research article evaluation of different culture media for … · 2020. 1. 27. · research article...

Research ArticleEvaluation of Different Culture Media for Improvement inBioinsecticides Production by Indigenous Bacillus thuringiensisand Their Application against Larvae of Aedes aegypti

Patil Chandrashekhar Devidas1 Borase Hemant Pandit1 and Patil Satish Vitthalrao12

1 School of Life Sciences North Maharashtra University PO Box 80 Jalgaon Maharashtra 425001 India2North Maharashtra Microbial Culture Collection Centre (NMCC) North Maharashtra UniversityPO Box 80 Jalgaon Maharashtra 425001 India

Correspondence should be addressed to Patil Satish Vitthalrao satishpatil7gmailcom

Received 19 August 2013 Accepted 11 November 2013 Published 2 February 2014

Academic Editors S R Gerrard S Narayanan R Tofalo and A I Vela

Copyright copy 2014 Patil Chandrashekhar Devidas et al This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

Production of indigenous isolate Bacillus thuringiensis 1199041199072 (Bt 1199041199072) was checked on conventional and nonconventional carbon andnitrogen sources in shake flasks The effects on the production of biomass toxin production and spore formation capability ofmosquito toxic strain were determined Toxicity differs within the same strain depending on the growth medium Bt 1199041199072 producedwith pigeon pea and soya bean flour were found highly effective with LC

50lt 4 ppm against larvae of Aedes aegypti These

results were comparable with bacteria produced from Luria broth as a reference medium Cost-effective analyses have revealedthat production of biopesticide from test media is highly economical The cost of production of Bt 1199041199072 with soya bean flour wassignificantly reduced by 23-fold The use of nonconventional sources has yielded a new knowledge in this area as the processdevelopment aspects of biomass production have been neglected as an area of research These studies are very important from thepoint of media optimization for economic production of Bacillus thuringiensis based insecticides in mosquito control programmes

1 Introduction

Microorganisms and microbial product with potential insec-ticidal activity can play an important role in controlling dis-eases by interrupting transmission mechanism by killinginsect vectors at community level [1] Worldwide efforts toscreen effective entomopathogenic microorganisms for con-trol of agriculturally and medically important insect pestshave yielded many Bacillus thuringiensis (Bt) isolates withvarious insecticidal properties [2] The Gram-positive bac-terium Bacillus thuringiensis is well known for its ability toform spores and crystal proteins with insecticidal activityagainst a wide variety of lepidopteran coleopteran anddipteran insects [3]The use of B thuringiensis as commercialbioinsecticides was due to the remarkable ability of thisbacterium to produce large quantities of insecticidal crystalproteins during large-scale fermentation In many countriesincluding developed ones wheremosquito borne diseases are

still a serious problem there is a need for large quantitiesof such microbial insecticides Recently more attention hasbeen drawn to the low-cost production of B thuringiensiswhich can be achieved through the optimization of cultureconditions using appropriate media [4 5] Few publishedreports are available on use of low-cost ingredients for devel-opment of media for B thuringiensis production Agroin-dustrial residues and byproducts like cheese whey soya beanmilk molasses chicken feather waste and paddy husk wastehave been used as ingredients [6ndash8] Edible seeds like mungbeans (Vigna radiata (L) R Wilczek) were used as majorsources of protein together with different combinations ofsoluble starch andor sugarcane molasses as major carbo-hydrate sources for the production of delta-endotoxin [9]Similarly soya bean flour groundnut cake powder and wheatbran extract (Triticum aestivum L) were separately used inlarge-scale production of B thuringiensis bioinsecticide [10]Media formulation and optimization are key considerations

Hindawi Publishing Corporatione Scientific World JournalVolume 2014 Article ID 273030 6 pageshttpdxdoiorg1011552014273030

2 The Scientific World Journal

in development of bioprocesses that can produce afford-able biological agents yet limited progress has been madein this area to satisfy market opportunities for affordablecommercial biological insecticide products It has been welldocumented that nutrient sources like carbon nitrogensources and macronutrients strongly influence the growthspore production and the toxicity associated with parasporalproteinaceous crystalline inclusions during sporulation andsynthesis of commercially useful metabolites in Bacillusspecies [11 12] Commonly used nutrient sources includea wide range of peptones extracts and hydrolysates manyof which are expensive for industrial-scale manufacture oflarge-volume products and have negative market accep-tance as animal byproducts [13 14] The study of growthmedium components affecting significantly the productionof biomass toxin production and spore formation is a steprequired to advance in the design of a low-cost culturemedium for the efficient production of all above responses

Therefore in the present work an attempt has been madeto determine the effects of several conventional and noncon-ventional carbon and nitrogen sources on the production ofbiomass toxin production and spore formation capability ofmosquito toxic strain Bt 119904V2 This study also determines thecost effectiveness of potential substrates in production of Btinsecticide

2 Material and Methods

21 Bacteria and Growth Bacillus thuringiensis 119904V2 waslocally isolated in India and tested for potential mosquitotoxic activity [1] Bt 119904V2 was maintained on nutrient agarslopes (HiMedia Mumbai) at 4∘C throughout the studyThe organism was grown in 50mL of nutrient broth withshaking for 24 hrs at 30∘CThis culture was further used as aninoculum (1vv) for a basal medium composed of 1 L waterand NaCl (025) Na

2HPO4(01) (MgSO

4(002) and

MnCl2(0005) and (pH 72)) The medium was autoclaved

for 30min at 121∘C

22 Media Preparation Conventional and nonconventionalcarbon and nitrogen sources were used as test materials

Carbon sources tested were glucose sucrose fructosecorn starch mannitol beet root pulp (Beta vulgaris L)banana (Musa paradisiaca L var Grand Naine) fruit pulpand mahua (Madhuca indica L) flower extract For carbonsources study basal medium was supplemented with yeastextract (05) and various carbon sources were incorporatedso as to correspond to 10 gL carbon concentration

Nitrogen sources tested were soya bean flour pigeon peaflour yeast extract malt extract beef extract egg albuminpowder casein powder ammonium sulphate and sodiumnitrate For nitrogen sources studies basal medium wassupplemented with glucose (10 gL) and various nitrogensources were incorporated at a final concentration of 05 gL

The Bt 119904V2 cells were grown for 48 hrs at 30∘C withshaking at 150 rpm in a basal medium supplemented withtest carbon andor nitrogen source The carbon and nitrogensources added to basal media constituents were investigated

to get maximum dry cell mass and to determine yield coeffi-cient protein content and toxicity to mosquito larvae

23 Preparation of Nonconventional Nutrients

231 Flower Extract of Mahua Flowers ofM latifolia L werecollected and dried in shade for 8 days at room temperature(28 plusmn 2∘C) 100 g dry flowers were soaked in 200mL hotdistilled water (95∘C) and incubated in shaker with 220 rpmat 29∘C for 2 h The extract filtered through muslin cloth wasused as a source of sugar in medium

232 Banana Pulp Banana pulp was prepared by blendingthe ripe banana in a mixer and reduced to puree Distilledwater was added in puree at ratio of 3 1 to make a final pureewhich could be poured

233 Beet Root Fresh beet roots were purchased from localmarket Pulp was prepared by blending the beet roots in amixer and reduced to puree Distilled water was added inpuree at ratio of 3 1 to make a final puree which could bepoured This pulp was further used as source of sugar innutrient medium

234 Pigeon Pea and Soya Bean Flour Pigeon pea and Soyabean flour were prepared by separately grinding beans finelyenough to pass through a 100-mesh

24 Cell Mass After fermentation was completed two sam-ples (50mL) were taken from each fermenter and thencentrifuged at 8000 rpm for 15min The supernatants werediscarded and the cell pellets were lyophilized Dry weightwas calculated and expressed in grams per liter (gL) Thesame sample was used for the toxicity test

241 Yield Coefficient The yield coefficient was expressed asthe ratio of carbon in the newly formed biomass to the carbonin the respective sugar source Total carbon estimation wasmeasured by the 3-5-dinitrosalicylic acid (DNS) modifiedmethod [15]

242 Protein Determination 1mL of culture medium wascentrifuged for 10min at 10000 g and the resulting pelletswere washed twice with NaCl (1mL) and twice with distilledwater These pellets were then suspended in 1mL of NaOH(50mML pH 125) in order to solubilize protein crystalsAfter 2 h of incubation at 37∘C total proteins in the super-natant were measured by using the method by Bradford [16]

243 Bacterial Growth Bt 119904V2 was inoculated in all the testmedia and allowed to grow under constant agitation in theshaker (200 rpm at 30∘C for 72 h) Culture samples from therespectivemediawere drawn (25mL) every 6 h till the end ofthe bacterial growth (72 h) The density of culture media wasmeasured (at 600 nm) using the UV-Vis spectrophotometer(Shimadzu Japan)The bacterial developmental stages (vege-tative to sporulation)weremonitored (LabomedMicroscopeIndia)

The Scientific World Journal 3

25 Toxicity Assay For the laboratory trial early fourthinstars larvae of Aedes aegypti were collected from city areaof Jalgaon (21∘210158405410158401015840N 76∘321015840310158401015840E elevation 209m) Theidentified larvae were kept in plastic enamel trays containingdechlorinated tap water They were maintained and all theexperiments were carried out at 28plusmn2∘C and 75ndash85 relativehumidity under 14 10 light and dark cycles Larvae were fedwith a diet of finely ground brewerrsquos yeast and dog biscuits(3 1) [1] Dry cell mass produced in different media wasassayed against early fourth instars larvae of Aedes aegyptiBioassay was performed by dissolving lyophilized Bt 119904V2powder in distilled water to get final different concentrationsof 15 10 75 5 25 1 and 05 ppm for 20 larvae in 50mL ofdistilled water Larval mortality was checked after 24 hrs ofincubation Each treatment was performed in three replicateseach In all cases the mortality of control larvae reared ona bacterial cell free diet (or water medium) and under thesame environmental conditions as the experimental larvaewas recorded and calculated by Abbott [17] formula

3 Statistical Analysis

The larvicidal activity of cell mass produced in each mediumat different concentrations of 15 10 75 5 25 1 and 05 ppmwas subjected to probit regression analysis The lethal con-centrations in ppm (LC

50 LC90) and the 95 confidence

intervals of LC50

(upper confidence limit and lower confi-dence limit) were calculated All conclusions are based onexperiments that are repeated in time to ensure repeatabilityof results Costs of the culture media were determined basedon the ingredient prices in the western region of India Mediawere compared based on their cost and potency againstAedesaegypti larvae

Experimental data were analysed by one-way analysisof variance (ANOVA) using statistical software Minitab forWindows version 13 Treatment means were separated byTukeyrsquos multiple comparison test at 120572 = 005

4 Result and Discussion

The present study aimed to maximize production of Bt basedbioinsecticide by ensuring high level of biomass protein andspore production from an indigenous isolate Bt 119904V2 withrelatively cheap nutrient sources Initially we screened 8 car-bon substrates (glucose sucrose fructose starch mannitolbanana beet root and mahua) based on biomass productioncapability at laboratory scale Amount of biomass producedby Bt 119904V2 on different carbon source was found to vary(Table 1) Out of eight test carbon sources and referencemedium (LB) only medium with glucose had the highestbiomass yield (630 plusmn 003 gL) in the basal productionmedium (Table 1) The second highest biomass yield (587 plusmn008 gL) was obtained in a medium with banana Consumedsugars were determined to test rate of carbon utilization andto determine yield coefficient Interestinglymore than 50ofinitial sugars (glucose and banana) were remaining withoutbeing consumed even after prolonged fermentation studies(72 h) These results are particularly interesting because

remaining residue could be revalued as economic nutrient innext fermentation batches or to fed animalsWeobserved thataddition of sucrose fructose starch mannitol and mahua ascarbon source had no significant positive effect on biomassyield These compounds were probably not preferred by theBt 119904V2 strain On the other hand glucose banana and beetroot produced biomass at par with LBmedium According toour results glucose should be added to the growth mediumin order to obtain positive effects on the growth of Bt 119904V2because glucose significantly stimulated biomass productionThis type of response indicates that the inclusion of car-bohydrates in a growth medium should be performed verycarefully Effect of these carbon sources was also analyzed bycalculating their yield coefficient (Y) it showed that glucoseis the best carbon source for biomass production (Table 1)We used glucose as an optimized carbon source in furtherstudies considering its pure form compared to banana pulpand cost in purification of banana pulp Our observation onbanana pulp utilization is a new one in the production of Btbased biopesticides Earlier Shyam et al [18] reported thatwaste ripe banana contains higher reducing sugars whichhelped in higher ethanol production using Saccharomycescerevisiae fermentation However banana could also be usedas an alternative to glucose after in detail factorial analysis ofbiomass production and cost analysis Cell mass and productformation bymicroorganisms can be described quantitativelyby yield coefficients expressed as the mass of cells or productformed per unit mass of substrate consumed With theyield coefficients the material balance equations for cellssubstrate and product can be straightforwardly formulated[19]

Since media components play a very important role indetermining the yield and insecticidal activity of the sporecrystal complex [20] effect of nitrogen sources on Bt 119904V2was evaluated for biomass yield toxin content and sporeproduction The usable form of Bt based product is in theform of spores Thus it is important to check spore produc-tion capacity of Bt 119904V2 on test media It was observed thatnatural nitrogen sources (pigeon pea and soya bean) supportmore biomass and toxin production than the syntheticnitrogen sources like ammonium sulphate and sodiumnitrate(Table 2)

Spore counts are known to be more accurate than drymass for yield determinations because drymass is affected bysuspended solids in the media This can be seen in mediumsupplemented with ammonium sulphate and sodium nitratewhere a dry mass was relatively obtained at par with LBmedium while the spore count was lower than expectedMedium supplemented with pigeon pea and soya bean flourhad the highest yield in terms of dry mass and spores permillilitre No strong apparent relationshipwas found betweenspore production and biomass yield The protein concentra-tion an indication of toxin production from all the testedmedia was quantified and there was a significant differencein the production of toxins (Table 2) between the mediawhich demonstrates that the toxins produced from each ofthe media was substrate specific

To determine a close correlation between the growth andproduction of bacterial agents wemeasured toxin to biomass


Table 1 Biomass production of Bt 1199041199072 in different carbon sources

Sugar source (gL) SC (gL) Biomass (gL) 119884 coefficientBC + glucose (10) 470 (002)e 630 (003)a 134BC + fructose (10) 712 (004)b 430 (009)c 060BC + mannitol (10) 802 (003)a 127 (005)e 014BC + sucrose (10) 528 (002)d 366 (007)d 070BC + starch (10) 793 (002)a 107 (002)e 013BC + banana (10) 482 (003)e 587 (008)b 122BC + beet root (10) 632 (008)c 510 (011)b 080BC + mahua (10) 676 (004)c 361 (004)d 053LB medium mdash 540 (003)b mdashSC Sugar consumed at the end of fermentation initial sugar concentration used in each test medium = 10 gmL BC basal medium for carbon source study(NaCl 25 gmL + Na2HPO4 1 gmL +MgSO4 02 gmL +MnCl2 005 gmL + yeast extract 5 gmL) Yield (119884) coefficient expressed as the ratio of carbon in thenewly formed biomass to the carbon in the respective sugar sourceData are presented as mean (SD)Means within a given column followed by the same letter are not significantly different Tukeyrsquos MRT (120572 = 005) 119875 lt 00001

Table 2 Comparative analysis of Bt 1199041199072 production in different nitrogen sources

Nitrogen source (gL) Biomass yield (gL) Toxin content (protein mgL) Spores (CFUmL) Toxinbiomass (mgg)BN + ammonium sulphate (5) 544 (005)c 160 (020)c 730 times 10

7 294BN + sodium nitrate (5) 490 (010)d 142 (030)e 910 times 10

6 289BN + egg albumin (5) 496 (050)d 155 (020)c 150 times 10

7 312BN + beef extract (5) 470 (003)d 113 (050)d 140 times 10

6 240BN + casein (5) 667 (005)b 184 (060)b 169 times 10

8 275BN + malt extract (5) 581 (006)c 154 (080)c 240 times 10

8 265BN + soybean (5) 654 (004)b 197 (020)b 112 times 10

9 301BN + pigeon pea (5) 745 (009)a 282 (070)a 224 times 10

9 378BN + yeast extract (5) 626 (008)b 167 (040)c 910 times 10

8 266LB medium (25) 540 (002)c 188 (020)b 132 times 10

9 348BN Basal medium for nitrogen source study (NaCl 25 gmL + Na2HPO4 1 gmL + MgSO4 02 gmL + MnCl2 005 gmL + glucose 10 gmL)data are presented as mean (SD) Means within a given column followed by the same letter are not significantly different Tukeyrsquos MRT (120572 = 005) 119875 lt 00001

ratio Pigeon pea containing media had the highest toxin tobiomass ratio (378) and spore production was also highSimilar trend was observed in all media with different nitro-gen sources used Ratio of toxin produced to the biomass yieldcould link well with the spore production Thus attentionshould therefore be directed not only towards fermentationswith high yields andor spore production media should beselected to obtain the high toxins per volume of biomassproduced

In growth curve experiment it was observed from the testculture media (pigeon pea and soya bean) that exponentialphase of Bt 119904V2 was initiated from the sixth hour onwards(Figure 1) Rapid multiplication of bacterial cells followedby an increase in culture density was observed This wasextended up to 48 h after which the Bt 119904V2 entered into thestationary phase of growth (48 to 72 h) Sporulation startedafter 48 h of growth and complete sporulationwas achieved in54 h and by 72 h the spores were found to have been releasedfrom the cells The Bt 119904V2 was able to digest the nutrientsfrom all the culture media completely by 72 h The growthpattern of Bt 119904V2 in pigeon pea and soya bean mediumwas higher than that from LB corroborating the results ofbiomass and spore production in the present study (Table 2)

Similar enhanced growth pattern of Bti was reported withchicken feather coconut cake andmanganese chloride basedcombination medium [21]

Toxicity tests (bioassays) with mosquito larvae and costanalysis were performed with Bt 119904V2 toxins produced frompigeon pea and soya bean media whereas LB was used as areference medium Table 3 represents comparative toxicitiesof Bt 119904V2 produced from LB pigeon pea and soya beanmedia against Aedes aegypti larvae Here the effect of toxin(asmeasured by LC

50and LC

90values) produced frompigeon

peamedia was increased bymore than 2-fold compared to LBmedia Toxins from soya bean media (LC

50317) had effects

close to LB media (LC50402)

The difference in bacteria mediated larvicidal efficacymay be due to the higher production of endotoxins (cry pro-teins) In the pigeon pea medium there is abundance of pro-teins and mixed salts which made it more suitable for growthand endotoxin productionThedifference in growth and toxicactivities of Bt 119904V2 in different media may be due to the dif-ferences in availability of growth nutrients It is interesting tonote that the toxicity obtained at the end of the fermentationdepends on the culture medium and operating conditions[22] Boulenouar et al [23] also observed that the different


Table 3 Toxic effect of Bt 1199041199072 on different media against IVth instars larvae of Aedes aegypti and comparative cost analysis for production oftoxin

Culture media (gL) LC50 plusmn SE (LCL-UCL) LC90 plusmn SE (LCL-UCL) Regression equation Cost per liter inUSD

Net differencelowast(in ratio)

LB medium (25) 402 plusmn 026 (357ndash465) 759 plusmn 064 (656ndash925) 119884 = 0438 + 237119883 161 mdashBN + pigeon pea (5) 157 plusmn 009 (137ndash176) 320 plusmn 017 (289ndash362) 119884 = 366 + 369119883 007 23BN + soyabean (5) 317 plusmn 016 (286ndash354) 594 plusmn 037 (531ndash685) 119884 = 0167 + 312119883 007 23LC50 lethal concentration that kills 50 of the exposed larvae LC90 lethal concentration that kills 90 of the exposed larvae SE standard error and LCL-UCL 95 upper and lower fiducial limits LC50 and LC90 expressed in ppmlowastRatio of cost of LB medium required for preparation of 1 L medium with the test medium

0

05

1

15

2

25

3

35

4

45

5

0 6 12 18 24 36 48 60 72Culture time (hours)

Opt

ical

den

sity

at600

nm

SB soyabeanPP pigeon peaLB Luria broth

Figure 1 Growth pattern of Bt 119904V2 produced in different media

strains of the same bacteria may show different growth andtoxic activities whichmay be due to the differences in growthrequirements of different strains In the past many attemptshave been made to culture Bacillus to produce toxin atcheaper cost which has provided similar mosquito toxicity asobserved in the present study [24ndash26]

The amount of raw materials used to prepare 1 liter ofculture medium was 5 g which is of negligible value oncost basis (Table 3) On the contrary production on LBmedium involved a cost of US $ 161 per liter (source ofprice estimation was followed as per commercial preparationof fermentation media) which is 23 times more costly thanpigeon pea and soya bean medium respectively How-ever transportation electricity and personnel cost are notincluded because these expenditures are incurred commonlyfor both culture media Moreover pigeon pea and soya beanhave grown in almost every part of India and most countriesof the world It has extensive usage but storage has beenits major problem as it is attacked by many pests Damaged

seeds not suitable for cultivation or as food can be used forthe production of fermentationmedia An alternative use forexample in fermentation medium may provide additionalrevenue to farmers particularly in India and countries thathave high levels of pigeon pea or soya bean production Inthe choice of materials for the production of media for Bthuringiensis three factors should be considered availabilitycost and how well the bacterium could utilize them Someculturemedia selected in the present work could represent aneconomical benefit for biopesticide production because theyallowedmaximumbiomass spore production and ultimatelyhigher toxin production levels at lower cost compared toconventional nutrient source The media preparations exam-ined thus separately proved adequate for cultivation of Bthuringiensis 119904V2 at least on a laboratory scale Medium withpigeon pea which provided the best fermentation mediumfor the growth and sporulation of the Bt 119904V2 strain in thisstudy can be considered for further development If theselow-cost Bt 119904V2 preparations are as successful in field trialsas they are in the laboratory they could be an important toolfor use in an integratedmosquito control programme in Indiaand other developing countries where the bioinsecticidescould be produced in regional laboratories in sufficientquantities to meet instant local demands during outbreak orto preserve for later use

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors are thankful to anonymous reviewers for criticalevaluation of the paper Dr S V Patil is thankful to RajivGandhi Science amp Technology Commission Mumbai Indiafor financial assistance (RGSTCFile-09DPP-05111)

References

[1] CD Patil S V Patil B K Salunke andR B Salunkhe ldquoInsecti-cidal potency of bacterial species Bacillus thuringiensis SV2 andSerratia nematodiphila SV6 against larvae of mosquito speciesAedes aegypti Anopheles stephensi and Culex quinquefasciatusrdquoParasitology Research vol 110 no 5 pp 1841ndash1847 2012


[2] J S Feitelson J Payne and L Kim ldquoBacillus thuringiensisinsects and beyondrdquoNature Biotechnology vol 10 no 3 pp 271ndash275 1992

[3] E Schnepf N Crickmore J Van Rie et al ldquoBacillus thuringien-sis and its pesticidal crystal proteinsrdquo Microbiology and Molec-ular Biology Reviews vol 62 no 3 pp 775ndash806 1998

[4] N Zouari A Dhouib R Ellouz and S Jaoua ldquoNutritionalrequirements of a strain of Bacillus thuringiensis subsp kurstakiand use of gruel hydrolysate for the formulation of a newmedium for 120575-endotoxin productionrdquoApplied Biochemistry andBiotechnology A vol 69 no 1 pp 41ndash52 1998

[5] S Ben Khedher S Jaoua and N Zouari ldquoImprovement ofbioinsecticides production by sporeless Bacillus thuringiensisstrains in response to various stresses in low cost mediumrdquoCurrent Microbiology vol 62 no 5 pp 1467ndash1477 2011

[6] L F A Alves S B Alves R M Pereira and D M F CapalboldquoProduction of Bacillus thuringiensis berliner var kurstakigrown in alternative mediardquo Biocontrol Science and Technologyvol 7 no 3 pp 377ndash383 1997

[7] S Poopathi K A Kumar L Kabilan and V Sekar ldquoDevelop-ment of low-cost media for the culture of mosquito larvicidesBacillus sphaericus and Bacillus thuringiensis serovar israelen-sisrdquoWorld Journal ofMicrobiology and Biotechnology vol 18 no3 pp 209ndash216 2002

[8] B Paul S Paul and M A Khan ldquoA potential economicalsubstrate for large-scale production of Bacillus thuringiensisvar kurstaki for caterpillar controlrdquo Biocontrol Science andTechnology vol 21 no 11 pp 1363ndash1368 2011

[9] S G Mummigatti and A N Raghunathan ldquoInfluence of mediacomposition on the production of 120575-endotoxin by Bacillusthuringiensis var thuringiensisrdquo Journal of Invertebrate Pathol-ogy vol 55 no 2 pp 147ndash151 1990

[10] G Prabakaran and K Balaraman ldquoDevelopment of a cost-effective medium for the large scale production of Bacillusthuringiensis var israelensisrdquoBiological Control vol 36 no 3 pp288ndash292 2006

[11] S Yan S Mohammedi R D Tyagi R Y Surampalli and J RValero ldquoGrowth of four serovar of Bacillus thuringiensis (varkurstaki israelensis tenebrionis and aizawai) in wastewatersludgerdquo Practice Periodical of Hazardous Toxic and RadioactiveWaste Management vol 11 no 2 pp 123ndash129 2007

[12] M K Gouda A E Swellam and S H Omar ldquoProduction ofPHB by a Bacillus megaterium strain using sugarcane molassesand corn steep liquor as sole carbon and nitrogen sourcesrdquoMicrobiological Research vol 156 no 3 pp 201ndash207 2001

[13] Y Nohata and R Kurane ldquoComplete definedmedium for large-scale production of polysaccharide bioabsorbent from Alcali-genes latus B-16rdquo Journal of Fermentation and Bioengineeringvol 83 no 1 pp 116ndash117 1997

[14] A Vuolanto N Von Weymarn J Kerovuo H Ojamo andM Leisola ldquoPhytase production by high cell density culture ofrecombinant Bacillus subtilisrdquo Biotechnology Letters vol 23 no10 pp 761ndash766 2001

[15] G L Miller ldquoUse of dinitrosalicylic acid reagent for determina-tion of reducing sugarrdquo Analytical Chemistry vol 31 no 3 pp426ndash428 1959

[16] M M Bradford ldquoA rapid and sensitive method for the quanti-tation of microgram quantities of protein utilizing the principleof protein dye bindingrdquoAnalytical Biochemistry vol 72 no 1-2pp 248ndash254 1976

[17] W S Abbott ldquoA method of computing the effectiveness of aninsecticiderdquo Journal of Economic Entomology vol 18 pp 265ndash267 1925

[18] K R Shyam M I Ganesh R Rajeswari and H HarikrishnanldquoUtilization of waste ripe Banana and peels for bio ethanolproduction using Saccharomyces cerevisiardquo Journal of Bioscienceand Research vol 2 no 2 pp 67ndash71

[19] JHong ldquoYield coefficients for cellmass andproduct formationrdquoBiotechnology Bioengineering vol 33 no 4 pp 506ndash507 1989

[20] H T Dulmage ldquoProduction of the spore-120575-endotoxin complexby variants of Bacillus thuringiensis in two fermentationmediardquoJournal of Invertebrate Pathology vol 16 no 3 pp 385ndash3891970

[21] S Poopathi and B Archana ldquoOptimization of medium compo-sition for the production of mosquitocidal toxins from Bacillusthuringiensis subsp israelensisrdquo Indian Journal of ExperimentalBiology vol 50 no 1 pp 65ndash71 2012

[22] M Ramırez-Lepe andM Ramırez-Suero ldquoBiological control ofMosquito larvae by Bacillus thuringiensis subsp israelensisrdquo inInsecticidesmdashPest Engineering chapter 11 InTech Press 2012

[23] N Boulenouar F Al-Quadan and H Akel ldquoEffect of variouscombinations of growth temperature pH and NaCl on intra-cellular activities of G6PDH and 6PGDH from four Bacillusstrains isolated from Jordanian hot springsrdquo Journal of BiologicalSciences vol 6 no 3 pp 586ndash590 2006

[24] M Shojaaddini S Moharramipour M Khodabandeh and ATalebi ldquoDevelopment of a cost effectivemedium for productionof Bacillus thuringiensis bioinsecticide using food barleyrdquo Jour-nal of Plant Protection Research vol 50 no 1 pp 9ndash14 2010

[25] K Yadav S Dhiman I Baruah and L Singh ldquoDevelopmentof cost effective medium for production of Bacillus sphaericusstrain isolated from Assam IndiardquoMicrobiology Journal vol 1no 2 pp 65ndash70 2011

[26] S Poopathi and S Abidha ldquoCoffee husk waste for fermentationproduction of mosquitocidal bacteriardquo Journal of EconomicEntomology vol 104 no 6 pp 1816ndash1823 2011

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in development of bioprocesses that can produce afford-able biological agents yet limited progress has been madein this area to satisfy market opportunities for affordablecommercial biological insecticide products It has been welldocumented that nutrient sources like carbon nitrogensources and macronutrients strongly influence the growthspore production and the toxicity associated with parasporalproteinaceous crystalline inclusions during sporulation andsynthesis of commercially useful metabolites in Bacillusspecies [11 12] Commonly used nutrient sources includea wide range of peptones extracts and hydrolysates manyof which are expensive for industrial-scale manufacture oflarge-volume products and have negative market accep-tance as animal byproducts [13 14] The study of growthmedium components affecting significantly the productionof biomass toxin production and spore formation is a steprequired to advance in the design of a low-cost culturemedium for the efficient production of all above responses

Therefore in the present work an attempt has been madeto determine the effects of several conventional and noncon-ventional carbon and nitrogen sources on the production ofbiomass toxin production and spore formation capability ofmosquito toxic strain Bt 119904V2 This study also determines thecost effectiveness of potential substrates in production of Btinsecticide

2 Material and Methods

21 Bacteria and Growth Bacillus thuringiensis 119904V2 waslocally isolated in India and tested for potential mosquitotoxic activity [1] Bt 119904V2 was maintained on nutrient agarslopes (HiMedia Mumbai) at 4∘C throughout the studyThe organism was grown in 50mL of nutrient broth withshaking for 24 hrs at 30∘CThis culture was further used as aninoculum (1vv) for a basal medium composed of 1 L waterand NaCl (025) Na

2HPO4(01) (MgSO

4(002) and

MnCl2(0005) and (pH 72)) The medium was autoclaved

for 30min at 121∘C

22 Media Preparation Conventional and nonconventionalcarbon and nitrogen sources were used as test materials

Carbon sources tested were glucose sucrose fructosecorn starch mannitol beet root pulp (Beta vulgaris L)banana (Musa paradisiaca L var Grand Naine) fruit pulpand mahua (Madhuca indica L) flower extract For carbonsources study basal medium was supplemented with yeastextract (05) and various carbon sources were incorporatedso as to correspond to 10 gL carbon concentration

Nitrogen sources tested were soya bean flour pigeon peaflour yeast extract malt extract beef extract egg albuminpowder casein powder ammonium sulphate and sodiumnitrate For nitrogen sources studies basal medium wassupplemented with glucose (10 gL) and various nitrogensources were incorporated at a final concentration of 05 gL

The Bt 119904V2 cells were grown for 48 hrs at 30∘C withshaking at 150 rpm in a basal medium supplemented withtest carbon andor nitrogen source The carbon and nitrogensources added to basal media constituents were investigated

to get maximum dry cell mass and to determine yield coeffi-cient protein content and toxicity to mosquito larvae

23 Preparation of Nonconventional Nutrients

231 Flower Extract of Mahua Flowers ofM latifolia L werecollected and dried in shade for 8 days at room temperature(28 plusmn 2∘C) 100 g dry flowers were soaked in 200mL hotdistilled water (95∘C) and incubated in shaker with 220 rpmat 29∘C for 2 h The extract filtered through muslin cloth wasused as a source of sugar in medium

232 Banana Pulp Banana pulp was prepared by blendingthe ripe banana in a mixer and reduced to puree Distilledwater was added in puree at ratio of 3 1 to make a final pureewhich could be poured

233 Beet Root Fresh beet roots were purchased from localmarket Pulp was prepared by blending the beet roots in amixer and reduced to puree Distilled water was added inpuree at ratio of 3 1 to make a final puree which could bepoured This pulp was further used as source of sugar innutrient medium

234 Pigeon Pea and Soya Bean Flour Pigeon pea and Soyabean flour were prepared by separately grinding beans finelyenough to pass through a 100-mesh

24 Cell Mass After fermentation was completed two sam-ples (50mL) were taken from each fermenter and thencentrifuged at 8000 rpm for 15min The supernatants werediscarded and the cell pellets were lyophilized Dry weightwas calculated and expressed in grams per liter (gL) Thesame sample was used for the toxicity test

241 Yield Coefficient The yield coefficient was expressed asthe ratio of carbon in the newly formed biomass to the carbonin the respective sugar source Total carbon estimation wasmeasured by the 3-5-dinitrosalicylic acid (DNS) modifiedmethod [15]

242 Protein Determination 1mL of culture medium wascentrifuged for 10min at 10000 g and the resulting pelletswere washed twice with NaCl (1mL) and twice with distilledwater These pellets were then suspended in 1mL of NaOH(50mML pH 125) in order to solubilize protein crystalsAfter 2 h of incubation at 37∘C total proteins in the super-natant were measured by using the method by Bradford [16]

243 Bacterial Growth Bt 119904V2 was inoculated in all the testmedia and allowed to grow under constant agitation in theshaker (200 rpm at 30∘C for 72 h) Culture samples from therespectivemediawere drawn (25mL) every 6 h till the end ofthe bacterial growth (72 h) The density of culture media wasmeasured (at 600 nm) using the UV-Vis spectrophotometer(Shimadzu Japan)The bacterial developmental stages (vege-tative to sporulation)weremonitored (LabomedMicroscopeIndia)






intervals of LC50




























50and LC



50317) had effects








0

05

1

15

2

25

3

35

4

45

5


Opt

ical

den

sity

at600

nm








Acknowledgments


References



































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology






intervals of LC50




























50and LC



50317) had effects








0

05

1

15

2

25

3

35

4

45

5


Opt

ical

den

sity

at600

nm








Acknowledgments


References



































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




















50and LC



50317) had effects








0

05

1

15

2

25

3

35

4

45

5


Opt

ical

den

sity

at600

nm








Acknowledgments


References



































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology






0

05

1

15

2

25

3

35

4

45

5


Opt

ical

den

sity

at600

nm








Acknowledgments


References



































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

research article evaluation of different culture media for … · 2020. 1. 27. · research article...

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