Você está na página 1de 26

Process Biochemistry 41 (2006) 323342 www.elsevier.

com/locate/procbio

Review

Recent advances in downstream processing and formulations of


Bacillus thuringiensis based biopesticides
Satinder K. Brar, M. Verma, R.D. Tyagi*, J.R. Valero
INRS-ETE, Universite du Quebec, 490, Rue de la Couronne, Quebec, Canada G1K 9A9

Received 30 March 2005; received in revised form 30 June 2005; accepted 8 July 2005

Abstract

Bacillus thuringiensis (Bt) tem sido usado extensivamente durante quatro dcadas em formulaes de biopesticidal devido
a seus registros de seguro de sade ambiental e humana. Thewidespread uso de Bt muitas vezes contestado pela produo
bem como os custos de formulao. Vrios meios de comunicao locais alternativos com xito substituram meios sintticos
de caros, mas a restrio real est incorporada nos custos de colheita e de formulao. Colheita de eficcia rege a
comercializao de um produto por que afetam a potncia e auxiliando na transformao durante o desenvolvimento de
formulaes. Formulao um elo crucial entre a produo e aplicao e dita economia, vida til mais longa, facilidade de
aplicao e eficcia de campo maior. Existem diversos fatores ambientais, como a ultravioleta radiao, chuva, pH,
temperatura e folhagem fisiologia, que nos impedem de eficcia de formulaes de Bt. Houve desenvolvimentos de
formulaes diferentes dependendo do destino da aplicao e viabilidade slida e lquida para superar os efeitos
ambientais adversos. Formulaes convencionais foram substitudas por verses avanadas, como microencapsulations e
microgrnulos para melhorar entomotoxicity residual. Este artigo analisa avanos recentes em processamento a jusante e
formulaes de biopesticidas Bt baseado incorporando o efeito de diferentes parmetros ambientais. Alm disso, provvel
incluso de formulaes de Bt alternativas de guas residuais fermentada (WW) e lamas de guas residuais (WWS) no futuro
tambm foi includo com entradas nas suas vantagens com alguns resultados salientes. # 2005 Elsevier Ltd. Todos os direitos
reservados.

Keywords: Bacillus thuringiensis; Biopesticides; Environmental effects; Formulations; Microencapsulations; Wastewater; Wastewater sludge

Contents
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 324

2. Harvesting techniques. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 324

2.1. Advances in downstream processing. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 325

3. What and why formulations?. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 325

3.1. Challenges in formulations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 326

3.2. Adjuvants/additives. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 326

3.3. Dry solid products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 326

3.3.1. Dusts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 326

3.3.2. Granules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 329

3.3.3. Briquettes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 329


S.K. Brar et al./Process Biochemistry 41 (2006) 323342 324
3.3.4. Wettable powders (WPs) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 329

3.4. Liquid suspensions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 330

3.4.1. Suspension concentrates (flowables) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 330

3.4.2. Emulsions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 331

3.4.3. Encapsulations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 331

3.5. Advances in formulations (for improved delivery) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 332

* Corresponding author. Tel.: +1 418 654 2617; fax: +1 418 654 2600. E-mail
address: tyagi@ete.inrs.ca (R.D. Tyagi).

1359-5113/$ see front matter # 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.procbio.2005.07.015
324 S.K. Brar et al./Process Biochemistry 41 (2006) 323342

3.6. Booster formulations (enhanced entomotoxicity) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 332

4. Environmental effects. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 332

4.1. Sunlight/UV radiation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 332

4.2. Rainfall and dew . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 333

4.3. pH . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 334

4.4. Temperature. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 334

4.5. Foliage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 334

5. Wastewater/wastewater sludge based Bt formulations. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 335

6. Recapitulation and research priorities. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 336

7. Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 336

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 336

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 336

1. Introduction do mercado em contraste com os pesticidas qumicos


fazem biopesticidas interessantes defensores para
Agricultura e florestas formam um importante recurso investigar.
para sustentar o sistema econmico, ambiental e social
global. Sua proteo contra pragas uma prioridade, e BT um Gram-positivo, que tem propriedades
devido ao impacto adverso de inseticidas qumicos, uso insecticidas (tambm chamado ' entomotoxicity ') que
de biopesticidas est aumentando [1]. Um nmero de afetam uma gama selectiva de ordens de insetos,
biopesticidas (bactrias, fungos, vrus, feromnios, nomeadamente bactrias formadoras de esporos,
extratos de plantas) foram j em uso para controlar vrios Lepidoptera, Diptera e Coleoptera [6,7]. Apesar de,
tipos de insetos responsveis pela destruio de florestas extensas pesquisas no campo da Bt biopesticidas, muitas
e culturas agrcolas. Bacillus thuringiensis (Bt) baseado formulaes no entregar efetivamente em campo devido
biopesticidas so especialmente de extrema importncia a estresse ambiental varivel (por exemplo, a silvicultura
e ocupam quase 97% do mundo biopesticida mercado e a agricultura). Outra razo pode ser a adopo de uma
[2,3]. Um pesticida biolgico eficaz somente se tem um abordagem integrada que pode desempenhar um papel
grande impacto potencial sobre a Praga alvo, tamanho do importante no desenvolvimento de Biopesticida, em
mercado, variabilidade de desempenho em campo, de outras palavras, alfaiataria fermentao e processos para
custo-eficcia, gabarito de usurio final e um nmero de produzir formulaes eficazes de maior potncia de
tecnolgico desafia ou seja, fermentao, formulao e colheita. Biopesticida pesquisa tem sido exaustivamente
sistemas de entrega [4,5]. Custo de desenvolvimento, detalhada em Burges [6], mas h avanos recentes, que
tempo e facilidade de registro e potencial de crescimento tiveram lugar em diante. Enquanto isso, de guas
S.K. Brar et al./Process Biochemistry 41 (2006) 323342 325
residuais (WW) e de guas residuais lamas (WWS) com enzimticos (proteases; chitinases; fosfolipases),
base em formulaes de Bt tambm precisam ser protenas insecticidas vegetativas e muitas incgnitas
elaborado e discutido devido a suas caractersticas virulentas junto com gases inertes / adjuvantes. Mais
positivas inerentes (discutidas mais tarde). cedo, lactose-acetona tcnica foi utilizada como um
mtodo para recuperar os esporos Bt com perdas
Engenharia gentica pode jogar um papel complementar mensurveis [14]. No entanto, uso de mtodos avanados
no desenvolvimento de formulaes mais eficazes, como ultracentrifugao, microfiltrao e vcuo filtrao
facilitando a maior produo de toxinas, ampliando o para separar slidos insolveis (ingredientes ativos) da
host gama e realando a germinao, esporulao e fraco solvel de lquido (inerte) do licor colheita,
ampliar o espectro de Bt, ainda o papel de formulaes resultou na recuperao eficiente do ingrediente ativo
com cepas convencionais no pode ser negligenciado (i.a.) de vrios produtos biotecnolgicos [15,16]. Co a
[8,9]. Microorganismos geneticamente modificados esto expresso de genes heterlogos quitinase em Bt tambm
alm do escopo deste artigo de reviso. Vrias reas do tem sido demonstrado para aumentar a atividade
ncleo precisam ser resolvidos antes das biopesticidas inseticida da bactria [17,18] e, portanto, recuperao
podem penetrar no mercado internacional: espectro de criteriosa de a.i. de caldo todo necessria aumentar a
atividade, a persistncia e a reciclagem, melhoria de potncia do produto.
formulaes com o uso de adjuvantes convencionais e
simples/aditivos que no so custos intensivos

[10]. Colheita de microorganismos da fermentao


submersa muitas vezes difcil devido baixa
concentrao de produtos, sua natureza termolbeis e em
alguns casos, pobre estabilidade. Adjuvantes de
Desde a entrada do primeiro produto comercial da Bt
estabilizao pode ter que ser incorporados na operao
chamado ' Sporeine ' na Frana em 1938, houve um
ps-colheita para evitar a mortalidade de esporos e/ou
aumento contnuo no desenvolvimento de produtos
germinao. Secagem rpida ou adio de produtos
avanados. Nesta luz, presente reviso discute vrios
qumicos biocidas especficos pode ser necessria para
avanos na Bt colheita tecnologias, bem como
prevenir o crescimento de contaminao microbiana no
formulaes. Alm disso, fatores ambientais, como raios
caldo ou centrifugar chorume [19]. Esse mtodo pode
UV, chuva, pH e temperatura que flagelam a estabilidade
do campo de formulaes de Bt so exploradas encontrar utilidade apenas para pequenos volumes de
juntamente com um WW/WWS alternativos com base lquido de caldo de carne. Mesmo, processo de flotao
em formulaes de Bt. de espuma foi aplicado para obter suspenses cristal-
enriquecido de Bt em que gelatina causou esporos para
2. tcnicas de coleta seletivamente ser arrastado na espuma e, assim,
separados de suspenses [20]. No entanto, esse mtodo
envolvido aps processos laboriosos passos incorporando
O caldo fermentado final do Bt composto por vrios produtos qumicos.
esporos, detritos celulares, corpos de incluso, enzimas e
outros slidos residuais, que precisa ser recuperado de
forma eficiente para ser utilizado na etapa de formulao
subsequente [11,12]. Geralmente, dependendo a
entomotoxicity desejada do produto final e escala de
produo, o processamento necessrio varia Alguns autores tm utilizado o mtodo de
significativamente. Fatores-chave que regem a escolha da pulverizao e secagem para caldo grandes volumes [21].
estratgia de colheita incluem taxa de transferncia de Essa secagem pode ser precedido por espessamento do
processo, caractersticas fsicas do produto e impurezas e lquido fermentao por centrifugao e filtrao
desejado de concentrao do produto final [13]. utilizando filtro SIDA como celite, superfloc, etc., para
reduzir a manipulao do volume [22,23]. O spray mais
seco, a gua removida da chorume caldo que atravessa
a entrada aquecida (150-200 8 C). O p resultante reveste
as paredes e recolhe o pulverizador secador [24]. Apesar
de uma perda fsica, ainda no mensurvel Bioatividade
Produtos mais comerciais de Bt contm protenas diminuda por pulverizar o processo de secagem, devido
insecticidas cristal (ICP), esporos viveis, sistemas
a exposio contnua dos componentes bioativos para as Tcnicas pr-existentes na indstria alimentar e
altas temperaturas [25]. biotecnologia poderiam ser adaptadas produo de
biopesticida com modificaes. Ultrafiltrao miclica
aprimorado (MEUF) foi uma tcnica recentemente
proposta para separar compostos orgnicos dissolvidos,
como thuringiensin de fluxos aquosos [29]. Neste
processo, surfactante foi adicionado para um fluxo
Uma recuperao eficiente do complexo ativo de aquoso contendo orgnico solute micelas de formulrio
esporo-cristal de Bt foi relatada por Rojas et al., usando para separar compostos para purificao subsequente de
uma centrfuga de disco-pilha ou um filtro de vcuo destino. A separao dependia da membrana de
rotativo com eficincia de recuperao de esporos ultrafiltrao (UF), tipo e concentrao de surfactante,
superior a 99% [26]. No entanto, a concentrao de conjunto de condies experimentais (tais como pH,
slidos secos produzidos por filtrao (31,5%) foi fora inica, temperatura e presso transmembrana, etc.)
superior a centrifugao (7,5%). Foi realizado estudo [30].
semelhante na Bt var Berliner usando uma centrfuga
contnua com taxa de recuperao de 85-90% e
diminuio da eficincia de separao com aumento nas
taxas de fluxo [27].

Remoo de produto in situ (ISPR), a recuperao de


um produto bioqumico da vizinhana de uma clula
durante a fermentao ativa tem sido sugerida como
possvel mtodo de remoo de protena em vrios
Na fase final aps a fermentao, lactose (5%) foi processos comerciais [31]. No entanto, no houve
adicionada s vezes como um crioprotetor para evitar nenhum aplicativo relatado do processo na produo de
aglutinao durante a armazenagem e co-precipitao Biopesticida ISPR. Microfiltrao de fluxo cruzado
lactose-acetona poderia ser usada como uma etapa (CFM) tem sido utilizada ultimamente para extrao de
sequencial para centrifugao para alcanar maior todo o tipo de protenas e colheita de levedura
eficincia de recuperao de d-endotoxina [28]. Os recombinante [32,33]. Lee et al usado CFM para extrao
produtos finais (p/suspenso) so devidamente de protenas resistentes ao ataque de proteases presentes
formulados como aquosa (flowable) ou concentrado, em na parede celular [34]. Mas, esse mtodo altamente
p, de pulverizao do leo ou granulam. No entanto, a especfico e tambm recomenda a recuperao do
literatura sobre mtodos de colheita muito escassa produto purificado e no pode encontrar a utilizao na
como a maioria da produo comercial Bt realizada recuperao biopesticida.
pelas indstrias e, portanto, o processo de separao
proprietrio e seguro.

Entre todos os avanos, centrifugao parece ser uma


alternativa vivel e com mais avanos em design e
Apesar de colheita considerada como um passo velocidade, poderia servir como o melhor equipamento
importante que pode aumentar ou suprimir a atividade de para processamento a jusante de biopesticidas. A
biopesticidal, ainda atuais tcnicas baseadas na viabilidade foi validada por estudos sobre processamento
centrifugao convencional, diferencial e gradiente de de centrfugo de restos de clulas e corpos de incluso de
densidade e secagem de pulverizador sofrem perdas recombinao Escherichia coli [35].
inerentes e ineficiente d-endotoxina recuperao.

Assim, o objetivo principal de uma boa tcnica de


2.1. avanos em processamento a jusante colheita para minimizar o nmero de operaes
unitrias envolvidas no processo, reduzindo custos de
validao de processo global e simplificando tambm a
facilidade e economia de automao de processos. Isto
S.K. Brar et al./Process Biochemistry 41 (2006) 323342 327
uma questo fundamental para o desenvolvimento de os biopesticidas, pontes de fermentao e campo de
uma unidade de produo integrada de Biopesticida que aplicao.
regem o custo total do produto.

3. O que e por que formulaes?

3.1. Desafios em formulaes

Os problemas de estabilidade de biopesticidas durante


a armazenagem e aps a aplicao estagnaram
biopesticida desenvolvimento a uma extenso grande Biopesticidas posar um dueto de diferentes
[36]. Nesta conjuntura, desenvolvimento de formulaes ofchallenges definir sua natureza inerente suspenses
pode desempenhar um papel fundamental, abordando de partculas; termicamente mais sensveis; propenso
quatro grandes objectivos que podem servir como pontos contaminao e pragas especficas e, portanto, precisam
de referncia para o sucesso: (1) estabilizar agentes ser concebidos em conformidade. Entretanto, a maioria
microbianos durante a distribuio e armazenamento; (2) dos desafios tem sido tratada no atual cenrio
ajuda a manipulao e aplicao do produto; (3) proteger biopesticida Bt, que mudou o
agente de factores ambientais adversos; (4) aumentar a conceptofchemicalpesticides ' autnomo '. Assim,
atividade de agentes microbianos em campo. extensiveexplorationof Bt biopesticidas e contnuos
Principalmente, composto por uma formulao a.i. avanos desde o incio do sculo XX contriburam
(fungos, bactrias, vrus, nematoides, etc.) e aditivos significativamente para a sua aceitao generalizada e
(vrios para manter o status quo da a.i.) para cumprir usam em comparao com outras biopesticidas.
objectivos de aforestated e dar forma aos biopesticidas.

326 S.K. Brar et al/processo bioqumica 41 (2006)


323-342
3.2. Adjuvantes/aditivos

Biopesticidas comerciais devem ser econmicas


produzir, ter estabilidade de armazenamento persistente,
alta atividade residual, ser fcil de manipular, misturar,
aplicar e fornecer consistentemente eficaz controle do
alvo pragas [37 40]. O objectivo global aprimorar o Eles so compostos quimicamente e biologicamente
uso de widescale de biopesticidas comerciais que podem ativos que podem alterar a formulao fsica e matar as
no ser to eficazes quanto suas contrapartes qumicas, espcies-alvo sem prejudicar outros insetos (ou seja,
mas com o tempo eles podem se tornar to eficazes. aumentar a sua seletividade; [42]) e reduzir a dose
Enquanto isso, os produtores podem ter que avaliar o seu necessria de Biopesticida eficaz [43]. Agncias de
nvel de controle de pragas para alcanar rendimentos de registro como nos agncia de proteo ambiental (US
colheita econmica [36]. EPA) regulam a incluso de certos ingredientes nas
formulaes de adjuvante e, portanto, testes de adjuvante
e / gases inertes contida dentro as listas 4A e 4B
Alm disso, a formulao aborda os problemas: composto por risco mnimo e no arriscar ingredientes
velocidade de matar; perda de atividade de campo atravs inertes, respectivamente [44]. Por exemplo, no passado,
da persistncia das condies ambientais prejudiciais, xileno foi usado como conservante em formulaes de
composto por luz solar, umidade adversa (seca ou Bt, mas seus impactos ambientais adversos mais tarde
molhada), chuva, vento, caractersticas da planta como levou retirada deste conceito [45,46]. Esse nus as
qumica de folhas e crescimento microbiano de cordas na incluso de adjuvantes principalmente
organismos concorrentes; Pobre palatabilidade e biodegradveis/aditivos realando eco-amizade de
modificao das tcnicas de aplicao, pelo uso de biopesticidas.
adjuvantes e gases inertes atravs de stable qumica [41].
Da, formulao, um pr-requisito obrigatrio para todos
Os fatores chave na seleo de apropriado adjuvantes Outro (s)
esto sumarizados na tabela 1 e detalhes do adjuvante Interaes de produto ou problemas de compatibilidade
diferente / aditivos com suas funes e exemplos so Ordem de incorporao na mistura de tanque
ilustrados na tabela 2. Largura de uso e zona tampo de avinicos

Modificado de Tu e Randall [212].


Seleo de adjuvante/aditivo rege-se pelo tipo de
formulao desejada e segue os mesmos critrios para
3.3. Dry solid products
todos mdia com base em formulaes de Bt. As
caractersticas e a composio de formulaes 3.3.1. Dusts
biopesticidal variam de acordo com o tipo de habitat Dusts are formulated by the sorption of an a.i. onto a
(folhagem solo gua; armazm; tamanho), patgeno finely ground, solid inert such as talc, clay, or chalk, with
(tipo; caractersticas; mecanismo de regenerao e particle size ranging from 50100 mm. Although, finer
fatores), reologia de materiais tcnicos (viscosidade, particles adhere better, at the same time, they pose serious
granulometria, densidade), espcie de inseto (alimentao inhalation hazard for the user and drift hazard for the
hbitos alimentares nicho; ciclo de vida), modo de aco sprayer. Couch and Ignoffo [48] have listed different types
(oral/contato); interaes hospedeiro- of inert fillers used with insect pathogens with different
pathogenenvironment (mudanas de comportamento, formulations normally containing <10% of microorganism
resistncia, estabilidade), modo de aplicao (areo; (a.i.) by weight.
terra) e taxa de aplicao (L/ha e kg/ha). Deve ser
enfatizado que o produto final determinado pelo meio
especfico utilizado na fermentao, as condies de
fermentao e ps-fermentao processamento.

Em termos gerais, formulaes podem ser


classificadas em slidos secos (p, grnulos, ps e
briquetes) e lquido (denominadas 'suspenses '; leo ou
base de gua e emulses) formulaes [47]. Diferentes
formulaes de Bt registrado no Canad / Amrica do
Norte com seus detalhes de potncia esto listados na
tabela 3. Esta lista no exaustiva, pois h numerosas
formulaes no registradas em todo o mundo,
especialmente nos pases em

Tabela 1
Fatores de seleo para escolher um adjuvante/aditivo

Tipo de formulao
Slido, lquido, encapsulado formas

Alvo (s)
Espcie de pragas (tipo)
Estgio do desenvolvimento
Crescimento denso ou esparso? (Baixa/alta volumes de spray?)
Barreiras penetrao (folhas cerosas, peludas ou grossas,
sedimentos?)
Mtodo de aplicao (area/terrestre; foliar pulverizador, boom e bico
pulverizador, hidrulica) e sincronismo
Volume aplicao taxa e spray tamanho espectro de gotculas

Meio ambiente
Condies do local (aquticas ou terrestres? Em reas sensveis?)
Condies atuais (temperatura do ar? Muito vento? Radiao
UV? Chuva?)
Qumica da gua (gua dura ou mole? PH baixo ou alto?)
S.K. Brar et al./Process Biochemistry 41 (2006) 323342 329
Table 2
Different types of adjuvants/additives used in microbial formulations
Adjuvants/additives Function(s) Example(s) Reference (s)
Dispersant Dispersion of formulation into Amylose; Aluminium silicate; [213,214]
dispersant medium Sodium starch glycolate;
Surfactants and wetters Enhance the emulsifying, dispersing, Ethoxylates (Tween/Triton series); [48,213]
spreading, sticking or wetting properties polyethylene glycol
of the biopesticide (includes spray
modifiers)
Stickers and spreaders Adhesion of pesticides onto the foliage Gelatin; gums; molasses; skimmed milk; [8,37,64,65,116,137,215
protecting from rain wash-off and spreading proprietary like Nufilm and chevron; 218]
evenly for maximum coverage vegetable gels; vegetable oils; waxes;
water-soluble polymers
Drift control Reduce spray drift, which most often Polyacrylamides, polysaccharides, [64,89,117,219,220]
agents/anti- results when fine (<50 mm diameter) and certain types of gums,
evaporants/ spray droplets are carried away from the sorbitol, sucrose, molasses,
humectant target area by breezes, including those polyglycol, molasses, glycerol
caused by the vehicle carrying the spray
equipment and control of foam while
mixing
Thickening agents Modify the viscosity of spray solutions and Water swellable polymers producing a [48,215,220]
are used to reduce drift, particularly for aerial particulate solution, hydroxyethyl
applications celluloses, and/or polysaccharide gums
pH Buffers Adjust or buffer pH; improve the dispersion Sodium phosphate; Potassium phosphate [214,221,222]
or solubilization in the formulation, control
its ionic state and increase adjuvant
compatibility
Defoaming and Reduce surface tension, physically burst the Dimethopolysiloxane-based; [58,223]
antifoam agents air bubbles, and/or otherwise weaken the silica; alcohol and oils
foam structure
UV radiation screens Protect from the deleterious effect(s) Congo Red; folic acid; lignin; molasses; p- [25,64,65,224228]
of sunlight by forming a protective aminobenzoic acid; alkyl phenols
layer on the formulations
Phagostimulants Stimulate feeding of formulations by pests Corn meal; sucrose; wheat germ; corn [104,115,228231]
germ; soya flour; casein, edible oil,
glutamate, molasses
Synergists Multiple modes of action; generally Sorbitol; sorbic acid; sodium phosphate; [98,232234]
complements various formulation stilbene; Tinopal; silicate; protease
components inhibitors, oleic acid, linoleic acid
Anti-microbial agents Suppresses the growth of other Sorbic acid; propionic acid; crystal violet [82,235]
microorganisms, retaining formulation purity
Carriers Aid in delivery of formulation to target Alginate; carrageenan; peat, [236243]
acrylate and acrylamide
supersorbents, diatomaceous
earth
Binders For binding the particulates in granules Gums; molasses; PVP, resins [66,82]
together
Suspending agents Keep the formulation in suspension Sorbitol; soya polysaccharides; [131,238]
starch glycolates; sucrose
Attractants Act as baits to attract target pests Pheromones, cucurbitacin and [244,245]
various alkaloids; plastisol (PVC
and cotton seed oil)
Multi-purpose Perform various functions at the same time Molasses; starch, lignin [107,113,119,246]
Particle size (0.550 mm), bulk density (0.50.6 g/cm 3)
and flowability are important control parameters [49].
During application, smaller particles collect on target
surfaces and large ones fall off and lack stickiness. Thus,
stickers (adherents) and desiccants (prevent caking) are
commonly employed. Bt dusts have been employed in
stored bulk grains to control surface-dwelling lepidopteran
pests [50]. Early work also reported higher control of
loopers, semi-loopers and cabbage pests with Bt dusts
when compared to conventional sprays [51]. The higher
control could be due to uniform coverage on underleaf (discussed later), use of dusts was restricted owing to
surface and adverse health impacts (respiratory) on the end user [52].
protection from sunlight, however, windy conditions would Still, their usage continues unregulated in developing
support liquid sprays. nations [53]. Recently, two formulations of Bacillus
Bt dusts have been widely used in the control of corn thuringiensis Berliner (wettable powder and dust) to
borer larvae. However, with the development of granules control the larvae of Lobesia botrana Denis and
Schiffermueler (Lepidoptera: Tortricidae) were tested in
328

Table 3
Products (formulations) registered for commercial use in Canada/North America
Industry/company Product Active ingredient(s) Entomotoxicity Registration Expiry Target pest(s)
year year
Valent Biosciences Dipel WP (Wettable Powder) 16000 BIU/mg 1972 2006 Spruce budworm; gypsy moth; bagworm; spring and fall
Corporation cankerworms and cabbagelooper

Thuricide 48LV (Liquid suspension) Bacillus thuringiensis 12.7 BIU/L 1984 2007 Bagworm; elm spanworm; fall spanworm; gypsy Moth; spring
berliner ssp. kurstaki and fall cankerworm; spruce budworm; jack pine budworm

Vectobac-200G Larvicide (Granules) 200 ITU/mg 1984 2007


Teknar Granules Larvicide 260 AAU/mg 1986 2004 Mosquitoes
Vectobac 200G (Granules) Bacillus thuringiensis 200 ITU/mg 1986 2007
israelensis
Vectobac 600L (aqueous suspension) 600 ITU/mg 1986 2004 Fungus gnats
Teknar HP-D Larvicide (aqueous suspension) 3000 AAU/mg 1986 2004 Mosquitoes & Black flies
Novodor Flowable Concentrate Bacillus thuringiensis 3.6% 1995 2004 Colorado potato and elm leaf beetle
ssp. tenebrionis
Foray 48B 12.7 BIU/L 1997 2004 Spruce budworm (Eastern & Western); gypsy moth; jackpine budworm;
eastern hemlock looper; whitemarked tussock moth and forest tent
caterpillar whitemarked tussock moth forest tent caterpillar and satin moth
Foray 48B A Low Volume 12.7 BIU/L 1997 2007
Foray 76B (aqueous concentrate) 20.0 BIU/L 1997 2007
Foray 96B 25.4 BIU/L 2003 2004
Dipel 2X DF (Dry Flowable Bacillus thuringiensis 32,000 IU/mg 2000 2007 spruce budworm; gypsy moth; bagworm; spring and fall
(Wettable Granules) berliner ssp. kurstaki S.K.Braretal./Pro
cankerworm; fall webworms; cessBioc
elm spanworm; hemistry41(2006)323342
tent caterpillar;
cabbage looper; leafroller and diamondback moth
Certis USA LLC Thuricide-HPC High Potency 4.2 BIU/L 1972 2007 Spruce budworm; gypsy moth; bagworm; spring & fall
Aqueous Concentrate cankerworms and cabbagelooper

AFA Environment Inc. Aquabac II XT (Liquid suspension) Bacillus thuringiensis israelensis 1.28 BITU/Kg 2003 2007
(Bacillus thuringiensis serotype H-14)
Aquabac 200G (10/14) (Granules) 0.20 BITU/Kg 2001 2006 Mosquitoes
Aquabac 200G (10/14) (5/8) 200 ITU/mg 2001 2006
AFA Environment Inc. Aquabac XT Bacillus thuringiensis israelensis 1200 ITU/mg 2001 2006 Mosquitoes and blackflies
Abbott Laboratories Ltd. Dipel 176 (Emulsifiable suspension) 16.9 BIU/L 1988 2007 Forest tent caterpillars; gypsy moth spruce budworms; hemlock looper
Woodstream Canada Safers BTK (Liquid concentrate) Bacillus thuringiensis berliner 12.7 BIU/L 1996 2006 Gypsy moth; tent caterpillar and cabbage looper
Corporation ssp kurstaki

AEF Global Inc. Bioprotec Aqueous Biological 12.7 BIU/L 2000 2007 Gypsy moth; eastern spruce budworm; western spruce
(aqueous suspension) budworm; jack pine budworm; forest tent caterpillar; eastern
hemlock looper; bagworm; elm spanworm; fall spanworm;
spring & fall cankerworm; satin moth and white marked tussock
moth
Bioprotec CAF 12.7 BIU/L 2001 2004
Aqueous Bioprotec HP 17,5000 IU/mg 2002 2004
Bioprotec ECO 12.7 BIU/L 2003 2007
Source: Modified from http://eddenet.pmra-arla.gc.ca; cited 07 June, 2004, Pest Management Regulatory Authority, Canada.
ITU, International Toxic Units; IU, International Units; BITU, Billion International Toxic Units; AAU, Aedes Aegypti Units (1 ITU = 2.5 AAU).
IU: it refers to standardized potency (by bioassay) of different marketed Bt products against Bt var. thuringiensis E-61 standard from Institut Pasteur, Paris, France, assigned a potency of 1000 International Units (IU) per mg. Bioassay is
carried out against mediterranean flour moth (Ephestia kuhniella) in Europe and in US, a primary reference standard of Bt HD-1-S-1971 strain with an assigned potency of 18000 IU/mg is being used, bioassayed against cabbage looper
(Trichoplusia ni).
S.K. Brar et al./Process Biochemistry 41 (2006) 323342 333

Greece. Dusts provided maximum kill in contrast to General advantages and disadvantages of different formulations
wettable powder [54]. 3.3.3. Briquettes
They are large blocks with sizes ranging from 100 to
3.3.2. Granules 250 mm and possess same carriers as in granules
The granules comprise discrete masses of 510 mm 3 presenting no drift problems. Bti based briquette
formulated by using carriers like clay minerals, starch formulations are largely utilized in public health sector for
polymers, dry fertilizers and ground plant residues [55]. control of mosquitoes.
Concentration of organisms in granules is 520%. There are Various formulations of Bti have given higher rates of
three typesofgranules:(1)exteriorgranules kill and sustained persistence, in several cases, ranging up
microbesattachedto outer surface of a carrier by a sticker; to two months in single application [68,72,73]. Briquettes
(2) exterior granules without stickers; (3) incorporated have been normally made using organic polymers like
granulesall constituents mixed into a paste to from matrix polyvinyl alcohol to give floatability causing sustained
and later sieved (after processingforgranulation) release of toxins over several months [7477]. Mostly
todesiredsize.Normally, they are employed in agricultural floating type formulations with carrier materials such as
crops, e.g., cabbage, corn, etc. [56]. wheat flour are common with Bti [75] and also buoyant
Granular ingredients are retained pasty so that the forms which have self-encapsulating abilities [78] and
material can be pressed through a granulation die. They are gypsum has been used to enhance sustained release of
made by mixing together all ingredients into a liquid carrier toxins [79].
and then extruded. There have beenvarious types of granules
wheat meal granules [57]; corn meal baits; granules 3.3.4. Wettable powders (WPs)
formed with gelatinized cornstarch or flour [25,5863]; They consist of 5080% technical powder, 1545%
casein [64]; gluten [65]; cottonseed flour and sugars [66]; filler, 110% dispersant and 35% surfactant by weight to
gelatin or acacia gum [67]; sodium alginate and paraffin achieve a desired potency formulation (measured in
[68]; diatomaceous earth [69] and semolina [70] commonly International Units). Fillers are hydrophilic and usually
employed as formulations. In a recent advancement in contain silica which resists cake formation and friability
granular formulations, solutions containing powder during grinding. However, silica content must be kept low
formulations of Bacillus thuringiensis var. israelensis (Bti) to avoid abrasion of formulation equipment [80].
or Bacillus sphaericus were transformed into ice pellets Dispersant must be added to retain suspension in
(named IcyPearls). This technique encompassed various dispersion; surfactants to overcome surface tension at
advantages over Bti sand granules: (1) Bti ice pellets melted liquidsolid interface (excess may lead to foam generation
on the water surface and released the microbial crystals; (2) and partitioning of spores therein). Normally, particles
therewas no loss of Bti by friction in the spraying settle rapidly, hence spray tanks fitted with agitators are
equipment; (3) the ice formulation resulted in increased usually used.
swath widths, significantly reducing application costs [71]. Among the dried formulations of biopesticides, much
However, practical diverse application of such formulations attention has been paid to WPs because of their longer
in tropical countries with peak temperatures remains a big shelf life, good miscibility with water and ease of
quest. application as sprays with conventional equipment [80].
Technology of granules has paved way to agglomerates Despite the advantages, literature is not abundant for Bt
or encapsulated versions which can give better protection WP products, only WP formulation for nuclear
against UV radiation, rain and wind. polyhedrosis virus of Anticarsia gemmatalis was shown to
be evaluated by Medugno et al. [81]. Recently, Teera-
Table 4 Arunsiri et al. [82] have reported development of Bt ssp.
Dry solid formulations Liquid suspensions
Advantages
Are ready-to-use types like dusts, granules, briquettes and Emulsion concentrates and suspension concentrates hold
applicable with simple equipments high a.i., bulk storage not necessary
Granules show less drift and applicable to hidden foliage too ULV concentrates can be used without mixing
WPs are easy to transport, store and apply when required; Encapsulated suspensions increase residual toxicity and decrease user
low risks of operator safety hazard
Cost effectivetransportation costs are low Development process is without harsh conditions of dryinghigher
recovery
Do not need high quantity of preservatives Used in agriculture, gardens and forests
Less expensive to apply
Disadvantages
Dusts are subject to drifts and pose user hazards Subject to deterioration on long storage
Some WPs could clog sprayers a.i. may settle out of emulsions and suspensions, at times
Formulation involves harsh spray drying stepsloss of a.i. Sometimes, require complex spray equipments, e.g., in forestry
Use restricted to gardens, agriculture and water streams
More expensive to apply
334 S.K. Brar et al./Process Biochemistry 41 (2006) 323342

aizawai based WP with 55% suspendibility, 24 s for balance (HLB) whereby lower HLB is suitable for water-
wetting time, and 5.69 104 CFU/mL of LC50 value against in-oil and vice versa for oil-in-water based suspensions
Spodoptera exigua larvae. The authors used fish meal as a [85,86].
growth substrate and locally available tapioca starch as a Formation of biopesticide concentrates in oil increased
sticker and vegetable oil as surfactant. Many Bt WP sedimentation and hence wet milling of product was
products that find use in gardens and agriculture could recommended to retain suspendibility. Biodegradable oils,
also be located on company website of Valent Biosciences for instance, vegetable oils are preferred and a detailed
and Novartis Corporation [83,84]. listing of ecologically benign oils is given by Underwood
Although there are lots of advantages rendered by dry [87]. The clear difference between water and oil based
formulations, yet they encompass various problems as formulations could be easily understood from Fig. 1.
well, as reported in Table 4. In forestry, generally, ultra low volume (ULV) sprays
(required dispersion rate of 20 BIU/ha in a final volume of
3.4. Liquid suspensions 2.5 L/ha) are preferred to deliver higher Bt concentrations
[88]. Earlier, the suspension concentrates were ready-to-use
3.4.1. Suspension concentrates (flowables) formulations requiring just the addition of sticker (0.06%)
They are suspensions of particulates in liquids, with which minimized spraying cost by eliminating mixing time
10 40% microorganism, 13% suspender ingredient, 1 and maximizing payload [88]. Some authors had also
5% dispersant, 38% surfactant, and 3565% carrier suggested addition of sorbitol as a dispersing agent, which
liquid (oil or water). The products are prevented from enhanced density of Bt formulations resulting in
settling due to reversible agglomeration by dispersants; concentrated suspensions (reducing loading costs) and
surfactants act as wetting agents and spreaders, and acting as an anti-freeze as well as anti-evaporant further
generally, non-ionic ones (derivatives of poloxyethylene structuring formulations [89]. The success rates of Bt
and polyoxypropylene) are preferred along with water formulations led to more than 70% use of Bt biopesticides
in

Fig. 1. Water and oil-based formulations on interaction with leaf (hydrophobic) surfaces. (A) Water soluble sunscreen; water droplet forms a large contact
angle on the hydrophobic surface. As the droplet dries the water-soluble sunscreen concentrates around the spore [6,119]. Water based formulations will
cause minimum drifts owing to density effects during field spray. (B) Oil soluble sunscreen; oil droplet forms a thin film and small contact angle on the
hydrophobic surface. As the oil droplet spreads over the surface, the oil-soluble sunscreen spreads with the oil exposing the spore [6]. (C) Water medium
and sunscreen is encapsulated to give a long term UV radiation protection (suggested approach).
soluble sunscreens [6]. Based on type of carrier liquid, North America in 1990s for forest Lepidoptera pests control
surfactants are characterized by hydrophilelipophile compared to less than 5% in 1981 [90]. However,
S.K. Brar et al./Process Biochemistry 41 (2006) 323342 335

nowadays, the trend is to achieve maximum kill with


minimum dosage (maximum efficacy per droplet) and in
this context, a study carried out
336 S.K. Brar et al./Process Biochemistry 41 (2006) 323342

in Quebec, Canada established 30 BIU/ha (BIU, billion diamondback moth even after 15 days. Elsewhere, Cheung
international unit) as the optimal dosage at 1.5 L/ha spray and Hammock [111] emulsified crystal and solubilized Bti
volume by using a high potency (20 BIU/L) product [91]. toxin with Freund incomplete adjuvant. The encapsulation
This strategy will help curb transportation costs enhancing of lipophilic material altered buoyancy of the toxin and
commercial viability of the Bt formulations. Liquid reversed sensitivities of the mosquito larvae towards Bti
formulations of B. sphaericus have also been successfully toxin. Attachment to latex beads was also considered to
employed in rice fields, ponds and other water sources enhance entomotoxicity of Bti formulations [112].
against mosquitoes from the genus Culex [92,93]. The starch encapsulated granules have been
successfully scaled up, but there were losses owing to
3.4.2. Emulsions decrease in residual activity due to rain and UV radiation
They comprise of liquid droplets dispersed in another [113]. Eventually, a modified method developed using
immiscible liquid (dispersed phase droplet size ranges from solvents showed sustained release of Bt over 12 days
0.1 to 10 mm), e.g., oil-in-water (normal emulsions) and [114]. When the formulations were tested in field, it was
water-in-oil (invert emulsions). The emulsions do not observed that there was no change in activity during dry
encounter sedimentation problems, but creaming and layer season, but during rains, rest of the formulations lost their
separation are common [94]. In biopesticide jargon, they are activity with respect to starch formulations [115]. To take
referred to as suspo-emulsions. As oil is external phase in into account pH effects normally encountered in field,
invert emulsions, losses due to evaporation and spray drift addition of gluten to the starch matrix aided in retaining
are minimal [95,96]. However, lower shelf stability and the larval mortality of O. nubilalis > 90% over a pH 511
phytotoxicity may affect the overall performance of the and also resisted 5 cm rain [116]. This provided a broader
emulsions. pH range for the formulations to act and protected d-
In 1980s, ULV sprays in forests comprised oil emulsions, endotoxin by competing with proteases at alkaline pH and
but lately it has been replaced with aqueous emulsions [6]. secondary plant compounds such as tannins [117]. Ramos
Presently, oil emulsions find restricted use in household or et al. [118] have discussed use of different biopolymers
agricultural sprays [44,97]. Probable reasons for their forgranular formulationsof Bt. There have been several
discontinued use in forests could be: increase in drift studies on incorporation of lignin into microcapsules to
velocity which may fall across large distances affecting further extend residual activity and it was observed that
habitations; creaming problem during storage and lower activity remained 75% after 7 days versus 53% for
biodegradability in contrast to water formulations. commercial Dipel formulation [119,120]. Moreover, the
maize flour microcapsule formulation offered increased
3.4.3. Encapsulations protection from UV radiation and improved rainfastness of
They are recent advances in bioinsecticidal formulations Bt during simulated rainfall. Likewise, Cote et al. [121]
and provide protection from extreme environmental reported an extended period of mortality of Choristoneura
conditions (UV radiation, rain, etc.) and enhanced residual rosaceana with bioencapsulated formulation (rice flour
stability due to slow release of formulations (sustained based) in comparison to conventional DiPel WP
delivery). They are usually liquid suspensions with formulation. Alginate coated Bti formulations were also
possibility of powders and granules too. Microbial used to control mosquitoes [122]. In some formulations,
propagules (e.g., Bt) are encapsulated in a coating (capsule) polyethylene glycol or ethylene glycol was used as an
made of gelatin, starch, cellulose and other polymers and encapsulating binder with anti-freeze properties for better
even microbial cells (also referred to as ghost dispersion [123125].
encapsulations) [98101]. Another recent advance in encapsulations is production
Initial encapsulations were an extension of chemical of hydrocapsules that are of shellcore type (water based),
pesticides and involved addition of clays, matrices such as consisting of a polymer membrane surrounding a liquid
polyvinyl propylene and polyvinyl alcohol [102,103]. This center. These shells are produced by using UV radiation
led to the exploitation of biopolymers to make the products initiated free-radical copolymerization of functionalized
eco-friendly [104106]. prepolymers (silicones, urethanes, epoxys, polyesters, etc.)
Conventionally, autoencapsulated (biological origin) and/or vinyl monomers such as acrylates for better
formulations against European corn borer (Ostrinia dispersion and UV radiation protection [126,127].
nubilalis), were made by mixing starch powder and sugar Encapsulation in the form of microcapsules has been
[104,107]. Fine, encapsulated products can be sprayed in extensively exploited to give smaller size, highly efficient
any volume as the pathogen is held tightly to additives fungal biopesticide formulations [122]. This technology
causing less wastage [108,109]. Bok et al. [110] suggested could be extended to Bt suspensions, which would
use of carbohydrate rich biopolymeric gels for construction enhance aerial dispersion onto foliage and feeding, by
of these matrices and showed 50% mortality of larvae.
S.K. Brar et al./Process Biochemistry 41 (2006) 323342 337

Liquid formulations have several advantages over dry lactose, sucrose and others [59,134]. With the progress in
formulations (Table 4), and especially encapsulated understanding of the actual and specific action of these
products lower the cost substantially due to close adjuvants, newer options ofamino acids,starch, ascorbic
juxtaposition of various mandatory additives like UV acid and non-conventional ones like cucurbitacins and
radiation screens and stickers in micro-quantities. In garbanzo beans were explored [60,135,136]. As interest
addition to these conventional categories, there are several escalated in these phagostimulants, some commercial ones
advances in formulations, which include sustained release like Coax, Entice, Gusto Konsume, Mo-Bait were
and use of synergists, or phagostimulants to enhance the introduced which increased feeding responses of pests
otherwise normal entomotoxicity. delivering higher entomotoxicity products in the field
[137,138]. Semiochemical-based toxic baits are another
3.5. Advances in formulations (for improved delivery) category of these types of formulations where a volatile
attractant or floral extract is
TheeffectivenessofBti,onaquaticorganisms,isgenerally addedtoformulationstofoolthepests.LanceandSutter[139]
dependent on bioavailability of the material which can be formulated a bait containing carbaryl, cucurbitacin, and
problematic in aqueous or sub-surface environments. For several non-pheromonevolatile attractants in dry,
example, entomopathogen may not remain in the region of bran/starch based carrier. Unfortunately, the formulation
interest, where the aquatic organisms are located, for a sank to the ground after 49days yieldingnegativeresults.
length Inyet another study, bait composed of a commercial
oftimesufficienttoprovidecompletetreatment.Akintosolid phagostimulant (Coax, Konsume, or Nu-Lure) and one of
formulations, controlled release formulations comprising several insect growth regulators (IGRs) was tested on fall
different carriers like polymers, charcoal, petroleum coke, army worm, Spodoptera frugiperda and this yielded
coke from coal, woody ring portions of corn cob, etc., can encouraging results especially with control rate of 90
be used with a coating of fatty acids and alcohols for 95% and reducing adult numbers than alternating swaths
regulating sustained release rate and profile into target [140,141]. Lately, addition of 675 mg/L monosodium
environment [128,129]. The proteinpolysaccharide glutamate to commercial formulation of Bt ssp. kurstaki,
complex (PPC) composition containing water-soluble (DiPel12X DF), lowered LC 50 from 450 to 150 mg/ L (P <
cellulose derivatives, seaweed polysaccharides such as 0.05, Lethal Ratio Significance Test), indicating its
alginate and carrageenin, seed mucilaginous potentialtoenhanceentomotoxicityandeconomyofBtbased
polysaccharides, complex plant exudate polysaccharides formulations [142].
such as gum Arabic, tragacanth, guar gum, pectin, ghatti, Moreover, lytic enzyme like chitinase could also increase
and microbially synthesized polysaccharides such as entomotoxicity by perforating the peritrophic membrane
xanthan gum have also been one of the choices for barrier in the larval midgut and thus increasing accessibility
controlled release formulations [130]. of Bt d-endotoxin molecule to its receptor on epithelial cell
In field application, issues like coverage and membranes [143]. Infact, synergism between chitinase and
application volume affect deposit structure of Bt Bt is as old as 1970s as also confirmed by growth studies of
formulations. Deposit efficacy has been a favorite and different subspecies of Bt on chitin [144]. Larvae of spruce
most controversial topic of researchers [89]. As droplet budworm, Choristoneura fumiferana, died more quickly
size is related to its radius by third order exponent, it when exposed to chitinase-Bt mixtures rather than Bt or
significantly affects deposits and in turn governs chitinase alone [145,146]. Similar observations were made
framework of new formulations. While application against the mortality of gypsy moth (Lymantria dispar)
methodologies producing large number of small droplets larvae and the toxic effect was correlated with enzyme
(spinning disk, rotary cage, etc.) often improve coverage, activity [147,148]. Different crude chitinase preparations
a concomitant increase in efficacy is not necessarily and endochitinases have constantly enhanced
observed [131,132]. entomotoxicity of Bt [149151].
There are other kinds of compounds that augment the
3.6. Booster formulations (enhanced entomotoxicity) entomotoxicity of Bt, antibiotics like zwittermicin, which
are similar in structure to the synergist molecule [152,153].
These formulations fall into a different class as there is Particles such as latex beads that adsorb solubilized
enhancement of entomotoxicity by either stimulant or polypeptides have even been used to raise the
synergistic action. It all began with preliminary gustatory entomotoxicity of Bti crystal proteins [154]. Additionally,
stimulation studies of Baculovirus based biopesticides for some optical brighteners like Tinopal LPW, known to be
bollworm control, which yielded encouraging three folds domain of viral biopesticides, when combined with Bt
increase in entomotoxicity [133]. Initial phagostimulant enhanced entomotoxicity and caused mortality of Colorado
studiesweremainlysugarbasedderivedfrompuresugarsviz. potato beetle larvae in relatively lesser time [155].
338 S.K. Brar et al./Process Biochemistry 41 (2006) 323342

Hence, addition of different phagostimulants, antibiotics, benzaldehyde, cinnamaldehyde, salicylaldehyde, methylene


optical brighteners and more recently chitinase producing Bt blue and yeast extract when employed as
hold the key to future Bt formulations. sunscreensforB.sphaericus,showedeliminationoflarvicidal
activity after 12 h UV irradiation [163].
Also, when efficacy of melanin for the protection of
4. Environmental effects mosquito larvicidal activity of Bt against UV radiation was
studied, the bioassays confirmed an important role of
As seen earlier, Bt formulations suffer short field melanin as a photoprotective agent [164]. The biggest
persistence owing to adverse environmental factors, which problem with melanin usage would be dissolution of the
needs to be understood completely before developing new pigment on exposure to rain. Recently, there has been a
formulations. study reported on Btk mutant producing melanin which on
irradiation at 254 nm and 366 nm, showed higher
4.1. Sunlight/UV radiation entomotoxicity than parent strain [165,166]. Thus,
utilization of such mutants with increased UV radiation
Natural sunlight, especially UV radiation portion of the resistance can aid in developing stable Bt formulations for
spectrum: UV-B (280310 nm) and UV-A (320400 nm) is field application.
mainly responsible for inactivation of insect pathogens. Based on the reported findings as outlined in Table 5,
Several authors have proposed the involvement of screening agents that absorb in 300400 nm range should
chromophores (exogenous, possibly endogenous too) enhance the persistence of foliar applied Bt products
derived from fermentation media, which after cell lysis, [161]. Several studies have been carried out with the
were adsorbed onto Bt crystal proteins [156158]. These incorporation of various UV radiation screens like Congo
chromophores, absorbing at 300380 nm, so far Red, folic acid, molasses, lignin, alginate, cellulose,
uncharacterized, passed excited, electronic-state energy to shellac yeast, p-amino benzoic acid with mixed results on
oxygen molecules, in turn converted to the highly reactive UV radiation protection [47,65,116,119,162,167172].
singlet, or free radical state. In this state, the singlet oxygen Certain fluorescent brighteners, especially compounds of
attacked indole side chains of tryptophan residues on the stilbene type, specifically, derivatives of 4,4 0-diamino-
protein toxin, resulting in loss of insecticidal activity 2,20-stilbene disulfonic acid and their salts enhanced
[157,158]. Cohen et al. [158] suggested the use of cationic biological activity up to 1000-fold and protected Bt from
moieties such as acriflavin and rhodamine B to transfer UV radiation exposure [173].
energy from excited tryptophan and act as UV radiation Further, UV radiation agents performed well with water
blocker. based Bt suspensions as discussed earlier. Recently, a
Effect of UV radiation also varies under different climate stateof-the-art technology has been developed involving
conditions,fore.g.,half-lifeofBtinUSAoncottonplantswas 30 double sheath a water-soluble protective coating and a
48 handinEgyptoncastorplants was1940 h[159,160]. Earlier hydrophobic oil carrier (suspending agent), for application
studies by Morris [161] used water soluble dyes, namely, DS to a target substrate. When such a formulation was applied
49 (benzophenone) and erio acid red (sodium salt of formyl- to a target substrate, oil component facilitated dispersal of
m-benzenedisulphonic acid) which increased the residual formulation over the substrate, resulting in adherence and
activity of Bt on white spruce trees by 2.9-fold. Dunkle and subsequent UV radiation protection [174].
Shasha [58,162] described a starch encapsulated Bt Nevertheless, encapsulation of biopesticidal materials
technique containing UV radiation screens against sunlight. in a matrix has been considered as the most effective form
Ragaei [160] utilized 2% solution of Congo Red which of formulation and as mentioned earlier, various coating
increased residual activity on castor plants 3.3-fold. Bartelt agents have been explored to achieve the same. Lately,
[59] demonstrated that if Coax (CCT, Lichtfield Park, AZ, there has been focus on utilization of protection as well as
USA), (a proprietary product used for corn borer larvae) was sustained release through microencapsulated formulations
incorporated in water dispersible granules; only 25% of the [175177]. Thus, UV radiation will have serious
entomotoxicity was lost in greenhouse tests. Subsequent repercussions on the product stability, which is considered
work carried out by McGuire et al. [104], documented a in development of advanced Bt formulations.
similar response by corn borer larvae under field conditions.
Bt granules (size, 150210 mm) with wheat meal used both
as a carrier and a feeding stimulant, against Earias insulana 4.2. Rainfall and dew
in cotton, gave excellent results against UV radiation, dew,
etc. [57]. The benefits of flour over starch include lower cost
and the protein may also act as a feeding stimulant, and/or a
sunlight screen [60]. Various organic compounds like
S.K. Brar et al./Process Biochemistry 41 (2006) 323342 339

Rainfall is another important natural component that carriers [180]. Such envelopes from the plant-adhering
would affect the persistence of biopesticides on foliage bacterium Pectobacterium cypripedi possess high sticking
leading to wash off before action. Behle et al. [116] abilities and have been used as effective carriers for
Table 5
Key findings on effects of UV radiation on entomopathogenic activity of Bt
Wavelength Test substance Effect Reference
(s)
250 nm Spores Lethal [247
249]
253.7 nm Spores; crystals Lethal to spores and no effect on crystals [250]
330 nm; 400 nm Spores; crystals Lethal [65,119]
300380 nm Crystal Inactivation of protein [58,156]
Sunlight (300750 nm) Spores Rapid inactivation [161,251]
Sunlight (300750 nm) Spores; crystals Spores more sensitive than crystals, but both inactivated in 3 days [168,252]
Visible light (400750 nm) Spores; crystals No effect [156,161]
reported that 3 cm of rain reduced efficacy by 20 percent chemical pesticides with 60% retention of toxicity after a
in Bt biopesticides. However, Ferro et al. [178] heavy simulated rain (84 mm) [181]. Therefore, in future
demonstrated that similar approach could be adopted for Bt based
little to no loss in activity of Bt ssp. tenebrionis was biopesticides.
observed after 2.5 cm of simulated rain if rain fell after the In toto, rains can significantly affect the activity of
insecticidal application had dried (approximately 15 min) biopesticides by wash-off before action and the need to
[178]. Heavy dew also decreased Bt efficacy on foliage. add stickers has to be studied comprehensively. Perhaps,
Rainfall may be even more important in product degradation encapsulated formulations could provide a better solution.
than light when foliage is shaded from direct sunlight
(forestry, whorl of corn plants) [179]. 4.3. pH
Several different approaches to formulation appear to
have improved the rainfastness of Bt products. LiteratureindicatesthatBtactivityisstableabovepH3and
Experimental cornstarch based formulations showed below 11 [80,182]. As the length of exposure increased,
increased activity following rainfall, possibly because of the sensitivity of Bt to extreme pHs also increased.
the ability of cornstarch to stick onto crop foliage [115] Similarly, as temperature increased, Bt sensitivity to
and similar results were shown by maize flour extreme pHs also increased. It is likely that the optimal
microcapsules [120]. Similarly, Pseudomonas fluorescens pH range for Bt is considerably narrower than pH 3 to 11
cells genetically engineered to produce Bt endotoxins under typical field conditions where high temperatures
showed improved rainfastness, possibly due to the frequently prevail [167].
presence of polysaccharides, proteins, and glycoproteins Normally, commercial Bt products are buffered in the
present in the Pseudomonas cell wall that help it adhere to region of pH 45, to inhibit growth of most microbial
crop foliage [6]. Conventional Bt products, in contrast, are contaminants and possible breakdown of crystal proteins
composed of lysed spores, cell debris and crystals, and by alkaline proteases that can lead to lowering of activity
thus cannot take advantage of any cell wall adhesiveness [131]. In practice, few end-users are plagued with highly
possessed by Bacillus cells. acidic water, but water pHs of 10 are unusual in some
This drawback has fuelled the development of new regions. For most commercial Bt products, end-users are
commercial products within the past several years. For advised to add buffering agents to their spray tanks when
foliar applications, Mycogen Corporations development water pH is higher than 9 due to utilization of
of the availablewater resources (already at alkaline pH).
CellCap7encapsulationsystemhasimprovedthestickinessof Recently, wheat gluten based formulations have been
spray applications, thus enhancing rainfastness [99101]. developed that could sustain the entire pH range 311
From a practical approach, it has been common practice [64,65].
amongproducerstoaddsugar,molasses,oilsorcommercially Effects of pH are mostly encountered while loading the
availablestickingagentstotheirBtspraytanks.Theimproved spray mixtures into containers (aluminum or iron) which
performance may be due to the sticking ability imparted could get corroded and afflict effective field application of
by these additives and there is a tendency to use multi- the biopesticides [6] and this necessitated use of buffering
purpose agents for better economy. Bacterial ghosts, agents. The pH has been also found to ruin the formulation
which represent empty cell envelopes of Gram-negative performance during storage (action of proteases) as well as
bacteria, have been application (tank corrosion) and post-application (foliage
appliedsuccessfullyasvaccinecandidatesoraspotentialdrug action discussed later) stage.
340 S.K. Brar et al./Process Biochemistry 41 (2006) 323342

4.4. Temperature bacterial persistence observed in laboratory tests conducted


on artificial diet or leaf discs does not translate to outdoor
Temperature plays an important role in shelf life as well field experiments. Hence, in addition to influencing the
as post-application persistence of Bt formulations. It has outcome of field tests, the effect of leaf growth and
been observed that temperatures lower than 10 8C and expansion should be taken into account when product rates,
higher than 30 8C may have deleterious effects on the application timing and application frequency protocol is
activity of bacterial pathogens over an extended period of developed, with higher rates and increased application
time [157]. This may be caused by degradation of the a.i. by frequencies recommended for fast growing agricultural
heat, or more likely, by reduced feeding of insects because crops.
of high or low temperature extremes [183]. In crop Certain chemicals present on the phylloplane and soil can
protection and vector control, temperature extremes are also affect the efficacy of product, for instance cotton
commonly encountered. For example, applications of Bti exudates Mg2+, Ca2+ and Mn2+ as carbonates and bicarbonates
made against mosquitoes, in Canada, are used in early raise the pH as high as 10 or 11, and this could affect
spring, when water temperatures are near 1015 8C when performance of the formulations [192]. As Bt endotoxin is a
larval feeding and digestive activity is low to allow digestive tract poison, leaf extract would have the least
significant activity of Bt spores and crystals [184]. The effect [6]. To overcome pH problems, casein, gluten and
feeding rates of mosquito larvae are similarly reduced by tannic acid based formulations were developed [64,65,193].
temperatures of 5 8C [185]. Thus, the most effective way to counteract foliage pH is by
Higher temperatures have also resulted in decreased applying well planned spray timing strategy and
activity of Bt products, especially in the tropics, where incorporating buffers either in the formulation or in the tank
temperatures frequently exceed 30 8C [161]. In addition to mix.
possible inactivation of the toxin, high temperatures resulted Extensive review by Joung and Cote [194] has
in decreased feeding rates for many insects and enhanced established that Bt would not have any significant impact on
deleterious effects of pH [182]. A method to counteract the the non-target microflora in soil, water and foliage under
effect of adverse temperatures is to select Bt strains active field conditions.
over a wide range of temperatures and use of encapsulated
formulations. Hence, both extremes of temperature can
decrease the residual activity of Bt formulations. 5. Wastewater/wastewater sludge based Bt formulations

4.5. Foliage Wastewater/wastewater sludge (WW/WWS) has been


successfully used as a raw material for Bt biopesticide
Biological activity of Bt formulations on foliage is production with lower process costs [195205]. However,
shortlived, with half-life ranging from 1 to 2 days on the formulation costs are yet to be accounted for in the
unshaded foliage [156] and 20 to 30 days on shaded foliage overall Bt production process. The WW/WWS based Bt
[186]. In addition to environmental factors (such as sunlight biopesticides encompass advantages and drawbacks over
and rain), two other factors leaf expansion and the commercial biopesticides. Two principal problems
presence of secondary plant compounds have a significant associated with the use of wastewater sludge are the
impact on persistence of Bt on foliage. Specifically, volatiles presence of toxic heavy metals and pathogens. As sludges
like aldehydes, ketones, carboxylic acids and their are completely sterilized prior to Bt fermentation, all
derivatives present on these leaves have an antibiotic effect pathogens are eliminated. Further, sludges used for Bt
on Bt spores and sometimes, cause of its inactivation or production meet the regulatory criteria prescribed by the
delay in normal growth [187189]. Quebec Environment Ministry [206] and about 50% of total
During evaluation of bacterial persistence, leaf growth sludge (1 million tonnes of dry sludge solids per year)
and expansion are normally overlooked parameters. For fast produced in Canada meets the regulatory criteria. This
growing plants such as leafy vegetables, tomatoes, potatoes mitigates the problem of metals. The quantity of Bt based
or cotton, it takes only a few days for bacterial formulations sludge to be sprayed in field (about 50 g for a potency of
to become significantly diluted by the development of new, 76B) would be very low compared to the amount of sludge
untreated leaf area [190]. In fact, it has been reported that Bt permitted for agriculture land application (1530 tonnes per
products appear to persist longer in forests (residual activity hectare depending on N, P, K content) and hence the risk of
of 2030 days) than in agricultural settings (hours to few metal contamination is almost nil [205].
days for row crops) [86,191]. The reasons are unclear but it The harvesting methods for WW/WWS based Bt
could be caused by shading from direct sunlight, as well as biopesticides may not require filter aids as sludge contains
lack of leaf expansion in forest fauna. This dilution effect flocs (average particle size of non-hydrolyzed, hydrolyzed
may be one of the most important reasons that improved sludge and was 17.8, 8.6, 4.1 and 2 mm, respectively),
S.K. Brar et al./Process Biochemistry 41 (2006) 323342 341

which will behave as adsorbents for spores and crystal activity (1.1 IU/mL) as compared to starch wastewater
protein during centrifugation and as protectants during (1.8 IU/mL) and soya (1.3 IU/mL). The high protease
adverse spray drying conditions [207,208]. However, activity in latter formulations would have caused
advanced harvesting techniques (for example, UF) might inactivation of crystal protein, hence decreased
need further research as membrane fouling and cake entomotoxicity and lower half-life.
formation could occur due to complex rheology [under Thus, UV resistance results gave better performance of
investigation in our laboratory]. Furthermore, higher the sludge based Bt formulations in comparison to half-
entomotoxicity was obtained in Bt fermented sludge life of conventional Bt formulations in field conditions
compared to semi-synthetic medium [200205]. This may ranging from 16 h to 2 days [169]. Additionally, domestic
require moderately concentrated broths for formulation sludge has been reported to have components with
amendment reducing solids concentration and hence chromopheric compounds or auxochromes (majority of
increasing suspendibility. fulvic, humic and hymathomelanic acids) with absorbance
Furthermore, during Bt formulation development, Bt at 350 nm [211]. These components would have acted as
fermented sludge produced chitinases, which would natural UV radiation screens in sludge based formulations.
enhance entomotoxicity (data unpublished) and there This warrants further studies to validate the effect of UV
would be no need to add external source of chitinases as radiation screens in formulations (conclusively, addition
done in several synthetic media derived formulations of UV radiation screens must further extend the residual
(discussed earlier). The WW/WWS based Bt formulations entomotoxicity). Hence, sludge based Bt biopesticides can
possess higher rainfastness property (preliminary studies be an important addition to Bt product profile as they
have yielded encouraging results, data unreported) as there would be more cost effective than the synthetic medium
is presence of extra-cellular polymeric substances (EPS) based biopesticides.
known to play an important role in biofilm formation
[209]. The EPS could act as stickers resulting in better
adherence. Moreover, due to the natural encapsulation of 6. Recapitulation and research priorities
spores and crystals in sludge flocs, the Bt formulations
were resistant to variable temperatures [210]. The biopesticide production is an integrated and
As is evident from Fig. 2, WW/WWS formulations interlinked process. Hence, rational biopesticide
even without addition of UV radiation screens performed development/delivery would comprise formulation type;
better than commercial formulation. The half-lives of synergy with application equipment; spatial targeting and
timing of application, in addition to use of smart
alternatives like costeffective media based formulations
(sludge based formulations, discussed earlier). Despite
extensive literature on formulations, no study has been
reported on either the formulations of Bt fermented
products based on waste/ agricultural by-products and/or a
complete process from fermentation until formulation.
Rheology of fermented broth will be decisive for
downstream processing steps to give a consistent product,
which will exert considerable influence on application and
ultimately, the efficacy. Despite considerable research, it is
not yet fully comprehensible if there is a dependence of
Fig. 2. Half lives for different Bt formulations (without UV radiation the biological properties of a microbial biopesticide
screen) on UV radiation exposure. formulation upon its physical properties, yet they are
formulations followed the order: raw sludge > interdependent [6]. Thus, development of formulations
hydrolyzed sludge > starch wastewater > soya and were must take into account the biotic (spore concentration and
reported to be (in days) 11.14 > 9.51 > 9.02 > 2.8. T0.5 entomotoxicity) and abiotic factors (UV radiation,
(half-life) of nonhydrolyzed sludge was higher probably temperature, pH, rain, foliage and others) for better
due to the flocs (average particle size, 17.8 mm) that delivery. Furthermore, different parameters and additives
provided protective sheath around crystal protein and for specific type of formulation (e.g., to avoid cake
spores reducing their inactivation and hence better formation in dry products, hygroscopic material must be
residual entomotoxicity. Hydrolyzed sludge was better avoided). Also, possibility of contamination and
than starch wastewater and soya probably due to two degradation of crystal protein by proteases during storage
reasons: higher floc size (8.6 mm) which formed a has to be minimized (pH changes) and hence strongly
protective sheath (discussed earlier) and lower protease acidic solutions are preferred. Distinct features suggest a
342 S.K. Brar et al./Process Biochemistry 41 (2006) 323342

practical place for both solid and liquid Bt formulations: [5] Copping L. The biopesticides manual. UK: British Crop Protection
in fact, both sell nearly equally. Council, 1998. p. 333.
[6] Burges HD, editor. Formulation of microbial biopesticides: beneficial
Within the limits of biological requirements of Bt, as well organisms, nematodes and seed treatments. Dordrecht, The Netherlands:
as the economic reality of production cost, the fermentation Kluwer Academic Publishers, 1998.
process has to be tailored to meet the needs of subsequent [7] Chapple AC, Downer RA, Bateman RP. Theory and practice of microbial
formulations. insecticide application. In: Lacey LA, Kaya HA, editors. Field manual of
techniques in invertebrate pathology. Dordrecht: Kluwer; 2000. p. 537.
[8] Parekh S, Vinci VA, Strobel RJ. Improvement of microbial strains and
fermentation processes. Appl Microbiol Biotechnol
7. Conclusions 2000;54:287301.
[9] Cords H, Fisher RA. Stable concentrated bacterial insecticide
Bt based biopesticidal formulations will find wider suspensions. US Patent 3,271,243; 1966.
application in future by adopting simple harvesting methods [10] Priest FG. Biological control of mosquitoes and other biting flies by
and robust and economical choice of various additives for Bacillus thuringiensis and Bacillus sphaericus. J Appl Bacteriol
different formulations. In general, there are two types of 1992;72:35769.
[11] Bernhard K, Utz R. Production of Bacillus thuringiensis for industrial
formulationssolid and liquid, including advances like
and commercial uses. In: Entwistle PF, Cory JS, Baily MJ, Higgs S,
encapsulations and both have a distinct application and editors. Bacillus thuringiensis: an environmental biopesticide: theory and
hence equal markets. Various environmental factors, namely, practice. Great Britain: Wiley Pubs; 1993. p. 255 67.
UV radiation, temperature, wind, pH and rain influence the [12] Rowe GE, Margaritis A. Bioprocess design and economic analysis for
field efficiency of Bt formulations. From late 1990s to the commercial production of environmentally friendly bioinsecticides
recently, Bt formulation trends have progressed in the from Bacillus thuringiensis HD-1 kurstaki. Biotechnol Bioeng
2004;86(4):37788.
direction of maximum efficacy per drop resulting in high
[13] Keller K, Friedmann T, Boxman A. The bioseparation needs for
potency concentrates requiring lower spray volumes. tomorrow. Trends Biotechnol 2001;19(11):43841.
Wastewaterand/wastewatersludgebasedformulationsmayhold [14] Dulmage HT. Insecticidal activity of HD-1, a new isolate of Bacillus
the key to various problems encountered by commercial thuringiensis var. alesti. J Invertebr Pathol 1970;15:2329.
medium based formulations aiding in two ways [15] Christy C, Vermant S. The state-of-the-art of filtration in recovery
sustainable reuse of wastes and enhanced penetration of Bt processes for biopharmaceutical production. Desalination 2002; 147:I-4.
[16] Darnon E, Morin E, Belleville MP, Rios GM. Ultrafiltration within
biopesticides into global pesticide market. This will also
downstream processing: some process design considerations. Chem Eng
greatly expand the repertoire of commercial Bt product Process 2003;42:299309.
types. [17] Lertcanawanichakul M, Wiwat C. Improved shuttle vectors for
expression of chitinase gene in Bacillus thuringiensis. Lett Appl
Microbiol 2001;2:1238.
Acknowledgements [18] Arora N, Ahmad T, Rajagopal R, Bhatnagar RK. A constitutively
expressed 36 kDa exochitinase from Bacillus thuringiensis HD-1.
Biochem Biophy Res Commun 2003;307:6205.
The authors are sincerely thankful to Natural Sciences [19] Soper RS, Ward MG. Beltsville Symposia in Agricultural Research. Biol
and Engineering Research Council of Canada (Grants Cont Crop Production 1981;5:16180.
A4984, STP235071, Canada Research Chair) for financial [20] Sharpe ES, Herman AI, Toolan SC. Foam flotation process for separating
support. We are also thankful to the Natural Sciences and Bacillus thuringiensis sporultion products. United States Patent
Engineering Research Council of Canada and Canadian 4,247,644; 1981.
[21] Zhou X, Chen S, Yu Z. Effects of spray drying parameters on the
Forestry Service for providing Ph.D. scholarship to Satinder
processing of a fermentation liquor. Biosyst Eng 2004;88(2):1939. [22]
K. Brar during the course of this research work. Bonnefoi A. Method for producing biological pest destroying agents. US
Patent 3,071,519; 1963.
[23]Cords H, Fisher RA. Stable concentrated bacterial insecticide suspensions.
References US Patent 3,271,243; 1966.
[24]Taborsky V. Small-scale processing of microbial pesticides. FAO
[1] Marrone PG. Microbial pesticides and natural products as alternatives. Agricultural Services Bulletin No. 96. Rome: Food and Agriculture
Outlook Agric 1999;28(3):14954. Organization of the United Nations; 1992.
[2] Cannon RJC. Prospects and progress for Bacillus thuringiensis based [25]Tamez-Guerra P, McGuire MR, Medrano-Roldan H, Galan-Wong LJ.
pesticides. Pestic Sci 1993;37:3315. Sprayable granule formulations of Bacillus thuringiensis. J Econ
[3] Chapple AC, Bateman RP. Application systems for microbial pesticides: Entomol 1996;89:142430.
necessity not novelty. In: Microbial Insecticides: Novelty or Necessity? [26]Rojas JV, Gutierrez E, De la Torre M. Primary separation of the
British Crop Protection Council Proceedings/Monograph Series No. 68; entomopathogenic products of Bacillus thuringiensis. Biotechnol Prog
1997, p. 18190. 1996;12:5646.
[4] Jacobsen BJ, Backman PA. Biological and cultural plant disease [27]Zamola B, Valles P, Meli G, Miccoli P, Kajfez F. Use of the centrifugal
controls: alternatives and supplements to chemicals in IPM systems. separation technique in manufacturing a bioinsecticide based on Bacillus
Plant Dis 1993;77:3115. thuringiensis. Biotechnol Bioeng 1981;XXIII: 107986.
S.K. Brar et al./Process Biochemistry 41 (2006) 323342 343

[28]Dulmage HT, Rhodes RA. Production of pathogens in artifical media. In: Adjuvants for Agrochemicals, Chemical Producers Distributors
Burges HD, Hussy NW, editors. Microbial control of insects and mites. Association, Memphis, TN; 1998. p. 45162.
New York: Academic Press; 1971. p. 50740. [50]McGaughey WH. Insect resistance to the biological insecticide Bacillus
[29]Tzeng YM, Tsun HY, Chang YN. Recovery of thuringiensin with thuringiensis. Science 1985;229:1935.
cetylpyridinium chloride using micellar-enhanced ultrafiltration process. [51]Sundara-Babu PC, Krishnan A. Preliminary studies on the effect of
Biotechnol Prog 1999;15:5806. Thuricide1 for control of the semi-looper, Achaea janata, on the castor
[30]Markels JH, Lynn S, Radke CJ. Cross-flow ultrafiltration of micellar bean plant, Ricinus communis. J Invertebr Pathol
surfactant solutions. AIChE J 1995;41(9):205866. 1970;15:12930.
[31]Agrawal A, Burns MA. Application of membrane-based preferential [52]Lynch RE, Robinson JF, Berry EC. European corn borer: yield losses and
transport to whole broth processing. Biotechnol Bioeng damage resulting from a simulated natural infestation. J Econ Entomol
1996;55(4):58191. 1980;73:1414.
[32]Persson A, Jonsson AS, Zacchi G. Use of microfiltration as first step in [53]Harris J, Dent D. Priorities in biopesticide research and development in
recovery of protein from a fermentation broth. Appl Biochem Biotechnol developing countries. Wallingford, UK: CABI Publishing, 2000.
2004;112(3):15162. [54]Ifoulis AA, Savopoulou-Soultani M. Biological control of Lobesia botrana
[33]Russotti G, Osawa AE, Sitrin RD, Buckland BC,Adams WR, Lee SS. Pilot (Lepidoptera: Tortricidae) larvae by using different formulations of
scale harvest of recombinant yeast employing microfiltration: a case Bacillus thuringiensis in 11 vine cultivars under field conditions. J Econ
study. J Biotechnol 1995;42:23546. Entomol April 2004;97(2):3403.
[34]Lee CT, Morreale G, Middelberg APJ. Combined in-fermenter extraction [55]Green JM. Adjuvant outlook for pesticides. Pestic Outlook. October 2000;
and cross-flow microfiltration for improved inclusion body processing. p. 1969.
Biotechnol Bioeng 2004;85(1):10313. [56]Mayer W, Wassmer C, Doerr S. Solid formulations. United States Patent
[35]Wong HH, ONeill BK, Middelberg AP. Centrifugal processing of cell 6,030,924; 2000.
debris and inclusion bodies from recombinant Escherichia coli. [57]Navon A, Keren S, Levski S, Grinstein A, Riven Y. Granular feeding baits
Bioseparation 1996;6(6):36172. based on Bacillus thuringiensis products for the control of lepidopterous
[36]Tsuji K. Recent trends in pesticide formulations. In: Foy CL, Pritchard DW, pests. Phytoparasitica 1997;25(Suppl.):101S10S.
Beestman GB, editors. Formulation science, vol. I. Association of [58]Dunkle RL, Shasha BS. Starch encapsulated Bacillus thuringiensis: a
Formulation Chemists; 1997. p. 5383. potential new method for increasing environmental stability of
[37]Cross JV, Poloneko DR. An industry perspective on registration and entomopathogens. Environ Entomol 1988;17:1206.
commercialization of biocontrol agents in Canada. Can J Plant Pathol [59]Bartelt RJ, McGuire MR, Black DA. Feeding stimulant for the corn borer
1996;18:44654. (Lepidoptera: Pyralidae): additives to a starch based formulation for
[38]Frey PM. Biocontrol agents in the age of molecular biology. Trends Bacillus thuringiensis. Environ Entomol 1990;19(1):1829.
Biotechnol 2001;19(11):4323. [60]Gillespie RL, McGuire MR, Shasha BS. Palatability of flour granular
[39]Matthews GA. Pesticide application methods, 2nd ed., Harlow: Longman formulations to European corn borer larvae (Lepidoptera: Pyralidae). J
Scientific and Technical, 1992. Econ Entomol 1994;87:4527.
[40]Hall FR, Chapple AC, Taylor RAJ. Downer RA. Biorational pest control [61]Tamez-Guerra P, Castro-Franco R, Medrano-Roldan H, McGuire MR,
agents formulation and delivery. In: ACS Symposium Series 595. 1995. Galan-Wong LJ, Luna-Olvera HA. Laboratory and field comparisons of
p. 6878. strains of Bacillus thuringiensis for activity against noctuid larvae using
[41]Hall FR, Menn JJ. Biopesticides: use and delivery. New Jersey, USA: granular formulations (Lepidoptera). J Econ Entomol 1998;91:8693.
Humana Press, 1999. [62]Yang C-C, Pan I-H, Chen M-H, Kao S-S, Tsai Y-S. Method for the
[42]Rodham DK, Wang Y, Cantwell JB, Winn PD, Foundling J. Formulating preparation of a biocidal formulation containing starch and Bacillus
microbial biocontrol agents. Pestic Sci 1999;55: 3402. thuringiensis. United States Patent 5,352,597; 1994.
[43]Behle RW, Tamez-Guerra P, Shasha BS, McGuire MR. Formulating [63]Shasha BS, McGuire MR. Adherent starch granules. United States Patent
bioinsecticides to improve residual activity. Formulations Forum99. 5,997,945; 1999.
Orlando, FL, USA; March 35, 1999. [64]Behle RW, McGuire MR, Shasha BS. Extending the residual activity of
[44]U.S. EPA. Inert ingredients in pesticide products: Policy statement; Bacillus thuringiensis with casein based formulations. J Econ Entomol
Revision and modification of lists, 52 FR 13305 and 54 FR 48314; 1989. 1996;89:1399405.
November 22. [65]Behle RW, McGuire MR, Gillespie RL, Shasha BS. Effects of alkaline
[45]Fortin C, Lapointe D, Charpentier G. Susceptibility of brook trout gluten on the insecticidal activity of Bacillus thuringiensis. J Econ
(Salvelinus fontinalis) fry to a liquid formulation of Bacillus Entomol 1997;90:35460.
thuringiensis serovar. israelensis (Teknar1) used for blackfly control. Can [66]Ridgway RL, Illum RR, Farrar Jr RR, Calvin DD, Fleischer SJ, Inscoe MN.
J Fish Aq Sci 1986;43:166770. Granular matrix formulation of Bacillus thuringiensisfor control of the
[46]Organic Materials Review Institute (OMRI). Comments on the National European corn borer (Lepidoptera: Pyralidae). J Econ Entomol
Organic Program Rule Proposed by the US Department of Agriculture 1996;89(5):108894.
TMD-94-00-2. Eugene, OR: OMRI; 1998. [67]Maldonado Blanco MG, Galan Wong LJ, Rodriguez Padilla C, Quiroz
[47]Rhodes DJ. Formulation of biological control agents. In: Jones DG, editor. Martinez H. Evaluation of polymer-based granular formulations of
Exploitation of microorganisms. London: Chapman and Hall; 1993. p. Bacillus thuringiensis israelensis against larval Aedes aegypti in the
41139. laboratory. J Am Mosq Control Assoc December
[48]Couch TL, Ignoffo CM. In: Burges HD, editor. Formulation of insect 2002;18(4):3528.
pathogens, in microbial control of pests and plant diseases. London: [68]Sulaiman S, Jeffery J, Sohadi AR, Yunus H, Busparani V, Majid R.
Academic Press; 1981. p. 62134. Evaluation of Bactimos wettable powder, granules and briquets against
[49]Hewitt AJ. The effect of tank mix and adjuvants on spray drift. In: mosquito larvae in Malaysia. Acta Trop 1990;47:18995.
McMullan PM, editor. Adjuvants for agrochemicals: challenges and [69]Quimby Jr PC, Caesar AJ, Birdsall JL, Connick Jr WJ, Boyette CD, Zidack
opportunities. Proceedings of the Fifth International Symposium on NK, et al. Granulated formulation and method for stabilizing biocontrol
agents. United States Patent 6,455,036; 2002.
344 S.K. Brar et al./Process Biochemistry 41 (2006) 323342

[70]Andersch W, Hain R, Kilian M. Granulates containing micro-organisms. [92]Lacey LA. Production and formulation of Bacillus sphaericus. Mosq News
United States Patent 5,804,208; 1998. 1984;1539.
[71]Becker N. Ice granules containing endotoxins of microbial agents for the [93]Cyanamid. Acrobe1 Biolarvacide: Technical Information, field trial
control of mosquito larvae-a new application technique. J Am Mosq summary, Wayne, New Jersey: American Cyanamid Company; 1992.
Control Assoc March 2003;19(1):636. [94]Narayanan KS, Ianniello RM, Pritchard DW. UV protective aqueous
[72]Skovmand O, Sanogo E. Experimental formulations of Bacillus sphaericus emulsion and emulsifiable solids for cosmetic and agrichemical
and B. thuringiensis israelensis against Culex quinquefasciatus and formulations. United States Patent 5,597,574; 1997.
Anopheles gambiae (Diptera: Culicidae) in Burkina Faso. J Med [95]Aven M, Hasui H, Motoyoshi M. Non-aqueous concentrated spreading oil
Entomol 1999;36:627. composition. United States Patent 6,387,848; 2002.
[73]Gunasekaran K, Prabakaran G, Balaraman K. Efficacy of a floating [96]Aven M. Non-aqueous suspension concentrate. United States Patent
sustained release formulation of Bacillus thuringiensis subsp. israelensis 6,165,940; 2000.
in controlling Culex quinquefasciatus larvae in polluted water habitats. [97]Langley J, Symes KC. Polymeric compositions and methods of producing
Acta Trop 2002;83:2417. them. US Patent 5,744,152; 1998.
[74]Levy R, Nichols MA, Miller Jr TW.. Culigel superabsorbent polymer [98]Bok SH, Lee HW, Son KH, Kim SU, Lee JW, Kim DY, et al. Process for
controlled-release system: application to mosquito larvicidal bacilli. In: preparing coated microbial pesticides and pesticides produced therefrom.
Proceedings and abstracts, Vth international colloquium on Invertebrate US Patent 5,273,749; 1993.
Pathology and Microbial Control; 1990.p. 107. [99]Barnes AC, Cummings SG. Cellular encapsulation of biological pesticides.
[75]Mittal PK. Biolarvicides in vector control: challenges and prospects. J United States Patent 4,695,462; 1987.
Vector Borne Dis 2003;40(12):2032. [100] Barnes AC, Cummings SG. Cellular encapsulation of pesticides
[76]Bradbury RS, Quinlan RJ, Most BH. Pesticidal composition for water produced by expression of heterologous genes. United States Patent
treatment. United States Patent 4,865,842; 1989. 4,695,455; 1987.
[77]Aly C, Mulla MS, Schnetter W, Xu BZ. Floating bait formulations increase [101] Barnes AC, Edwards DL. Cellular encapsulation of biologicals for
effectiveness of Bacillus thuringiensis var. israelensis against Anopheles animal and human use. United States Patent 4,861,595; 1989.
larvae. J Am Mosq Control Assoc December [102] Ahmed SM, Nagamma MV, Majumdar SK. Studies on granular
1987;3(4):5838. formulation of Bacillus thuringiensis Berliner. Pestic Sci 1973; 4:1923.
[78]Goldberg LJ. Carrier and dispersal mechanism for a microorganic larvicide. [103] Baker CA, Brooks AA, Greenlay AZ, Hennis JMS. Encapsulation
United States Patent 4,187,290; 1980. method. European Patent 0320483; 1987.
[79]Yang KW, Junginger GJ, Mockridge RG, Pearce, III RC. Compositions of [104] McGuire MR, Shasha BS, Lewis LC. Field evaluations of granular starch
gypsum pesticide briquets. United States Patent 5,747,054; 1998. formulations of Bacillus thuringiensis against Ostrinia nubilalis
[80]Jones KA, Burges HD. Formulation of bacterial, viruses and protozoa to (Lepidoptera: Pyralidae). J Econ Entomol 1990;83:220710.
control insects. In: Burges HD, editor. Formulation of microbial [105] Baker CA, Brooks AA, Greenley RZ, Henis JM. Encapsulation of
biopesticides: beneficial micro-organisms, nematodes and seed biological material in non-ionic polymer beads. United States Patent
treatments. Dordrecht, Netherlands: Kluwer Academic Publishers; 1998. 5,089,407; 1992.
p. 7127. [106] Clark FM, Offit PA, Speaker TJ. Aqueous solven encapsulation method,
[81]Medugno CC, Ferraz JMG, Maia AHN, Freitas CCL. Evaluation of a apparatus and microcapsules. United States Patent 6,531,156; 2003.
wettable powder formulation for the nuclear polyhedrosisvirusof [107] Shasha BS, Dunkle RL. Starch encapsulation of entomopathogens. US
Anticarsia gemmatalis (Lep.: Noctuidae). Pest Sci 1997;51:1536. Patent 4,859,377; 1989.
[82]Teera-Arunsiri A, Suphantharika M, Ketunuti U. Preparation of spray-dried [108] Eden J, Trksak R, William R. Starch based particulate encapsulation
wettable powder formulations of Bacillus thuringiensis-based process. US Patent 4,755,397; 1988.
biopesticides. J Econ Entomol 2003;96(2):2929. [109] Shasha BS, McGuire MR. Adherent, autoencapsulating spray
[83]Valent Biosciences website, http://www.valent.com/agricultural_ products/, formulations of biocontrol agents. US Patent 506,197; 1991.
cited, 2 January, 2004. [110] Bok SH, Kim SU, Kwon YK. Bioencapsulated biopesticides and process
[84]Novartis Corp. website, http://www.novartis.com/, cited, 2 January, 2004. for the manufacture thereof. Canadian Patent CIPO 2,118,267; 1994.
[85]Hazen JL. Adjuvantsterminology, classification, and chemistry. Weed [111] Cheung PYK. Hammock BD. Micro-lipid-droplet encapsulation of
Technol 2000;14:77384. Bacillus thuringiensis subsp. israelensis d-endotoxin for control of
[86]Witt WW. Adjuvants. Agripedia: University of Kentucky College of mosquito larvae. Appl Environ Microbiol 1985;50(4):9848.
Agriculture. http://www.ca.uky.edu/agripedia/pls404/adjuvant.htm; 2001,
[112] Schnell DJ, Pfannenstiel MA, Nickerson KW. Bioassay of solubilized
cited 3 November, 2003.
Bacillus thuringiensis var. israelensis crystals by attachment to latex
[87]Underwood AK. Adjuvant trends for the new millennium. Weed Technol beads. Science 1984;223:11913.
2000;14:76572.
[113] Shasha BS, McGuire MR. Slow release formulations of pesticides. In:
[88]Valero JR. Three years of experimental aerial application of Bacillus Chasin DG, Bode LE, editors. Pesticide formulations and application
thuringiensis at 2.01/ha against Choristoneura fumiferana, Information systems. Philadelphia: American Society for Testing and Materials, 1992.
Report LAU-X-89B. Quebec, Canada: Forestry Canada, 1989.
[114] McGuire MR, Shasha BS. Adherent starch granules for encapsulation of
[89]Smirnoff WA, Valero JR. Characteristics of a highly concentrated Bacillus insect control agents. J Econ Entomol 1992;85(4):142533.
thuringiensis formulation against spruce budworm, Choristoneura
[115] McGuire MR, Shasha BS, Lewis LC, Nelson TC. Residual activity of
fumiferana (Lepidoptera: Tortricidae). Can Ent 1983; 115:4434.
granular starch encapsulated Bacillus thuringiensis. J Econ Entomol
[90]Cibulsky RJ, Devisetty BN, Melchior GL, Melin BE. Formulation and 1994;84:6317.
application technologies for microbial pesticides: review of progress and
[116] Behle RW, McGuire MR, Shasha BS. Effects of sunlight and simulated
future trends. J Test Eval 1993;21:5003.
rain on the residual activity of Bacillus thuringiensis formulations. J
[91]Bauce E, Carisey N, Dupont A, van Frankenhuyzen K. Bacillus Econ Entomol 1997;90:150616.
thuringiensis subsp. kurstaki aerial spray prescriptions for balsam fir
[117] Navon A. Control of Lepidopteran pests with Bacillus thuringiensis. In:
stand protection against spruce budworm (Lepidoptera: Torstricidae). J
Entwistle PF, Cory JS, Bailey MJ, Higgs S, editors. Bacillus
Econ Entomol 2004;97(5):162434.
S.K. Brar et al./Process Biochemistry 41 (2006) 323342 345

thuringiensis, an environmental biopesticide: theory and practice. New [139] Lance DR, Sutter GR. Field tests of a semiochemical-based toxic bait for
York: Wiley; 1993. p. 12546. suppression of corn rootworm beetles (Coleoptera: Chrysomelidae). J
[118] Ramos LM, McGuire MR, Galan Wong LJ. Utilization of several Econ Entomol 1992;85:96773.
biopolymers for granular formulations of Bacillus thuringiensis. J Econ [140] Chandler LD. Use of feeding stimulants to enhance insect growth
Entomol 1998;91:110913. regulator-induced mortality of fall army worm (Lepdioptera: Noctuidae)
[119] Shasha BS, McGuire MR, Behle RW. Lignin-based pest control larvae. Florida Entomol 1993;76:31626.
formulations. US Patent 575,0467; 1998. [141] Chandler LD. Comparison of insecticide-bait aerial application methods
[120] Tamez-Guerra P, McGuire MR, Behle RW, Shasha BS, Galan-Wong LJ. for management of corn rootworm (Coleoptera: Chrysomelidae).
Assessment of microencapsulated formulations for improved residual Southwestern Entomol 1998;23:14759.
activity of Bacillus thuringiensis. J Econ Entomol 2000;93:21925. [142] Pszczolkowski MA, Brunner JF, Doerr MD, Brown JJ. Enhancement of
[121] Cote JC, Vincent C, Son KH, Bok SH. Persistence of insecticidal Bacillus thuringiensis with monosodium glutamate against larvae of
activity of novel bioencapsulated formulations of Bacillus thuringiensis obliquebanded leafroller (Lep.: Tortricidae). J Appl Entomol
var. kurstaki against Choristoneura rosaceana [Lepidoptera: Tortricidae]. 2004;128(7):474.
Phytoprot 2001;82:7382. [143] Kramer KJ, Muthukrishnan S. Insect chitinases: molecular biology and
[122] Winder RS, Wheeler JJ, Conder N, Otvos IS, Nevill R, Duan L. potential uses as biopesticides. Insect Biochem Mol Biol
Microencapsulation: a strategy for formulation of inoculum. Biocont Sci 1997;27(11):887900.
Technol 2003;13:15569. [144] Smirnoff WA, Valero JR. Determination of the chitinolytic activity of
[123] Lew CW, Branly K, Gaytan J. Encapsulation with water soluble polymer. nine subspecies of Bacillus thuringiensis. J Invertebr Pathol
United States Patent 5,643,351; 1997. 1977;30:2656.
[124] Lew CW, Branly K, Gaytan J. Encapsulation with water soluble polymer. [145] Smirnoff WA. Three years of aerial field experiments with Bacillus
United States Patent 5,599,583; 1997. thuringiensis plus chitinase formulation against the spruce budworm. J
[125] Owaga M, Inoue M, Ohtsubo T, Kawada H. Solid insect growth Invertebr Pathol 1974;24(3):3448.
regulating compositions. United States Patent 5,707,639; 1998. [146] Morris ON. A 2-year study of the efficacy of Bacillus
[126] Lechelt-Kunze C, Simon J, Zitzmann W, Kalbe J, Muller H-P, Koch R. thuringiensischitinase combinations in spruce budworm (Choristoneura
Biological material embedded in hydrogels, a process for the embedding fumiferana) control. Can Entomol 1976;108:322533.
thereof, and its use as artificial seed. United States Patent 6,164,012; [147] Daoust RA. Mutualistic interactions in the microbiological control of
2000. Lymantria dispar (L). PhD Dissertation. Amherst: University of
[127] Toreki W, Manukian A, Strohschein R. Hydrocapsules and method of Massachusetts; 1978.
preparation thereof. United States Patent 6,780,507; 2004. [148] Gunner HB, Met MZ, Berger S. Chitinase producing Bt strains. In:
[128] Kotz ME, Van der Zwan JJ. Controlled release agglomerated carrier. Grimble DG, Lewis FB, editors. Microbial control of spruce budworms
United States Patent 4,563,34; 1986. and gypsy moths. U.S. Forestry Service, GTR-NE-100; 1985. p. 1028.
[129] Levy R. Controlled delivery compositions and processes for treating [149] Martins EMF. Guzzo SD, Palazzo DA, Harakava R. Metabolic
organisms in a column of water or on land.United States Patent alterations detected in Natal orange trees infected by Xylella fastidiosa.
6,001,382; 1999. Summa Phytopathol 1999;25:28893.
[130] McArdle B. Herbicidal and insecticidal proteinpolysaccharide delivery [150] Wiwat C, Thaithanun S, Pantuwatana S, Bhumiratana A. Toxicity of
compositions and methods for controlling plant and insect populations. chitinase- producing Bacillus thuringiensis ssp. kurstaki HD-1 (G)
United States Patent 5,747,416; 1998. toward Plutella xylostella. J Invertebr Pathol 2000;76(4): 2707.
[131] Dubois NR, Reardon RC, Mierzejewksi K. Field efficacy and deposit [151] Liu M, Cai QX, Liu HZ, Zhang BH, Yan JP. Yuan ZM Chitinolytic
analysis of Bacillus thuringiensis, Foray 48B, against Gypsy moth. J activities in Bacillus thuringiensis and their synergism effects on
Econ Entomol 1993;86(1):2733. larvicidal activity. J Appl Microbiol 2002;93:3749.
[132] Ebert TA, Taylor RAJ. Downer RA, Hall FR. Deposit structure and [152] Manker DC, Lidster WD, Starnes RL, MacIntosh SC. Potentiator of
efficacy of pesticide application. 1. Interactions between deposit toxicant Bacillus pesticidal activity. Patent Coop. Treaty WO94/09630; 1994.
concentration and deposit number. Pestic Sci 1999; 55:78392. [153] Broderick NA, Goodman RM, Raffa KF, Handelsman J. Synergy
[133] Ignoffo CM, Hostetter DL, Smith DB. Gustatory stimulant, sunlight between zwittermicin A and Bacillus thuringiensis subsp. kurstaki
protectant, evaporation retardant: three characteristics of a microbial against gypsy moth (Lepidoptera: Lymantriidae). Environ Entomol
insecticidal adjuvant. J Econ Entomol 1975;69(2):20710. 2000;29(1):1017.
[134] Allsopp PG, Sugars. amino acids, and ascorbic acid as phagostimulants [154] Visser B, van Workum M, Dullemans A, Waalwijk C. The mosquitocidal
of larvae of antitrogus parvulus and lepidiota negatoria (Coleoptera: activity of Bacillus thuringiensis var. israelensis is associated with Mr
scarab aeidae). J Econ Entomol 1992;85:10611. 230,000 and 130,000 crystal proteins. FEMS Microbiol Lett
[135] Bartlet E, Parsons D, Williams IH, Clark SJ. The influence of 1986;30:2114.
glucosinolates and sugars on feeding by the cabbage stem flea beetle, [155] Martin PAW. A stilbene optical brightener can enhance bacterial
Psylliodes chrysocephala. Entomol Exp Appl 1994;73:7783. pathogenicity to gypsy moth (Lepidoptera: Lymantriidae) and Colorado
[136] Lopez Jr JD, Shaver TN, Lingren PD. Evaluation of feeding stimulants potato beetle (Coleoptera: Chrysomelidae). Biocont Sci Technol
for adult Helicoverpa zea. In: Proceedings, Beltwide Cotton Production 2004;14(4):37583.
and Research Conferences, National Cotton Council; 1994. p. 9204. [156] Pusztai M, Fast P, Gringorten L, Kaplan H, Lessard T, Carey PR. The
[137] Farrar Jr RR, Ridgway RL. Enhancement of activity of Bacillus mechanism of sunlight mediated inactivation of Bacillus thuringiensis
thuringiensis Berliner against four Lepidopterous insect pests by nutrient crystals. Biochem J 1991;273:437.
based phagostimulants. J Entomol Sci 1995;30: 2942. [157] Ignoffo CM. Environmental factors affecting persistence of
[138] Lopez Jr JD, Lingren PD. Feeding response of adult Helicoverpa zea entomopathogens. Florida Entomol 1992;75:51625.
(Lepidoptera: Noctuidae) to commercial phagostimulants. J Econ [158] Cohen E, Rozen H, Joseph T, Braun S, Margulies L. Photoprotection of
Entomol 1994;87:16538. Bacillus thuringiensis var. kurstaki from ultraviolet irradiation. J
Invertebr Pathol 1991;57:34351.
346 S.K. Brar et al./Process Biochemistry 41 (2006) 323342

[159] Beegle CC, Yamamoto T. Invitation paper (C.P. Alexander Fund): history [180] Paukner S, Kohl G, Jalava K, Lubitz W. Sealed bacterial ghosts novel
of Bacillus thuringiensis Berliner research and development. Can targeting vehicles for advanced drug delivery of water-soluble
Entomol 1992;124:587616. substances. J Drug Target 2003;11:15161.
[160] Ragaei M. Studies on the effect of Bacillus thuringiensis on the greasy [181] Hatfaludi TS, Liska M, Zellinger D, Ousman JPa, Szostak M, Ambrus
cutworm Agrotis ypsilon (Rott.). PhD Thesis. University of Cairo; 1990. ARD, et al. Bacterial ghost technology for pesticide delivery. J Agric
[161] Morris O. Protection of B. thuringiensis from inactivation by sunlight. Food Chem 2004;52:562734.
Can Entomol 1983;115:121527. [182] Nishiitsutsuji-Uwo J, Ohsawa A, Nishimura MS. Factors affecting the
[162] Dunkle RL, Shasha BS. Response of starch encapsulated Bacillus insecticidal activity of delta endotoxin of Bacillus thuringiensis. J
thuringiensis containing UV screens to sunlight. Environ Entomol Invertebr Pathol 1976;29:1629.
1989;18:103541. [183] Han EN, Bauce E. Dormancy in the life cycle of the spruce budworm:
[163] Cokmus C, Sayar AH, Sacilik SC, Osmanagaoglu O , Berber I. physiological mechanisms and ecological implications. Recent Res Dev
Effects of UV-light on Bacillus sphaericus and its protection by Entomol 2000;3:4354.
chemicals. J Basic Microbiol 2002;40(4):21521. [184] Molloy DP. Progress in the biological control of black flies with Bacillus
[164] Liu YT, Sui MJ, Ji DD, Wu IH, Chou CC, Chen CC. Protection from thuringiensis israelensis, with emphasis on temperate climates. In:
ultraviolet irradiation by melanin of mosquitocidal activity of Bacillus deBarjac H, Sutherland DJ, editors. Bacterial control of mosquitoes and
thuringiensis var. israelensis. J Invertebr Pathol 1993;62:1316. black flies. New Brunswick, NJ: Rutgers University Press; 1990. p. 161
[165] Patel KR, Wyman JA, Patel KA, Burden BJ. A mutant Bacillus 86.
thuringiensis producing a dark-brown pigment with increased UV [185] Becker N, Margalit J. Use of Bacillus thuringiensis israelensis against
resistance and insecticidal activity. J Invertebr Pathol 1996; 67:1204. mosquitoes and blackflies. In: Entwhistle PF, Cory JS, Bailey MJ, Higgs
[166] Saxena D, Ben-Dov E, Manasherob R, Barak Z, Boussiba S, Zaritsky A. S, editors. Bacillus thuringiensis, an environmental biopesticide: theory
A UV tolerant mutant of Bacillus thuringiensis subsp. kurstaki producing and practice. Chichester, England: John Wiley and Sons; 1993. p. 147
melanin. Curr Microbiol 2002;44:2530. 70.
[167] Salama HS, Morris ON. The use of Bt in developing countries. In: [186] Beckwith RC, Stelzer MJ. Persistence of Bacillus thuringiensis in two
Entwistle PF, Cory JS, Bailey MJ, Higgs S, editors. Bacillus formulations applied by helicopter against western spruce budworm
thuringiensis, an environmental biopesticide: theory and practice. (Lepidoptera: Tortricidae) in north central Oregon. J Econ Entomol
Chichester, England: John Wiley and Sons; 1993. p. 23754. 1987;80:2047.
[168] McGuire MR, Shasha BS, Eastman CE, Oloumi-Sadeghi H. Starch and [187] Smirnoff WA. Effects of volatile substances released by foliage of Abies
flour based sprayable formulationseffect on rainfastness and solar balsamea. J Invertebr Pathol 1972;19(1):325.
stability of Bacillus thuringiensis. J Econ Entomol 1996;89:8639. [188] Maksymiuk B. Occurrence and nature of antibacterial substances in
[169] Ragaei M. Laboratory evaluation of shellac as ultraviolet screen for the plants affecting Bacillus thuringiensis and other entomogenous bacteria.
Bacillus thuringiensis var. entomocidus against Spodoptera littoralis J Invertebr Pathol 1970;15(3):35671.
larvae. Anz. Schadlingskunde, Pflanzenschutz, Umweltschutz [189] Ferry N, Edwards MG, Gatehouse JA, Gatehouse AMR. Plant-insect
1998;71:1324 (article available in English). interactions: molecular approaches to insect resistance. Curr Opin
[170] Hobbs DG, Campbell BJ, Lidster WD. Ultra violet radiation lignin Biotechnol 2004;15:15561.
protected pesticidal compositions. US Patent 5,994,266; 1999. [190] Ali A, Young SY. Effects of rate and spray volume of Bacillus
[171] Wirtz KR, Lebo SE, Sanford ME. Method for providing a stable thuringiensis var. kurstaki on activity against Heliothis virescens and
protective coating for UV sensitive pesticides. United States Patent persistence in cotton terminals. J Econ Entomol 1993;86: 7358.
5,939,089; 1999. [191] Fuxa JR. Fate of released entomopathogens with reference to risk
[172] De Vlieger JJ, Smits PH. Composition for controlling pests, process for assessment of genetically engineered microorganisms. Entomol Soc Am
the preparation thereof and use thereof in crop protection. United States Bull Winter 1989.
Patent 5,807,566; 1998. [192] Gringorten JL, Mile RE, Fast PG, Soho SS, van Frankenhuyzen K.
[173] Shapiro M, Dougherty E, Hamm JJ. Compositions and methods for Suppression of Bacillus thuringiensis d-endotoxin activity by low
biocontrol using fluorescent brighteners. United States Patent 5,124,149; alkaline pH. J Invertebr Pathol 1992;60:4752.
1992. [193] GibsonDM, Gallo LG, Krasnoff SB, Ketchum RE. Increased efficacy of
[174] Leland JE, Mullins DE, Vaughn LJ. Water soluble UV-protective Bacillus thuringiensis subsp. kurstaki in combination with tannic acid. J
coatings for biological pesticides and process for making same. US Econ Entomol April 1995;88(2):2707.
Patent 20040038825; 2004. [194] Joung KC, Cote JC. A review of the environmental impacts of the
[175] Yu JY, Lee WC. Microencapsulation of pyrrolnitrin from Pseudomonas microbial insecticide Bacillus thuringiensis. Agriculture and AgriFood
cepacia using gluten and casein. J Ferment Bioeng Canada, Technical Bulletin No. 29, 2000. 16 pages. Copies available at:
Horticultural R&D Centre web site at http://res2.agr.ca/ stjean/crdh.htm.
1997;84(5):4448.
[195] Sachdeva V, Tyagi RD, Valero JR. Production of biopesticides as a novel
[176] Ramstack JM, Herbert PF, Strobel J, Atkins TJ. Preparation of
method of wastewater sludge utilization/disposal. Water Sci Technol
biodegradable microparticles containing a biologically active agent. US
2000;42:2116.
Patent 5,650,173; 1997.
[196] Tirado-Montiel ML, Tyagi RD, Valero JR. Wastewater treatment sludge
[177] Fowler JD, Feinstein BE. Insecticidal matrix and process for preparation
as a raw material for the production of Bacillus thuringiensis based
thereof. US Patent 5,885,603; 1999.
biopesticides. Water Res 2001;35(16):380716.
[178] Ferro DN, Slocombe AC, Mercier CT. Colorado potato beetle
[197] Tirado-Montiel ML, Tyagi RD, Valero JR, Surampalli RY. Production
(Coleoptera: Chrysomelidae): residual mortality and artificial
biopesticides using wastewater sludge as a raw materialeffect of
weathering of formulated Bacillus thuringiensis subsp. tenebrionis. J
process parameters. Water Sci Technol 2003; 48(8):23946.
Econ Entomol 1997;90:57482.
[198] Lachhab K, Tyagi RD, Valero JR. Production of Bacillus thuringiensis
[179] van Frankenhuyzen K, Nystrom C. Residual toxicity of a high potency
biopesticides using wastewater sludge as a raw material: effect of
formulation of Bacillus thuringiensis to spruce budworm. J Econ
inoculum and sludge solids concentration. Process Biochem
Entomol 1989;82:86872.
2001;37(2):197208.
S.K. Brar et al./Process Biochemistry 41 (2006) 323342 347

[199] Tyagi RD, Foko VS, Barnabe S, Vidyarthi AS, Valero JR. Simultaneous [219] Carr ME, Doane WM, Wing RE, Bagley EB. Starch encapsulation of
production of biopesticide and alkaline proteases by Bacillus biological active agents by a continuous process. US Patent 5,183,690;
thuringiensis using sewage sludge as rawmaterial. Water Sci Technol 1993.
2001;46(10):24754. [220] Morris ON, Converse V, Kanagaratnam P. Chemical additive effects on
[200] Vidyarthi AS, Desrosiers M, Tyagi RD, Valero JR. Foam control in the efficacy of Bacillus thuringiensis Berliner subsp. kurstaki against
biopesticide production from sewage sludge. J Ind Microbiol Biotechnol Mamestra configurata (Lepidoptera: Noctuidae). J Econ Entomol
2000;25:8692. 1994;88:81524.
[201] Vidyarthi AS, Tyagi RD, Valero JR. Effect of surface active agents on [221] McMullan PM. Utility adjuvants. Weed Technol 2000;14:7927.
the production of biopesticides using wastewater sludge as a raw [222] Montermini A, Nanni C, Boselli M. Integrated defence of poplar: two
material. Water Sci Technol 2001;44(10):2539. years trials against Hyphantrea cunea (Drury) with a new microbial
[202] Vidyarthi AS, Tyagi RD, Valero JR, Surampalli RY. Studies on the formulation of Bacillus thuringiensis var. kurstaki distributed by
production of B. thuringiensis based biopesticides using wastewater helicopter. ANNPBCPC second international symposium on pes-
sludge as a raw material. Water Res 2002;36(19): 485060. ticide application techniques, Strasbourg; 2224 September 1993.
[203] Yezza A, Tyagi RD, Valero JR, Surampalli RY. Bioconversion of p. 4336.
industrial wastewater and wastewater sludge into Bacillus thuringiensis [223] Ejiofor AO, Okafor N. Formulation of a flowable liquid concentrate of
based biopesticides in pilot plant fermentor. Biores Technol, in press. Bacillus thuringiensis serotype H-14 spores and crystals as mosquito
[204] Yezza A, Tyagi RD, Valero JR, Surampalli RY, Smith J. Scale-up of larvicide. J Appl Bacteriol 1991;71:2028.
biopesticide production processes using wastewater sludge as a raw [224] Yang C-C, Pan I-H, Chen M-H, Kao S-S, Tsai Y-S. Anti-ultraviolet
material. J Ind Microbiol Biotechnol 2004; doi:10.1007/s10295- biocidal composition. United States Patent 5,427,787; 1995.
0040176-z. [225] Smith KL, Herbig SH. Labile insecticide compositions. United States
[205] Yezza A, Tyagi RD, Valero JR, Surampalli RY. Production of Bacillus Patent 5,750,126; 1998.
thuringiensis based biopesticides in batch and fed-batch cultures using [226] Lebo Jr. SE, Detroit WJ. Method for microencapsulation of agriculturally
wastewater sludge as a raw material. J Chem Technol Biotechnol active substances. United States Patent 5,552,149; 1996.
2004;80:50210. [227] Bohm HA, Friend DR. Microencapsulated insecticidal pathogens. United
[206] MENV. Guide sur la valorisation des matie`res residuelles fertilisantes: States Patent 4,948,586; 1990.
Crite`res de references et normes reglementaires. Direction du Milieu [228] McGuire MR, Streett DA, Shasha BS. Evaluation of starch encapsulation
Rural, Environnement Quebec, Canada; 2004. 138 pp. for formulation of grasshopper (Orthoptera: Acrididae) entompox
[207] Brar SK, Verma M, Tyagi RD, Valero JR, Surampalli RY. Sludge based viruses. J Econ Entomol 1991;84:16526.
Bacillus thuringiensis biopesticides: viscosity impacts. Water Res [229] Shapiro M, Vaughn JL. Enhancement in activity of homologous and
2005;39:300111. heterologous baculoviruses infectious to cotton bollworm (Lepidoptera:
[208] Brar SK, Verma M, Tyagi RD, Valero JR, Surampalli RY. Efficient Noctuidae) by an optical brightener. J Econ Entomol
centrifugal recovery of Bacillus thuringiensis biopesticides from 1995;88(2):2659.
fermented wastewater and wastewater sludge, Water Res 2005, [230] Munson D, Lew CW, Gaggero JM, Branly K. Aqueous solven
submitted for publication. encapsulation method, apparatus and microcapsules. US Patent
[209] Sutherland IW. Exopolysaccharides in biofilms, flocs and related 5,571,522; 1996.
structures. Water Sci Technol 2001;43(6):7786. [231] Alexander SR, Aref AA, Smith MS. Palatable solid pesticidal
[210] Brar SK, Verma M, Tyagi RD, Valero JR, Surampalli RY. Development compositions of ethylene and vinyl acetate copolymer. US Patent
of sludge based stable aqueous Bacillus thuringiensis formulations. 5,135,744; 1992.
Water Sci Technol 2004;50(9):22936. [232] Tamez-Guerra P, McGuire MR, Behle RW, Shasha BS, Pingel RL.
[211] Manka J, Rebhun M, Mandelbaum A, Bortinger A. Characterization of Storage stability of Anagrapha falcifera nucleopolyhedrovirus in spray-
organics in secondary effluents. Environ Sci Technol 1974; 8(12):1017 dried formulations. J Invertebr Pathol 2002;79(1): 716.
20. [233] Berto P, Dickburt C, Lepoivre P. Biopesticide compositions. European
[212] Tu M, Randall JM. Weed control methods handbook. Ohio, USA: The Patent 1,238,587,A2; 2002.
Nature Conservancy, 2003. [234] Gaudet, MD, Puritch GS. Fatty acid salt enhancement of bacterial
[213] Lisansky SG, Quinlan RJ, Tassoni G. The Bacillus thuringiensis insecticide. US Patent 4,826,678; 1989.
production handbook. Newbury: CPL Press, 1993. p. 124. [235] Marshall LGI. Biological control agent biocarriers and method of
[214] Prabakaran G, Padmanabhan V, Balaraman K. Development of a self formation. US Patent 5,888,500; 1999.
floating slow release formulation of Bacillus thuringiensis var. [236] Yardin MR, Kennedy IR, Thies JE. Development of high quality carrier
israelensis and its larvicidal activity. Indian J Exp Biol January materials for field delivery of key microorganisms used as biofertilisers
2001;39(1):824. and biopesticides. Rad Phys Chem 2000;57: 5658.
[215] Angus TA. Use of methyl cellulose in laboratory tests of bacterial [237] Vidyasekaran P, Muthamilan M. Development of formulations of
pathogens of insects. Can Ent 1954;86:206. Pseudomonas fluoroscens for control of chickpea wilt. Plant Dis
[216] Angus TA. Potential usefulness of vinyl latices as stickers. Can Ent 1995;79(8):7826.
1959;(XCI):2545. [238] Drop data analysis.
[217] Jankevica J, Zarins I. Biological control of Malacosama neustria L. http://www.dropdata.net/download/PestiSci_L3_6.PDF, cited 4 March,
population with Latvian isolate of nuclear polyhedrosis virus. In: Evans 2004.
HF, editor. Microbial insecticides: novelty or necessity? Symposium
[239] Levy R. Flowable insecticidal delivery compositions and methods for
Proceedings No. 68 British Crop Protection Council. 1997. p. 2858.
controlling insect populations in an aquatic environment. US Patent
[218] Tamez-Guerra P, Garcia-Gutierrez C, Medrano-Roldan H, GalanWong 4,985,251; 1991.
LJ, Sandoval-Coronado CF. Spray dried microencapsulated Bacillus
[240] Levy R. Terrestrial delivery compositions and methods for controlling
thuringiensis formulations for the control of Epilachna varivestis.
insect and habitat-associated pest populations in terrestrial environments.
Mulsant Southwest Entomol 1999;24:3748.
US Patent 4,983,390; 1991.
348 S.K. Brar et al./Process Biochemistry 41 (2006) 323342

[241] Levy R. Insecticidal delivery compositions and methods for controlling a [247] Raun ES, Sutter GR, Revelo MA. Ecological factors affecting the
population of insects in an aquatic environment. US Patent 5,824,328; pathogenicity of Bacillus thuringiensis var. thuringiensis to the European
1998. corn borer and fall armyworm. J Invertebr Pathol 1966;8:36575.
[242] Henderson; Jack A. Insecticide carriers and insecticides. US Patent [248] Cantwell GE, Franklin BA. Inactivation by irradiation of spores of
5,326,560; 1994. Bacillus thuringiensis var. thuringiensis. J Invertebr Pathol 1966;8:256
[243] Puritch GS, McHarg D, Bradbury R, Mason W. Supersorbent material as 8.
pesticide potentiator. US Patent 5,037,654; 1991. [249] Griego VM, Spence KD. Inactivation of Bacillus thuringiensis spores by
[244] Losel P, Penners G, Weissmuller J. Insecticidal attract-and-kill ultraviolet and visible light. Appl Environ Microbiol 1978; 35(5):906
formulations. US Patent 5,707,638; 1998. 10.
[245] McKibben GH, Dickens JC, Smith JW. Plastic bait composition for [250] Burges HD, Hillyer S, Chanter DO. Effect of ultraviolet and gamma rays
attracting and killing crop pests. US Patent 5,290,556; 1994. on the activity of delta endotoxin protein crystals of Bacillus
[246] Sundaram A, Leung JW, Devisetty BN. Rainfastness of Bacillus thuringiensis. J Invertebr Pathol 1975;25:59.
thuringiensis deposits on conifer foliage. In: Berger, Bala, Devisetty and [251] Morris ON. Ed. Report of the 1989 CANUSA Cooperative Bacillus
Hall, editors. Pesticide Formulations and Application Systems, vol. 13, thuringiensis Spray Trials. FMDX-40. Ontario, Canada: Forest Pest
ASTM STP 1183. Philadelphia: American Society for Testing and Mgmt. Inst., Sault Ste. Marie; 1980.
Materials; 1993. p. 22741. [252] Ignoffo CM, Garcia C. UV photo-inactivation of cells and spores of
Bacillus thuringiensis and effects of peroxidase on inactivation.
Environ Entomol 1978;7:2702.

Você também pode gostar