-30 formulation (FH-4); (a) Rheological prole at dierent pHs; (b) rheological prole at dierent temperatures; and (c)
Biological ecacy (P, particle size); bars shown with dierent letters are signicantly dierent at P 6 0.05. Error bars for standard deviation in graphs have
been omitted for clarity. Points and lines designated P correspond to the particle sizes on the right axis in Fig. 3a and b.

particle size; 34 to 24 lm) showed a decline at 40 and 50 C (P < 0.05) (Fig. 3c). The Tx was lowered probably due to
after 30 d storage (Fig. 3b). There was a possibility that at degradation of crystal protein by protease at pH 6 and
higher temperatures, shear-induced interactions increased 6.5 and higher temperatures as Bt proteases were reported
which resulted in oc structure break-up causing a particle to be thermophilic (Tyagi et al., 2001) as well as weakening
size decline after 120 d. Similarly, certain inter and intra of spore cortex at temperature extremes (Burges, 1998).
particle interactions such as hydrogen bonding and van There may also be consequent eect on other virulence fac-
der Waals forces would have probably decreased causing tors and thus decreasing the eective Tx.
a change in the orientation of EPS ocs leading to the vis- Although the spore survival (7788%, P < 0.05) (Table 5)
cosity decrease. was not so high for FH-4, yet it was selected as optimal for-
The Tx at pH 6 and 6.5 and temperatures 30 and 50 C mulation based on physical and Tx stability. FH-4 formula-
decreased gradually from 19550 to 16550 SBU ll1 tion showed higher suspendibility ranging from 69% to 94%
682 S.K. Brar et al. / Chemosphere 67 (2007) 674683
at dierent pHs and 6286% at various temperatures (Table
5). Thus, the percent spore survival decreased and suspendi-
bility increased with increase in pH. This suggested that at
higher pH, the oc interactions were repulsive leading to
enlarged oc structure and possible attack by proteases (dis-
cussed earlier) causing an increased in suspendibility.
3.4.2. Contamination, corrosion and caking
There was no growth of pathogenic microorganisms
(Salmonella, Staphylococccus, yeast and mould, total and
fecal coliforms and Enterococcus) at any pH and tempera-
ture. Likewise, no corrosion was observed in the tested pH
range through 24 h. Corrosion plays an important role in
Bt sprays as often the loading containers are made up of
aluminium or iron (Burges, 1998).
No sedimentation and caking of FH-4 formulation was
observed during 24 months storage. Lower sedimentation
and caking are desirable for shelf stability as well as applica-
tion of formulations (Burges, 1998). Additionally, when par-
ticle size prole of FH-4 (stable hydrolyzed sludge
formulation) was plotted against commercial Bt product,
Foray 76B (Fig. 4), maximum volume percent of particles
of FH-4 formulation overlapped with Foray and smaller per-
Fig. 4. Particle size prole of stable hydrolyzed sludge formulation and commercial Bt formulation (Foray 76B).
centage fell outside the range, yet below the standard norm
(recommended particle size 625lm). This will result in better
synergy with application equipment easing eventual eld
application. Thus, the stable Bt fermented hydrolyzed sludge
formulations will form a strong proponent of the Bt biopes-
ticides repertoire increasing their marketability.
Furthermore, the optimal combination of sodium
monophosphate (buer), sorbitol (suspending agent) and
sodium metabisulte (suspending as well as anti-microbial
agent) is being explored to reduce the utilization of propi-
onic/sorbic acid as anti-microbial agent which will reduce
the overall cost of the formulations. Interestingly, during
preliminary studies, when propionic and sorbic acid con-
centration was decreased from 0.5% and 0.4% w v1 to
0.3 and 0.2% w v1, respectively, there was no microbial
contamination perhaps aided by anti-microbial action of
sodium metabisulte.
4. Conclusions
The rheology study of hydrolyzed sludge during Bt fer-
mentation and development of stable formulations thereof
led to following conclusions:
 S.K. Brar et al. / Chemosphere 67 (2007) 674683
1. During fermentation, hydrolyzed sludge showed non-
Newtonian pseudoplastic behaviour.
2. The rheological models during fermentation and formu-
lation showed varying degree of condence of ts.
3. The power law constants, consistency index increased
with a peak at 12 h and later decreased, whereas, the
ow behaviour index increased continuously and
attained constancy during stationary phase and fol-
lowed exponential law.
4. The stable formulation followed power law and the for-
mulation pseudoplasticity decreased as compared to Bt
fermented broth, enhancing the ow properties of
former.
5. The stable formulation comprising sorbitol, sodium
monophosphate and sodium metabisulte approached
Newtonian behaviour, but viscosity decreased with
shear rate.
6. A cocktail of suspending agents, namely, sorbitol,
sodium monophosphate and sodium metabisulte in
the ratio 2.2:1:1 was optimal and suspendibility ranged
from 69% to 94%.
7. The stable formulation showed consistent physical and
biological proles at pHs 4 to 4.5 and 4 to 30 C.
Acknowledgements
The authors sincerely thank the Natural Sciences and
Engineering Research Council of Canada (Grants A4984,
STP235071, and Canada Research Chair) for nancial sup-
port. We are also thankful to NSERC, CFS and SOPFIM
for providing scholarship to Satinder K. Brar.
References
APHA, AWWA, WPCF, 1998. In: Clesceri, L.S., Greenberg, A.E., Eaton,
A.D. (Eds.), Standard Methods for Examination of Water and
Wastewaters, 20. American Public Health Association, Washington,
DC, USA.
Barnabe, S., Verma, M., Tyagi, R.D., Valero, J.R., 2005. Culture Media
for Increasing Biopesticide Producing Microorganisms Entomotoxic-
ity, Methods of Producing Same, Biopesticide Producing Microorgan-
isms So Produced and Method of Using Same. US patent
International application PCT/CA2005/000235.
Brar, S.K., Verma, M., Tyagi, R.D., Valero, J.R., Surampalli, R.Y.,
Banerji, S., 2004. Development of sludge based stable aqueous Bacillus
thuringiensis formulations. Water Sci. Technol. 50, 229236.
683
Brar, S.K., Verma, M., Tyagi, R.D., Valero, J.R., Surampalli, R.Y.,
2005a. Sludge based Bacillus thuringiensis biopesticides: viscosity
impacts. Water Res. 39, 30013011.
Brar, S.K., Verma, M., Barnabe, S., Tyagi, R.D., Valero, J.R., Surampalli,
R.Y., 2005b. Impact of Tween-80 during Bacillus thuringiensis
fermentation of wastewater sludges. Process Biochem. 40, 2695
2705.
Brar, S.K., Verma, M., Tyagi, R.D., Valero, J.R., Surampalli, R.Y.,
2006a. Ecient centrifugal recovery of Bacillus thuringiensis biopesti-
cides from fermented wastewater and wastewater sludge. Water Res.
40, 13101320.
Brar, S.K., Verma, M., Tyagi, R.D., Valero, J.R., Surampalli, R.Y.,
2006b. Screening of dierent adjuvants for wastewater/wastewater
sludge based Bacillus thuringiensis formulations. J. Economic Ento-
mol. 99, 10651079.
Burges, H.D. (Ed.), 1998. Formulation of Microbial Biopesticides:
Benecial organisms Nematodes and Seed Treatments. Kluwer Aca-
demic Publishers., Dordrecht, The Netherlands.
Hansen, B.M., Salamitou, S., 2000. Virulence of Bacillus thuringiensis. In:
Charles, J.F., Delecluse, A., Nielsen-LeRoux, C. (Eds.), Entomopath-
ogenic Bacteria: From Laboratory to Field Application. Kluwer
Academic Publishers., Dordrecht, The Netherlands, pp. 4164.
Hwang, H.J., Kim, S.W., Xu, C.P., Choi, J.W., Yun, J.W., 2004.
Morphological and rheological properties of the three dierent species
of basidiomycetes Phellinus in submerged cultures. J. Appl. Microbiol.
96, 12961305.
Kwon, K.K.L., Hyun, S.J., Sung, Y.Y., Joung, H.L., Jung, H.L., Hong,
K., 2002. Isolation and identication of biolm-forming marine
bacteria on glass surfaces in Dae-Ho Dike, Korea. J. Microbiol.,
260266.
Lisansky, S.G., Quinlan, R.J., Tassoni, G., 1993. The Bacillus thuringiensis
Production Handbook. CPL Press, Newbury, p. 124.
Maybury, J.P., Hoare, M., Dunnill, P., 2000. The use of laboratory
centrifugation studies to predict performance of industrial machines:
studies of shear-insensitive and shear-sensitive materials. Biotechnol.
Bioeng. 67, 265273.
MENV, 2004. Guide sur la valorisation des matieres residuelles fertili-
santes: Criteres de references et normes reglementaires. Direction du
Milieu Rural, Environnement Quebec, Canada, p. 138.
Tenney, M.W., Stumm, W., 1965. Chemical occulation of microorgan-
isms in biological waste treatment. J. Water Pollut. Cont. Fed. 37,
13701388.
Tyagi, R.D., Foko, V.S., Barnabe, S., Vidyarthi, A.S., Valero, J.R., 2001.
Simultaneous production of biopesticide and alkaline proteases by
Bacillus thuringiensis using sewage sludge as raw material. Water Sci.
Technol. 46, 247254.
Vidyarthi, A.S., Tyagi, R.D., Valero, J.R., Surampalli, R.Y., 2002. Studies
on the production of Bacillus thuringiensis based biopesticides using
wastewater sludge as a raw material. Water Res. 36, 48504860.
Yezza, A., Tyagi, R.D., Valero, J.R., Surampalli, R.Y., Smith, J., 2004.
Scale-up of biopesticide production processes using wastewater sludge
as a raw material. J. Ind. Microbiol. Biotechnol. 31, 545552.