SCALE-UP OF BIOPESTICIDE PRODUCTION PROCESSES USING WASTEWATER SLUDGE AS A RAW MATERIAL THIT K QUY M SN XUT THUC TR SU SINH HC S DNG BN NC THI LM NGUYN LIUAbstract Studies were conducted on the production of Bacillus thuringiensis (Bt)-based biopesticides to ascertain the performance of the process in shake flasks, and in two geometrically similar fermentors (15 and 150 l) utilizing wastewater sludge as a raw material. The results showed that it was possible to achieve better oxygen transfer in the larger capacity fermentor. Viable cell counts increased by 3855% in the bioreactor compared to shake flasks. As for spore counts, an increase of 25% was observed when changing from shake flask to fermentor experiments. Tm tt Cc nghin cu tin hnh v sn xut thuc tr su sinh hc da vo vi khun Bacillus thuringiensis (Bt) xc nh hiu sut ca qu trnh ln men trong bnh lc, v trong hai hnh thc ln men tng t (15 v 150 lt) s dng bn nc thi lm nguyn liu .Kt qu cho thy n c th t c s vn chuyn oxy tt hn trong cc ni ln men cng sut ln hn. S lng t bo sng c tng 38-55% trong cc l phn ng so vi bnh lc. S bo t tng 25% c quan st khi thay i t bnh lc ln men th nghim. Spore counts were unchanged in bench (15 l) and pilot scale (5.35.5 e+08 cfu/ml; 150 l). An improvement of 30% in the entomotoxicity potential was obtained at pilot scale. Protease activity increased by two to four times at bench and pilot scale, respectively, compared to the maximum activity obtained in shake flasks. The maximum protease activity (4.1 IU/ml) was obtained in pilot scale due to better oxygen transfer. The Bt fermentation process using sludge as raw material

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was successfully scaled up and resulted in high productivity for toxin protein yield and a high protease activity. S bo t m c khng thay i quy m nh (15lt) v quy m th im (5,35,5 e+08cfu / ml; 150 lt). Tng 30% kh nng nng cao tnh c i vi su thu c quy m th im. Hot ng phn gii ca protease tng 2-4 ln quy m nh v quy m th im, tng ng, so vi hot ng ti a t c trong bnh lc. Cc hot ng protease ti a (4,1 IU/ml) thu c quy m th im do s vn chuyn oxy tt hn. Qu trnh ln men ca Bt s dng bn thi lm nguyn liu c thu nh thnh cng v kt qu l cho hiu sut cao cc protein c t v hot ng ca protease. Keywords Protease .Bacillus thuringiensis. Wastewater sludge .Sporulation. Entomotoxicity T kho Protease - Bacillus thuringiensis - X l nc thi bn - hnh thnh bo t - Tnh c i vi su.A. Yezza. R. D. Tyagi (&) INRS Eau, Terre et Environnement (INRS-ETE), 2800 Rue Einstein, CP 7500, Sainte-Foy, QC, G1V4C7, Canada E-mail: tyagi@inrs-ete.uquebec.ca Tel.: +1-418-6542617 Fax: +1-418-6542600 J. R. Valero Canadian Forestry Service, Centre Foresterie des Laurentides, 1055 du P.E.P.S, 3800, Sainte-Foy, QC, G1V4C7, Canada R. Y. Surampalli. J. Smith United States Environmental Protection Agency, 17-2141, Kansas City KS 66117, USA A. Yezza. R.D.Tyagi INRS Eau, Terre et Environnement (INRS-ETE), 2800 Rue Einstein, CP 7500, Sainte-Foy, QC, G1V4C7, Canada Email: tyagi@inrs-ete.uquebec.ca in thoi: +1-418-6542617 Fax: +1-418-6542600 J. R. Valro Dch v Lm nghip Canada, Trung tm Foresterie des Laurentides, du PEPS 1055, 3800, SainteFoy, QC, G1V4C7, Canada

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R. Y. Surampalli. J. Smith C quan Bo v mi trng Hoa K, 17-2141, thnh ph Kansas, KS 66117, USA

Introduction The production of Bacillus thuringiensis (Bt)-based biopesticides using wastewater sludge as a raw material has been successfully achieved in our laboratory [20, 25, 28, 29, 32, 33]. All these latter studies were conducted in shake flasks and/or bench scale bioreactors (10 l working volume). However, in order to develop suitable technology for possible commercialization, it was essential to carry out tests in pilot fermentors. Gii thiu Vic sn xut vi khun Bacillus thuringiensis (Bt) da trn thuc tr su sinh hc s dng bn thi lm nguyn liu t c thnh cng trong phng th nghim ca chng ti [20, 25, 28, 29, 32, 33]. Tt c nhng nghin cu sau ny c thc hin trong bnh lc v / hoc nui cy quy m nh (10 lt th tch). Tuy nhin, c th pht trin cng ngh ph hp cho thng mi ha , n cn thit phi thc hin cc th nghim trong quy m ln men th im. The purpose of scale-up is the selection of design conditions and operational procedures to ensure that the effect of different variables on the process is the same in units of different size. The final objective is to bring a fermentation process to its economic fruition, and therefore the technical aspects are important in so far as they can be translated into economic indicators such as yield, productivity, capital investment, and unit production cost, the combined evaluation of which eventually decides the economic viability of the process. Mc ch xy dng quy m sn xut l vic la chn cc iu kin v qu trnh hot ng m bo rng nh hng ca cc yu t khc nhau n quy trnh l nh nhau trong n v c kch thc khc nhau. Mc tiu cui cng l mang li mt qu trnh ln men c thnh qu kinh t, do v mt k thut l quan trng cho n nay v chng c th to ra cc ch s kinh t nh sn lng, nng sut, vn

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u t v n v chi ph sn xut, tng kt nh gi m cui cng quyt nh kh nng kinh t ca qu trnh. Up-scaling fermentation processes from lab-scale to commercial units is challenging due to the difficulty in assessing the factors affecting the scale-up process during cultivation. It is well known that microorganisms are more susceptible to environmental variables, such as pH, temperature, dissolved oxygen (DO) and composition of raw materials, at large scale. These variables have a substantial effect on cell growth and metabolite production [13]. M rng quy m cc qu trnh ln men t quy m trong phng th nghim n cc n v thng mi l mt thch thc do s kh khn trong vic nh gi cc yu t nh hng n qu trnh m rng quy m trong thi gian ln men. iu cng c bit rng cc vi sinh vt d b bin i vi cc bin mi trng, chng hn nh pH, nhit , oxy ha tan (DO) v thnh phn nguyn liu, vi quy m ln. Cc yu t ny c nh hng ng k n tng trng t bo v s trao i sn phm [13]. Also, particular attention must be paid to the oxygen transfer rate (OTR) and heat transfer rate. It is critical to ensure that, at large scale cultivation, the Bt process has adequate oxygen transfer and cooling capacity [35]. Other operational factors can and do change in scale-up, such as quality of mixing, shear stress, selection of cheaper media, foam control, physiological state of the inoculum, and sterilization of culture medium [14]. Ngoi ra, phi ch c bit n tc truyn ti oxy (OTR) v tc truyn ti nhit. iu l rt quan trng m bo cho ln men quy m ln, qu trnh Bt chuyn oxy y v lm mt cng sut [35]. Cc yu t tc ng khc v c th lm thay i v xy dng quy m, chng hn nh cht lng ca s o trn, la chn cc mi trng r hn, kim sot bt, tnh trng nhim truyn sinh l, v kh trng mi trng nui cy [14]. To ensure successful scale-up of fermentation processes, it is essential to determine appropriate scale-up criteria and the factors that reflect critical4

parameters of the fermentation process. Bt fermentation is a strong aerobic fermentation. The most utilized criteria for scale-up of aerobic fermentations maintaining geometric similarity are based on empirical or semi-empirical equations, which correlate the volumetric oxygen transfer coecient (KLa) and the volumetric airow rate per unit working volume (Q/V). m bo xy dng thnh cng quy trnh ca qu trnh ln men, chnh l iu cn thit xc nh cc tiu ch ph hp cho xy dng quy trnh v cc yu t phn nh cc thng s quan trng ca qu trnh ln men. Ln men Bt l mt qu trnh ln men hiu kh mnh m. Cc tiu ch s dng m rng qu trnh ln men hiu kh duy tr s ng phn hnh hc da trn phng trnh thc nghim hoc bn thc nghim m tng quan vi h s chuyn th tch oxy (K La) v tc lun chuyn dng th tch khng kh / lng n v lm vic (Q/V). Geometric similarities are the ratio between liquid height and vessel diameter, or the ratio between impeller diameter and vessel diameter. Geometric similarity is important from the practical point of view, in that it simplies prediction of largescale fermentor performance [16]. ng phn hnh hc l t l gia chiu cao cht lng v ng knh mch, hoc t l gia ng knh lm vic v ng knh bnh. ng phn hnh hc l rt quan trng t quan im thc t, trong n n gin ho vic d on xy dng quy m ln men hiu qu [16]. The primary aim of our investigation was to ensure reproducibility of results obtained at bench (15 l) and pilot (150 l) scale. Reproducible results are vital to a systematic analysis of the inuence of process development parameters on fermentation performance on a large scale. Mc tiu kho st chnh ca chng ti l m bo kh nng ti sn xut kt qu thu c quy m nh (15 lt) v quy m th im (150 lt). Kt qu ti sn xut l rt quan trng phn tch h thng cc yu t nh hng trong qu trnh pht trin cc thng s v hiu sut ln men quy m ln. Materials and methods5

Bacillus thuringiensis strain B. thuringiensis var. kurstaki HD-1 (ATCC 33679) (Btk) was obtained from ATCC and maintained in the Canadian Forestry Service Lab (Ste-Foy, QC, Canada). An active culture was maintained by streak inoculating nutrient agar slant plates [tryptic soy agar (TSA) = 3.0% (v/v) tryptic soya broth (TSB; Difco, Detroit, Mich.) +1.5% Bacto- agar (Difco)], incubating at 30C for 48 h and then storing at 4C for future use. Vt liu v phng php Chng Bacillus thuringiensis B. thuringiensis var. kurstaki HD-1 (ATCC 33679) (Btk) c thu thp t ATCC v duy tr trong phng th nghim Dch v Lm nghip Canada (Ste-Foy, QC, Canada). Mi trng c duy tr bi vt cy trn thch nghing dinh dng [thch nc tng trypsin (TSA) = 3,0% (v/v) nc tng luc thit trypsin (TSB; Difco, Detroit, Michigan) 1,5% Bacto-agar (Difco)] , 30C trong 48 gi v sau bo qun 4C s dng trong tng lai. Inoculum preparation The inoculum was prepared in two steps as reported by Lachhab et al. [20]. Aliquots of bacteria from the slant were used to inoculate 500 ml Erlenmeyer flasks containing 100 ml sterilized TSB (3% v/v). After 812 h incubation at 30C under shaking conditions (250 rpm), 2% (v/v) of this broth was used as seed culture to inoculate an Erlenmeyer flask containing the same medium (sludge in this case) as used for Bt fermentation The flasks were then incubated in a rotary shaking incubator at 30C with an agitation speed of 250 rpm for about 1014 h. All the media used for inoculum preparation were adjusted to pH 7 before autoclaving. Finally, a 2% (v/v) inoculum of the actively growing cells (in exponential phase) of the pre-culture was transferred to the experimental shake flask and fermentor. Cy truyn chng

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Cy truyn chng c chun b trong hai bc theo bo co ca Lachhab v cng s [20]. Lng xc nh ca cc vi khun t ng thch nghing c s dng cy 500 ml bnh Erlenmeyer cha 100 ml TSB tit trng (3% v / v). Sau khi 8-12 gi 30C trong iu kin lc (250 vng / pht), 2% (v / v) nc dng ny c s dng nh mi trng ht ging cy mt bnh Erlenmeyer c cha cng mt mi trng (bn trong trng hp ny) c s dng cho ln men Bt. Cc bnh sau c gi trong mt t lc 30C vi mt tc giao ng t 250 vng / pht trong khong 10-14 gi. Tt c cc mi trng c s dng chun b cy truyn c iu chnh vi pH 7 trc khi hp. Cui cng, cy truyn 2% (v / v) ca cc t bo pht trin tt (trong giai on theo cp s nhn) ca mi trng trc c chuyn cho cc bnh lc th nghim v ln men. Raw material Secondary sludge from a wastewater treatment plant [Communaute Urbaine du Quebec (CUQS)] was used (Table 1). The sludge was allowed to settle to increase the solids concentration to 25 g/l. Nguyn liu Bn th cp t mt nh my x l nc thi [Communaut Urbaine du Quebec (CUQS)] c s dng (Bng 1). Bn ny c php c nh tng nng cht rn n 25 g / l. Table 1 Physical and chemical characteristics of Communaute Urbaine du Quebec (CUQS) sludge Bng 1 c im vt l v ha hc ca bn Communaut Urbaine du Quebec (CUQS) Characteristics Physical characteristics Total solids (g / l) Volatile solids (g / l) Suspended solids (g / l) Volatile suspended solids (g / l) Value 29 19 25 18 c im c im vt l Tng cht rn (g / l) Cht rn d bay hi (g / l) Cht rn l lng (g / l) Cht rn l lng d bay hi (g Gi tr 29 19 25 18

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c im Gi tr / l) pH 5.7 pH 5.7 Chemical characteristics c tnh ha hc Total carbon (%,dry TS) 40.43 Tng s carbon (%, kh TS) 40.43 Total nitrogen (%,dry TS) 5.25 Tng nit (%, kh TS) 5.25 Ammonical nitrogen (mgN / kg) 632 Ammonical nit (mgN / kg) 632 Total phosphorus (MGP / kg) 10,525 Tng phot pho (MGP / kg) 10,525 Orthophosphates (MGP / kg) 5,830 Orthophosphates (MGP / kg) 5,830 Metals (mg / kg) Kim loi (mg / kg) Al 3 + 16,445 Al 3 + 16,445 2+ 2+ Ca 18,778 Ca 18,778 a CD 3.3 a CD 3.3 Cr 94 Cr 94 Cu + 271 Cu + 271 Fe 12,727 Fe 12,727 Mg 2,556 Mg 2,556 Mn 182 Mn 182 K+ 2,563 K+ 2,563 Pb 67 Pb 67 Zn 551 Zn 551 The physical characteristics of the sewage sludge, such as total sludge solids (TS), volatile solids (VS), suspended solids (SS) and volatile suspended solids (VSS), were determined according to standard methods [4]. The chemical characteristics of the sludge, such as total carbon and nitrogen, were analyzed with an NA 1500NCS analyser (Carlo Erba, Milan, Italy). Other chemical components in the sludge, e.g., ammonical nitrogen, total phosphorus and phosphate as PO4, were analysed with a Technicon Analyser II (Technicon, Tarrytown, N.Y.). Metal (Al, Cd, Cr, Cu, Fe, Mn, Pb, Zn, Mg and Ca) concentrations were analysed by atomic absorption spectrometry using a Spectra AA-20 (Varian Techtron, Springvale, Victoria, Australia).

Characteristics

Value

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Cc c im vt l ca bn thi, chng hn nh tng cht rn bn (TS), cht rn d bay hi (VS), cht rn l lng (SS) v cht rn l lng d bay hi (VSS), c xc nh theo phng php chun [4]. Cc c tnh ha hc ca bn, chng hn nh cacbon, tng s nit, c phn tch vi 1500-NCS NA (Carlo Erba, Milan, Italy). Thnh phn ha hc khc trong bn, v d, ammonical nit, tng s phot pho v phosphate l PO4, c phn tch vi Analyser Technicon II (Technicon, Tarrytown, New York). Kim loi (Al, Cd, Cr, Cu, Fe, Mn, Pb, Zn, Mg v Ca) nng c phn tch bng quang ph hp th nguyn t s dng Spectra AA20 (Varian Techtron, Springvale, Victoria, Australia). Fermentation procedure Shake flask experiment A 2% (v/v) inoculum of the pre-culture was used to inoculate a 500 ml Erlenmeyer flask containing 100 ml sterilized sludge. The flask was then incubated at 30C1 in a shaking incubator at 250 rpm for 48 h. Qu trnh ln men Th nghim bnh lc Cy truyn 2% (v / v) ca mi trng c s dng cy 500 ml bnh Erlenmeyer cha 100 ml bn tit trng. Sau bnh 30C 1 trong t lc 250 vng / pht trong 48 h. Bench and pilot-scale fermentor Fermentations were carried out in two stirred-tank bioreactorsbench- and pilotscalewith accessories and automatic control systems for DO, pH, antifoam, impeller speed, aeration rate and temperature. Details of fermentor dimensions are summarized in Table 2. The computer program used (Fix 5.5; Intellution, Norwood, Mass.) allowed automatic set-point control and registration of all stated parameters. Ln men quy m nh v th im Ln men c thc hin trong hai tank khuy h thng lng lc v quy m th im, vi cc thit b v h thng iu khin t ng cho DO, pH, antifoam, tc9

cnh khuy, tc thng kh v nhit . Thng tin chi tit v kch thc ln men c tm tt trong Bng 2. Cc chng trnh my tnh c s dng (Fix 5,5; Intellution, Norwood, Massachusetts) cho php t ng thit lp im kim sot v ci t ca tt c cc thng s nu. Table 2 Dimensions of bench- and pilot-scale bioreactors Bng 2 Kch thc ca h thng nui cy quy m nh v quy m th im. Bioreactor Bench Pilot scale 150 100 3 0.25 0.5 2 0.75 1.5 H thng nui cy Tng khi lng (l) Th tch nui cy(l) B cnh khuy (su cnh , tua-bin Rushton) ng knh khuy, D (m) Tank c ng knh, D Quy m Quy m nh 15 10 3 0.1 th im 150 100 3 0.25 0.5 2 0.75 1.5

scale Total volume (l) 15 Working volume (l) 10 Impeller (six-blade 3 Rushton turbines) Stirrer diameter, D I 0.1 (m) Tank diameter, D t 0.20 (m) Dt/DI 2 Liquid height HL, H L 0.3 (m) HL/Dt 1.5

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0.20 (m) Dt/DI 2 Chiu cao ct lng HL, 0.3 H L (m) HL/Dt 1.5

t

Before each sterilization cycle, the pH-electrode was calibrated using buffers of pH 7 and 4 (VWR, Mississauga, ON, Canada). The DO probe was calibrated to zero (with 1 N sodium sulfite solution) and 100% (saturated with air). Sludge was added to the fermentor, and polypropylene glycol (PPG; Sigma, Oakville, ON, Canada) (0.1% v/v) solution was added to control foam during sterilization. The fermentor was then sterilized in situ at 121C for 30 min. Trc khi mi chu k kh trng, cc in cc pH, c hiu chun bng cch s dng b m ca pH 7 v 4 (VWR, Mississauga, ON, Canada). Kim tra thm d

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DO c nh c n khng (vi 1 N sodium sunfit natri) v 100% (bo ha vi khng kh). Bn c ln men, v polypropylene glycol (PPG; Sigma, Oakville, ON, Canada) (0,1% v / v) gii php l c kim sot bt trong qu trnh kh trng. sau ln men c tit trng ti ch 121C trong 30 pht. After sterilization, the fermentor was cooled to 30C. The DO probe was recalibrated to zero by sparging N2 gas, and to 100% saturation by sparging air at maximum agitation rate (150 rpm for pilot-scale fermentor and 500 rpm for benchscale fermentor). The fermentor was then inoculated (2% v/v inoculum) with actively growing cells of the pre-culture. Fermentations were carried out under constant volumetric airflow rate per unit volume (Q/ V=0.3 vvm). Sau khi tit trng xong, ln men c lm lnh 30C. Kim tra thm d DO c hiu chun li bng khng bi s sc kh N2, v bo ha 100% sc khng kh mc hot ng ti a (150 vng / pht cho ln men quy m th im v 500 vng / pht cho ln men quy m nh). Sau ln men c truyn (2% v / v cht truyn) vi cc t bo pht trin tt ca mi trng trc. Ln men c thc hin theo t l khng i lung khng kh th tch / lng n v (Q / V = 0,3 vvm). The agitation speed was varied to maintain DO values above 20% of saturation, which ensured oxygen concentrations above the critical level [6]. We found that if the oxygen concentration is allowed to decrease below 2024% of saturation, the entomotoxicity value was markedly reduced (data not shown). The temperature was controlled at 30C1 by circulating hot or cold water through the jacket using a circulation pump. Tc vn ng c thay i duy tr gi tr DO trn 20% bo ha, m nng oxy m bo trn mc gii hn [6]. Chng ti thy rng nu nng oxy gim 20-24% di bo ha, gi tr tnh c i vi su gim ng k (d liu khng c hin th). Nhit c kim sot 30C 1 ca tun hon nc nng hoc lnh thng qua cc lp gi nhit bng cch s dng mt my bm lu thng.11

The pH was controlled at 7.00.1 using either 4 N NaOH or 6 N H2SO4 via computer-controlled peristaltic pumps. Foaming during fermentation was controlled using both a mechanical foam breaker and a chemical antifoam agent (PPG in 1:10 aqueous emulsion). DO and pH were continuously monitored by means of polarographic DO probe (Mettler Toledo, Newark, Del.) and pH sensor (Mettler Toledo) respectively. Samples were collected periodically to monitor changes in total viable cells (VC), spore count (SC), proteolytic activity (PA) and entomotoxicity (Tx). pH c kim sot 7,0 0,1 bng cch s dng 4 N NaOH hoc 6 N H2SO4 thng qua my tnh iu khin my bm nhu ng. Cht to bt trong qu trnh ln men c kim sot bng cch s dng c mt my ct c kh v bt antifoam mt tc nhn ha hc (PPG nh dch nc 1:10). DO v pH c lin tc theo di bng phng tin thm d DO polarographic (Mettler Toledo, Newark, Del) v cm bin pH (Mettler Toledo) tng ng. Cc mu c thu thp nh k theo di nhng thay i trong tng s t bo c hiu qu (VC), s bo t (SC), hot ng phn gii protein (PA) v tnh c i vi su (Tx). Estimation of cell and spore count To determine VC and SC, samples were serially diluted with sterile saline solution (0.85% NaCl). Appropriately diluted samples (0.1 ml) were plated on TSA plates and incubated at 30C for 24 h to form fully developed colonies. For SC, appropriately diluted samples were heated in a silicone bath at 80C for 10 min and then chilled on ice for 5 min [31]. This heat/cold shock lysed the vegetative cells and liberated those spores already formed in the bacterial cells [34]. VC and SC titers were estimated by counting colonies grown on nutrient medium. c lng t bo v s bo t xc nh VC v SC, mu c tun t pha long vi dung dch mui v trng (0,85% NaCl). Mu pha long thch hp (0,1 ml) c u trn a TSA v 30C trong 24 gi hnh thnh cc khun lc pht trin y . i vi SC, pha long mu thch hp c nung nng trong mt bn cha silicone 80C12

trong 10 pht v sau chp lnh n trong 5 pht [31]. Sc nhit nng / lnh lm cho dung gii cc t bo bnh thng v gii phng cc bo t hnh thnh trong cc t bo vi khun [34]. chun VC v SC c c tnh bng cch m khun lc pht trin trn mi trng dinh dng. For all counts, the average of at least three replicate plates was used for each dilution tested. For enumeration, 30300 colonies were counted per plate. The results were expressed in colony forming units per milliliter (cfu/ml). In order to establish the reliability and reproducibility of the plate count technique, ten independent samples were drawn (at the same time) from the shake flask experiment and were serially diluted and plated. Each dilution was plated on three different plates, colonies were counted and the standard deviation calculated. The standard deviation thus calculated was 6%. i vi tt c cc php m, trung bnh c t nht ba mu lp li cc a c s dng cho tng pha long th nghim. i vi php m, 30-300 khun lc c m trn mi a. Kt qu c th hin khun lc hnh thnh vi n v ml (cfu / ml). thit lp tin cy v kh nng ti sn xut c ca k thut m khun lc trn a , mi mu c lp c rt ra (ng thi) t th nghim bnh lc v c tun t pha long v trn a. Mi pha long c vo ba mng khc nhau, khun lc c tnh v tnh ton lch chun. Do lch chun tnh l 6%. Bioassay The Tx of the fermentation products was measured by bioassay as the relative mortality to spruce budworm larvae (Choristoneura fumiferana, Lepidoptera: Tortici- dae; responsible for the destruction of conifer forests) induced by the fermentation product compared to that induced by the standard preparation Foray 76B (Abbott Labs, Chicago, Ill.). Bioassays were carried out using the diet incorporation method [7]. In this technique, 1.5ml of serially decimal diluted sample was mixed with 30 ml molten (usually 55C) agar-based diet and distributed into 20 bioassay tubes (1 ml/tube).13

Th nghim sinh hc Cc Tx ca cc sn phm ln men dit cc loi u trng ph hoi n cy vn sam c chng minh bi th nghim sinh hc c t l t vong tng i (Choristoneurafumiferana, cnh vy, Tortici-dae, chu trch nhim v vic ph hy rng cy l kim) gy ra bi cc sn phm ln men so vi gy ra bi vic chun b tiu chun Foray 76B (Abbott hicago,Ill.). Th nghim sinh hc c thc hin bng phng php kt hp ch n ung [7]. Trong k thut ny, 1,5 ml mu pha long thp phn tun t c trn vi 30 ml thch m (thng l 55C) da trn ch n ung v phn phi thnh 20 ng th nghim (1 ml/ng). Three sets of controls (diet with sterilized water and sterilized production medium) were also included in the procedure to correct the mortality of larvae due to the sludge. One third-instar larva of eastern spruce budworm, kindly supplied by the Laurentian Forestry Centre (Ste-Foy, QC, Canada) was placed in each vial after the diet solidied. The vials were incubated at ambient temperature for 1 week. Samples with >10% mortality in controls were dis-carded and the whole procedure was repeated. Ba b iu khin (ch n ung bng nc kh trng v kh trng mi trng sn xut) cng c bao gm trong quy trnh ng t l t vong ca u trng do bn. Ba im u trng ng ch ca pha ng l u trng ph hoi n cy vn sam c cung cp do Trung tm Lm nghip Laurentian (Ste-Foy, QC, Canada) c t trong mi chai thuc sau ch n c nh. Cc l c nhit phng trong 1 tun. Mu vi t l t vong> 10% trong kim sot c loi b v ton b quy trnh c lp li. The Tx of the Bt preparation was calculated as mortality observed relative to the standard preparation and expressed in terms of billions (109) of international units of toxicity per liter (BIU/l). The standard deviation for Tx measurement was less than 5%. Cc Tx ca vic chun b Bt c tnh nh quan st thy t l t vong lin quan n vic chun b tiu chun v c th hin di dng hng t (10 9) ca cc n14

v quc t ca cc c tnh trn mi lt (BIU/l). lch chun cho Tx o c t hn 5%. Proteolytic activity assay Proteolytic activity was determined according to Kunitz [19] with minor modications. Samples collected from the fermentor were centrifuged at 7,650 g for 20 min at 4C. The supernatant thus obtained was appropriately diluted with borate buer, pH 8.2. Alkaline protease activity was assayed by incubating 1 ml diluted enzyme solution with 5 ml casein for 10 min at 37C in a constanttemperature water-bath. The reaction was termi-nated by adding 5 ml 10% trichloroacetic acid (TCA).This mixture was incubated for 30 min to precipitate the total non-hydrolyzed casein. Th nghim hot ng phn gii protein Hot ng phn gii protein c xc nh theo Kunitz [19] vi nhng thay i nh. Cc mu thu c t ln men c ly tm 7650 g trong 20 pht 4C. Cc phn ni pha trn thu c pha long mt cch thch hp vi b m borat, pH 8.2. Hot ng ca protease kim c th nghim bi 1ml dung dch pha long enzyme vi 5ml casein trong 10 pht 37C trong iu kin nhit khng i. Phn ng ny c kt thc bng cch thm vo 5ml acid 10% tricloaxetic (TCA). Hn hp ny c trong 30 pht kt ta casein thy phn khng hon ton. Parallel blanks were pre-pared with inactivated casein. At the end of the incubation period, samples and blanks were ltered through Whatman 934-AH paper. The absorbance of the ltrate was measured at 275 nm. Results were validated by treating a standard enzyme solution (Subtilisin Carls-berg, Sigma-Aldrich-Canada) of known activity in the same way and under the same conditions. One proteolytic activity unit was dened as the amount of enzyme preparation required to liberate 1 mol (181 g) tyrosine from casein per minute in pH 8.2 buer at 37C. Statistical treatment of the results showed maximum deviations of 5%.

15

Mu i chng song song c chun b vi casein bt hot.Vo cui thi k bnh, mu v mu i chng c lc qua giy Whatman 934-AH. Vic hp th ca lc c o 275nm. Kt qu c xc nhn bng cch x l mt gii php enzyme tiu chun (Subtilisin Carlsberg, Sigma-Aldrich-Canada) ca hot ng c bit n trong cng mt cch v trong cng iu kin. Mt n v hot ng phn gii protein c nh ngha l tng s enzyme cn thit gii phng 1

mol (181 g) tyrosine t casein mi pht trong b m pH 8,2 37C. Thngk th nghim ca cc kt qu cho thy lch ti a 5%. Determination of KLa, oxygen uptake rate and OTR The volumetric oxygen transfer coecient (KLa) was measured in bench- and pilot plant-fermentors. In this work the measurement of KLa is based on the dynamic gassing out method [2]. KLa was determined during fermentation at the sampling times. During batch fermentation, the mass balance of the DO concentrations in the cultivation medium was dened thus:= OTR OUR

Xc nh KLa, t l hp th oxy v OTR Cc h s chuyn th tch oxy (KLa) c o phng th nghim v nh my ln men th im. Trong qu trnh o K La l da trn phng php thot kh ng [2]. KLa c xc nh trong qu trnh ln men ti cc ln ly mu.Trong qu trnh ln men, s cn bng khi lng ca nng DO trong mi trng canh tc c nh ngha nh sau:= OTR OUR The oxygen transfer rate (OTR) = KLa(C* - CL), and OUR = QO2X, where KL is the

liquid phase mass transfer coecient, a the gas liquid interfacial area, KLa the volumetric oxygen transfer coecient, C* the sat- urated oxygen concentration, and CL the DO concen- tration in the medium. OUR was measured by shutting o airow:

16

= - QO2X Vic chuyn giao tc oxy (OTR) = KLa(C* - CL), v OUR = QO2X, trong KL

l giai on chuyn giao h s khi lng cht lng, a cht kh lng gia khu

vc, K La h s th tch chuyn oxy, C* nng oxy bo ha, v C L nng DO trong mi trng. OUR c o bng cch ngt dng khng kh: = - QO2X Oxygen concentration in the fermentation broth was converted from % air saturation to mmol O2/l as fol-lows: the DO electrode was calibrated in medium at 30C and then transferred to air-saturated distilled water at known temperature and ambient pressure. This reading, together with the known saturation concentration of oxygen in distilled water (0.07559 mmol/l at 30C) (100%), was used to estimate the saturation concentration of oxygen in cultivation medium at 30C. Nng oxy trong nc dng ln men c chuyn i t bo ha khng kh % n mmol O2/l nh sau: cc in cc c hiu chun DO trong mi trng 30C v sau chuyn cho khng kh bo ha nc ct nhit c bit n v p sut xung quanh. iu ny c, cng vi nng bo ha oxy trong ting nc ct (0,07559mmol/l 30C) (100%), c s dng c tnh nng bo ha oxy trong mi trng canh tc 30C.

Results and discussionEnhancement of viable cell and spore count

VC and SC proles, Tx potential and proteolytic activity were very similar in shake ask, bench and pilot scale fermentor (Figs. 1, 2, 3). A short lag phase was observed in shake ask as well as in bench and pilot fermentors;

17

Fig. 1 Total viable cells (VC), spore counts (SC), entomotoxicity (Tx) and protease activity (PA) during fermentation in shake asks this could be attributed to good adaptation of Bt in the pre-culture stage. The preculture stage allowed the bacteria to synthesize specic enzymes for the degradation of the organic matter present in sludge, and use the generated energy for cellular development [20].

Kt qu v tho lunNng cao s tn ti ca t bo v s bo t Bn cht VC v SC, Tx c tim nng v hot ng phn gii protein rt ging nhau trong bnh lc, quy m nh v quy m ln men th im (Figs 1, 2, 3). Giai on ngn ca pha lag c quan st trong bnh lc cng nh quy m nh v ln men th im.

18

Hnh. 1 Tng s cc t bo tn ti(VC), s bo t (SC), tnh c i vi su (Tx) v hot ng ca protease (PA) sut qu trnh ln men trong bnh lc.

iu ny c th tng trng cho kh nng thch ng tt ca Bt trong giai on trc ln men. Giai on trc ln men cho php cc vi khun tng hp enzym c th cho s phn hy ca cc hp cht hu c trong bn, v s dng nng lng c to ra pht trin t bo [20]. Irrespective of the scale of fermentation, exponential growth occurred during the rst 910 h (Figs. 1, 2, 3): the maximum values of specic growth rates (max) based on total VC count were 0.354, 0.409 and 0.569/h in shake ask, bench scale fermentor and pilot scale fermentor, respectively (Table 3). Figures 1, 2 and 3 also

19

Fig. 2 a VC, SC, Tx and PA during fermentation in a bench-scale fermentor. b Proles of dissolved oxygen (DO), stirrer speed, volumetric oxygen transfer coecient (KLa), oxygen transfer rate (OTR) and oxygen uptake rate (OUR) in bench-scale fermentor (10 l working volume)

Fig. 3 a VC, SC, Tx and PA during fermentation in a pilot-scale fermentor. b DO, stirrer speed, KLa, OTR and OUR proles in pilot scale fermentor (100 l working volume)

Khng phn bit quy m ca qu trnh ln men, tng trng theo cp s nhn xy ra trong 9-10 gi u tin (Figs. 1, 2, 3): gi tr ti a ca tc tng trng c th (max ) da trn tng s VC l 0,354, 0,409 v 0,569 / h trong bnh lc, ln men quy m nh v quy m ln men th im, tng ng (Bng 3). Con s 1, 2 v 3 cng

20

Hnh. 2 a VC, SC, Tx v PA trong qu trnh ln men quy m ln men nh. b Bn cht ca oxy ha tan (DO), tc khuy, h s vn chuyn th tch oxy (KLa), t l chuyn oxy (OTR) v t l hp thu oxy (OUR) quy m ln men nh (10 lt).

21

Hnh. 3 a VC, SC, Tx v PA trong thi gian ln men quy m th im. b DO, khuy tc , KLa, bn cht OTR v OUR trong ln men quy m th im (100 lt)

show that it is dicult to demarcate vegetative growth and spore production; spore concentration increased from zero time. The presence of spores in the sample drawn just after inoculation (zero time) indicated that there were spores in the inoculum. However, the increase in total VC count (spore and vegetative cells) showed that active growth continued for 910h followed by a slower growth (Figs. 1, 2, 3). Exponential growth per- sisted for about 6 h. cho thy rng n rt kh phn ranh gii tng trng t nhin v sn xut bo t; tp trung bo t tng t khng gi. S hin din ca cc bo t trong mu c rt ra ngay sau khi bm chng (khng gi) ch ra rng c cc bo t trong cht bm. Tuy nhin, vic tng s lng tng VC (bo t v t bo t nhin) cho thy22

hot ng tng trng tip tc trong 90-10 gi tip theo l mt s tng trng chm hn (Figs 1, 2, 3). Tng trng theo cp s nhn tn ti trong khong 6 h. The VC count increased by 3855% in the bioreactors compared to in shake asks (Table 3). The reason for this increase might be the comparatively high lmax value and controlled conditions of pH and DO in bioreactors. The higher max in the fermentor could be attributed to better oxygenation capacity of the medium at the bench and pilot scale, in agreement with the conclusions of others [1, 36] working with gruel and molasses as raw material, respectively. S m VC tng 38-55% trong cc phn ng sinh hc so vi trong bnh lc (Bng3). L do cho s gia tng ny c th l tng i cao max v iu kin kim sot ca pH v DO trong nui cy lng lc. Gi tr max trong ln men c th c quy cho kh nng oxy ha tt hn ca mi trng quy m nh v quy m th im, trong s tng thch vi kt lun ca ngi khc [1, 36] vi nguyn liu l cho v mt ng lm nguyn liu th, tng ng. Table 3: Fermentation parameters and performance of the Bacillus thuringiensis (Bt) process at dierent production scales. SS Sus-pended solids, max maximum growth rate, VC total viable cells, SC spore count, Tx entomotoxicity, PA proteolytic activity. Bng 3: Cc thng s ln men v hiu sut ca vi khun Bacillus thuringiensis (Bt) trong qu trnh sn xut cc quy m khc nhau. Cht rn SS, max tc tng trng ti a, tng s VC cc t bo c th tn ti, s bo t SC, tnh c i vi su Tx, PA hot ng phn gii protein.

23

Flask

Bench scale 300-500

Pilot scale Tc

Bnh

Quy m nh 300-500

Quy m th im 100-200

Agitation (rpm) 250

100-200

khu y (rpm) Sc kh (vvm) SS (g / l) max (mi gi) VC (48 h) (cfcu/ml) SC (48 h) (cfu/ml) Tx (48 h) (10 9 IU/l) Max PA (IU/ml)

250

Aeration (vvm) SS (g/l) 25

0.3 25 0.409 5,97 e+08 5,37e+ 08 12

0.3 25 0.567 6,67e+ 08 5,53e +08 13

25 0.354

0.3 25 0.409

0.3 25 0.567

max (per hour) 0.354 VC (48 h) (cfcu/ml) SC (48 h) (cfu/ml) Tx (48 h) (10 9 IU/l) Max PA (IU/ml) 4,3 e+08 3,97e+08 10 1,2 at 24h

4,3 e+08 5,97 e+08 6,67 e+08 3,97e+08 5,37 e+08 5,53 e+08 10 1,2 lc 24 gi 12 2,6 lc 36gi 13 4,1 lc 30gi

2,6 at 36 h 4,1 at 30 h

An increase of about 35% in SC was observed going from shake ask to fermentor experiments (Table 3). It is important to point out that sporulation was not affected by the change of scale under controlled conditions. The SC was approximately the same at bench and pilot scale (5.35.5 e+08 cfu/ml); this could be due to non-limiting oxygen conditions. Tng khong 35% trong SC c quan st t khi lc bnh ln men th nghim (Bng 3). iu quan trng l ch ra rng hnh thnh bo t khng b nh hng bi s thay i v quy m trong iu kin kim sot. Cc SC xp x nh nhau quy m nh v th im (5,3-5,5 e+ kin hn ch oxy. Enhancement of Tx potential08

cfu /ml), iu ny c th l do iu

24

Tx potential was measured from 12 h of growth and showed an almost linear increase with fermentation time. Early spore production (Figs. 1, 2, 3) resulted in a concomitant increase in Tx value irrespective of scale. Tx values were not measured before 12 h; however, there could be production of Tx in the medium before 12 h along with spore production. Tx obtained at the end of active growth (12 h) was substantial, 50% of maximum (at the end of fermentation) in shake asks and bench scale fermentors and 69% in pilot scale fermentors (Figs. 1, 2, 3). This is contrary to the ndings of many authors who stated that, during exponential growth, cells do not contain the crystal toxin antigen and are nontoxic [21]. Tng cng tim nng Tx Tx tim nng c o t 12h ca s tng trng v cho thy s gia tng gn nh tuyn tnh vi thi gian ln men. Sm sn xut bo t (Hnh 1, 2, 3) dn n s gia tng ng thi gi tr Tx khng phn bit quy m. Tx gi tr khng o trc 12h, tuy nhin, c th c sn xut Tx trong mi trng trc khi 12 h cng vi sn xut bo t. Tx thu c phn cui ca s tng trng tch cc (12h) l ng k, 50% ti a ( cui qu trnh ln men) vo bnh lc v quy m ln men nh v 69% trong quy m ln men th im (Hnh 1, 2, 3). iu ny tri vi nhng pht hin ca nhiu tc gi ni rng, trong qu trnh tng trng theo cp s nhn, cc t bo khng cha c t khng nguyn tinh th v khng c hi [21]. One of the reasons for this discrepancy could be slow Bt growth in the sludge medium, which might have helped the spores to mature properly and provided sucient time for the formation of endotoxin before cell lysis occurred [33]. Aronson [5] also showed that a crystal toxin antigen appears in the cells strictly during spore engulfment (stage III) and cortex synthesis (stage IV). This could explain the substantial Tx value obtained after 24 h fermentation; Tx values were 70%, 75% and 77% in shake ask, bench scale fermentor and pilot scale fermentor, respectively (Figs. 1, 2, 3). The balance of the Tx potential appeared

25

during the remainder of the fermentation course (2448 h), where there was no substantial increase in SC. Mt trong nhng l do cho s khc bit ny c th lm chm qu trnh tng trng ca Bt trong mi trng bn, c th gip cc bo t trng thnh ng v cung cp thi gian cho s hnh thnh ca ni c t trc khi ly gii t bo xy ra [33]. Aronson [5] cng cho thy mt khng nguyn c t tinh th xut hin trong cc t bo ng trong giai oan hnh thnh bo t (giai on III) v tng hp v (giai on IV). iu ny c th gii thch nhng gi tr Tx ng k thu c sau 24h ln men; Tx c gi tr l 70%, 75% v 77% trong bnh lc, ln men quy m nh v quy m th im, tng ng (Hnh 1, 2, 3). S cn bng v tim nng Tx xut hin trong phn cn li ca qu trnh ln men (24-48h), ni khng c s gia tng ng k trong SC. The highest entomocidal activity was also found near the end of cultivation, as shown in Figs. 1, 2 and 3. A 30% improvement in Tx potential was obtained at pilot scale as compared to shake asks. In general, the high- er max values obtained in bench and pilot fermentors (Table 3) resulted in higher VC, SC and Tx, as fast-growing cells carry higher energy, leading to higher sporulation and Tx potency [29, 32]. Cc tnh c hot ng cao nht c tm thy gn cui canh tc, nh trong Hnh 1, 2 v 3 Mt ci tin 30% ca tim nng Tx thu c quy m th im so vi bnh lc. Nhn chung, gi tr max cao hn t c quy m nh v ln men th im (Bng 3) kt qu VC, SC v Tx cao hn, nh pht trin cc t bo nhanh chng mang nng lng cao hn, dn n hnh thnh bo t cao hn v tim nng Tx [29, 32]. Proteolytic activity Bacillus are typical carrion dwellersthey are well adapted to digest varied and complex substrates. From the onset of sporulation, Bt synthesizes dierent types

26

of proteases required to hydrolyze complex proteins in order to satisfy its nutritional needs [8, 10]. Hot ng phn gii protein Bacillus l sinh vt k sinh in hnh, chng thch nghi phn gii cc hp cht phc tp khc nhau. Khi bt u hnh thnh bo t, Bt tng hp cc loi protease cn thit khc nhau thy phn protein phc tp p ng nhu cu dinh dng ca chng [8, 10]. The evolution of PA produced at dierent production scales is illustrated in Figs. 1, 2 and 3. The maximum PA observed was 1.2 (24 h), 2.6 (36 h) and 4.1 (30 h) IU/ml in shake ask, bench and pilot scale fermentors, respectively. At all production scales, a decline in PA was recorded after attaining the maximum level. Thismight be due to inactivation of proteases due to inter-actions with other compounds in the culture medium. S tin trin ca PA quy m sn xut khc nhau c minh ha trong Hnh 1, 2 v 3. Gi tr PA ti a quan st l 1,2 (24h), 2.6 (36h) v 4.1 (30h) IU/ml trong bnh lc, ln men quy m nh v quy m th im, tng ng. Ti tt c cc quy m sn xut, mc gim ca PA c ghi li sau khi t mc ti a. iu ny c th l do s bt hot protease tng tc vi cc hp cht khc trong mi trng ln men. PA increased by 2 and 3.4 times at bench and pilot scale, respectively, compared to the maximum activity obtained in shake asks (Table 3). This could be due to an increase in oxygen transfer, and pH control.Tyagi et al.[30] obtained proteolytic activities in fermentors that were four times higher than those obtained in shake asks using sewage sludge as raw material for Bt production. Meunier [22] showed that changes in pH during growth of Bt in shake ask experiments could inactivate proteolytic enzymes. PA tng t 2 n 3,4 ln quy m nh v th im, tng ng, so vi hot ng ti a t c trong bnh lc (Bng 3). iu ny c th l do s gia tng vn chuyn oxy, v kim sot pH. Tyagi v cng s [30] thu c cc hot ng27

phn gii protein trong ln men t bn ln so vi nhng ngi thu c trong bnh lc bng cch s dng bn thi lm nguyn liu cho sn xut Bt. Meunier [22] cho thy rng thay i pH trong qu trnh pht trin ca Bt trong cc th nghim bnh lc c th bt hot cc enzym thy phn protein. Moreover, the maximum PA (4.1 IU/ml) was obtained in pilot scale without aecting the production of -endotoxin. These results are in line with those of Tyagi and co-workers [30], who showed that a substantial amount of alkaline proteases, as well as Bt toxins, could be produced simultaneously using sludge as raw material without aecting the Tx of the nal fermentor broth. Moreover, a formulation devoid of protease oers a more stable product. Therefore, it is preferable to eliminate the protease in the supernatant by centrifugation and use the residual solids containing Bt cells, spores and endotoxintions, and the protease in the supernatant could be recovered for further use [36,37]. Hn na, PA ti a (4,1 IU/ml) c thu thp quy m th im m khng nh hng n vic sn xut - ni c t. Cc kt qu ny ph hp vi Tyagi v ng nghip [30], ch ra rng mt s lng ng k cc protease kim, cng nh c t Bt, c th c sn xut ng thi s dng bn thi lm nguyn liu th m khng nh hng n Tx trong ln men chnh. Hn na, hn ch to ra cc protease cung cp mt sn phm n nh hn. V vy, n thch hp hn loi b cc protease trong phn ni trn mt bng ly tm v s dng cc cht rn c cha Bt t bo, bo t v ni c t. iu ny s lm gim nhng tc ng tiu cc ca protease Bt, v cc protease phn ni trn mt c th c thu hi s dng tip [36,37]. OUR, OTR and K L a pattern Proles of KLa, OUR, and OTR in bench and pilot scale fermentors are presented in Figs. 2 and 3, respectively. During the rst stage of fermentation (rst 6 h of growth), a decrease in DO was noticed due to the higher OUR resulting from active growth of Bt (exponential growth). Thereafter (1015 h of fermentation), the oxygen consumption rate decreased and the oxygen concentration increased28

and remained relatively constant until the end of fermentation. In wastewater sludgemedia, KLa increased at the beginning of fermentation and then uctuated somewhat irregularly during the rest of the fermentation due to changes in impeller speed and metabolite production. M hnh OUR, OTR v K La Bn cht ca K La, OUR, v OTR quy m ln men th im v quy m nh c trnh by trong Hnh 2 v 3, tng ng. Trong giai on u ca qu trnh ln men (6h tng trng), DO gim c ch do OUR cao hn thu c t tng trng tch cc ca Bt (m tng trng). Sau (10-15h ln men), t l tiu th oxy gim v nng oxy tng ln v vn tng i n nh cho n khi kt thc qu trnh ln men. Trong mi tng nc thi bn, K La tng vo lc bt u ln men v sau bin ng bt thng trong phn cn li ca qu trnh ln men do thay i tc iu khin v sn xut cht chuyn ha. Increasing the agitation and aeration rates is the tra ditional approach used to enhance OTR. Increased agitation produces more gas dispersion, and gas dispersion produces higher oxygen transfer [23]. However, in pilot scale, even at low stirrer speed, KLa values were higher than in bench-scale fermentors (Figs. 2, 3). This could be due to the small size of air bubbles produced due to hydrostatic pressure in the pilot-scale bioreactor. In fact, bubble characteristics have been found to strongly aect the value of KLa [15]. Juarez and Orejas [17] also found an increase in KLa with decreased bubble size. Tng t l tc khuy v thng kh l phng php truyn thng c s dng tng cng OTR. Tng tc khuy trong sn xut lm phn tn kh nhiu hn, v kh phn tn trong sn xut cao hn vn chuyn oxy [23]. Tuy nhin, trong quy m th im, ngay c tc khuy thp, K La gi tr K La cao hn quy m ln men nh (Hnh 2, 3). iu ny c th l do kch thc nh ca bt kh c sn xut do p lc thy tnh trong cc phn ng lng lc quy m th im. Trong thc t, c im bt kh c tm thy mnh nh hng n gi tr ca

29

KLa [15]. Juarez v Orejas [17] cng cho thy s gia tng KLa bt kh vi kch thc gim. OUR values were useful determinants of the dierent phases of the Bt growth cycle during batch fermentation [9]. The peaks of KLa, OUR and OTR occurred at the same time, 6 h after inoculation (Figs. 2 and 3), then dropped sharply. The sharp decrease in KLa, OUR and OTR could be identied as marking the end of the exponential growth phase. OUR and OTR in pilot scale fermentor are higher than those in bench scale (Figs. 2, 3), and this resulted in a higher specic growth rate in pilot scale (Table 3). This is in accord with the obser- vation of Rowe et al. [24], who observed that OUR in-creased with max. OUR l yu t quyt nh gi tr hu ch ca cc giai on khc nhau ca chu k tng trng Bt trong hng lot qu trnh ln men [9]. Cc nh ca KLa, OUR v OTR xy ra cng mt lc, 6 gi sau khi bm chng (Hnh 2 v 3), sau gim mnh. Cc mc gim mnh trong KLa, OUR v OTR c th c xc nh l nh du s kt thc ca giai on tng trng theo cp s nhn OUR v OTR trong ln men quy m th im l cao hn so vi quy m nh (Hnh 2, 3), v iu ny dn n mt s tng trng cao hn t l c th v quy m th im (Bng 3). iu ny l ph hp vi s quan st ca Rowe v cng s [24], nhng ngi quan st thy rng OUR gia tng vi max Dierences in KLa, OUR and OTR between the two fermentor scales can be attributed to changes in the interfacial area (a), which is aected by the volume of liquid, liquid height in the vessel, the dimensions of the fermentation vessel, bubble size, and the pattern and vigor of agitation. S khc nhau trong K La, OUR v OTR gia hai quy m ln men c th c quy cho nhng thay i trong khu vc gia (a), l b nh hng bi khi lng, chiu cao cht lng, cht lng trong bnh, cc kch thc ca bnh ln men, kch thc bt, v kiu ln men v mnh ca tc khuy. Correlation between K La and proteolytic activity

30

OTR and pH are the key variables in the production of alkaline proteases [3, 18]. A correlation has been found between the K La and PA in submerged fermentation. Oxygen transfer is enhanced at larger scales because higher supercial velocities are achieved when the dimensional ratios of the vessels are held constant [15, 27]. S tng quan gia KL a v hot ng phn gii protein OTR v pH l cc ch tiu quan trng trong vic sn xut cc protease kim [3,18]. Mt s tng quan c tm thy gia cc K La v PA ln men chm. S vn chuyn oxy c tng cng cc quy m ln hn v vn tc cao hn b ngoi t c khi cc t l kch thc ca bnh c n nh [15, 27]. The increase of OTR in the pilot scale fermentor in- creased the PA (Figs. 2, 3). At pilot scale fermentor, aeration was achieved by a gas sparger consisting of a perforated ring located at the bottom of the reactor. This sparger supplied a smaller bubble size than the bench-scale fermentor (air supply by perforated tube). The smaller bubble size could also have been created through higher hydrostatic pressure in the pilot-scale fermentor. The benet of small bubbles is the slow rising velocity, which keeps air bubbles in the liquid longer, allowing more time for the oxygen to dissolve [11]. Furthermore, small air bubbles provided a higher interfacial area (a) than large air bubbles [26]. S gia tng ca OTR trong ln men quy m th im lm tng PA (Hnh 2, 3). Ti ln men quy m th im, thng kh t c bi h thng sc kh gm mt vng khoan nm di cng ca l phn ng. H thng sc kh ny cung cp mt kch thc bt nh hn so vi quy m ln men nh (khng kh cung cp bi ng khoan). Kch thc bt nh hn c th c to ra thng qua p lc thy tnh cao hn quy m ln men th nghim. Li ch ca bt nh l lm chm tng vn tc, m gi bt kh trong cht lng di hn, cho php nhiu thi gian hn cho oxy ha tan [11]. Hn na, bt khng kh nh cung cp cho khu vc gia cao hn (a) so vi bt kh ln [26]. Stirrer speed could also aect PA. A higher agitation rate in bench scale (300500 rpm) compared to pilot scale (100190 rpm), increased oxygen transfer in the31

vessel, but did not improve enzyme production. This could be due to strong shear rates, which could lead to deterioration of the cells and hence the enzyme yield [26]. Tc khuy cng c th nh hng n PA. Mt t l cao hn v tc khuy quy m nh (300-500 vng/pht) so vi quy m th im (100-190 vng/ pht), tng chuyn oxy trong bnh, nhng khng nh hng n sn xut enzym. iu ny c th l do tc va chm mnh, dn n s suy gim ca cc t bo v do sn lng enzyme [26]. Process performance The weak performance (max,VC, SC, Tx and PA) recorded in shake asks compared to fermentors could be explained by the fact that Bt fermentation in shake asks presents certain limitations at the level of control of major fermentation parameters including pH, oxygen transfer, considerable evaporation and poor mixing. Humphrey [14] revealed that the cotton plug could be a factor limiting oxygen transfer. Quy trnh thc hin Cc hot ng yu km (max,VC, SC, Tx v PA) c ghi li trong bnh lc so vi ln men c th c gii thch bi thc t l Bt ln men trong bnh lc trnh by nhng hn ch nht nh cp kim sot cc thng s qu trnh ln men chnh bao gm pH, s vn chuyn oxy, thng kh ng k v s khuy trn km. Humphrey [14] cho thy cc nt bng c th l mt yu t hn ch chuyn oxy. It is clear that under controlled conditions of pH and DO, a signicant enhancement of VC, SC, Tx potential and PA are observed. The dierences observed between the bench and pilot-scale runs were presumably related to the mixing and ow characteristics of each fermentor. In general, the mixing time is not the same in reactors of dierent scale [12]. R rng l trong iu kin c kim sot pH v DO, mt yu t quan trng ca VC, SC, tim nng Tx v PA c quan st. S khc bit quan st gia cc th im quy m nh cha v th im chy c cho l lin quan n cc c tnh32

pha trn v lu lng ca mi ln ln men. Nhn chung, thi gian pha trn l khng ging nhau trong l phn ng quy m khc nhau [12]. Wastewater sludge contains a large quantity of proteins, which results in intense foaming during exponential growth of Bt. However, adequate control of agitation and aeration with a well-optimized sludge solids concentration can avoid problems related to foam formation during fermentation. Higher aeration rates often encounter the problem of excessive foaming, especially during exponential growth of Bt using sludge as raw material [31]. X l nc thi bn c cha mt lng ln protein, kt qu l to bt mnh trong qu trnh tng trng theo cp s nhn ca Bt. Tuy nhin, kim sot y tc khuy v thng kh vi nng ti u ha cng nh cc cht rn bn c th trnh cc vn lin quan n hnh thnh bt trong qu trnh ln men. T l thng kh cao hn thng gp phi vn ca qu nhiu cht to bt, c bit trong tng trng theo cp s nhn ca Bt s dng bn lm nguyn liu [31]. For large-scale production of Bt, the supply of raw material is not problematic as wastewater sludge is abundantly available. However, this complex medium is characterized by an inconsistent and unknown composition, especially regarding the availability of assimilable nitrogen and carbon sources, which may lead to a great variability between fermentation batches. i vi sn xut quy m ln ca Bt, vic cung cp nguyn liu khng c vn nh nc thi bn l di do sn c. Tuy nhin, mi trng phc tp ny c c trng bi mt thnh phn cha bit, c bit l v s sn c ca nit ng ha v cc ngun carbon, c th dn n mt bin i ln gia cc m ln men. However, keeping the solids concentration at an optimum level of 2025 g/l [20] in the fermentor will give a fairly constant process performance. The eect of variability of sludge composition on Bt growth and toxin synthesiswas veried by collecting sludge samples from the CUQ wastewater treatment plant at dierent times (to cover seasonal, day and night and other possible variations) for a period

33

of 1 year. These samples were used to grow Bt under similar conditions in shake asks. Tuy nhin, gi cho nng cht rn mt mc ti u 20-25g/l [20] trong ln men s cung cp cho qu trnh ln men lin tc mt cch ng u. nh hng ca s bin i cc thnh phn bn vo s tng trng Bt v tng hp c t c xc minh bng cch thu thp cc mu bn t cc nh my x l nc thi CUQ vo cc thi im khc nhau ( trang tri theo ma, ngy v m v cc bin th c th khc) trong thi gian 1 nm. Nhng mu ny c s dng pht trin Bt trong iu kin tng t trong bnh lc. It was found that, utilizing the optimal suspended solids concentration of 2025 g/l, the variation in Tx value was between +9 and -13% (unpublished data). Another way to combat the variation in sludge characteristics and its consequences on fer- mentation could be the use of dewatered sludge. Dewa- tered sludge can be stored over time without changes in composition. However, dewatered sludge furnished 20% lower Tx in comparison to fresh sludge at the same solids concentration (25 g/l) [33]. C th thy rng, s dng ti u cc cht rn l lng nng 20-25g/l, s bin ng v gi tr Tx c gia +9 v -13% (khng cng b d liu). Mt cch khc chng li s bin ng v c im bn v hu qu ca n i vi qu trnh ln men c th l s dng bn long. Bn long c th c lu tr theo thi gian m khng thay i trong thnh phn. Tuy nhin, hm lng bn long Tx 20% thp hn so vi bn ti cng nng cht rn (25g /l) [33]. In conclusion, the performance and reproducibility of the process at two dierent production scales has been demonstrated. The scale-up study has conrmed the optimal conditions derived from the small-scale opti- mization study, resulting in higher Tx potential and a high PA. Thus, this process provides solutions for safe wastewater sludge disposal and production of high potency and low cost biopesticides.

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Tm li, hiu sut v kh nng ti sn xut ca qu trnh ny c chng minh ti hai quy m sn xut khc nhau. Nghin cu quy m xc nhn cc iu kin ti u xut pht t vic nghin cu ti u ha quy m nh, dn n tim nng Tx cao hn v PA cao. Nh vy, qu trnh ny cung cp cc gii php x l an ton bn nc thi v sn xut hiu qu cao v chi ph thuc tr su thp. Acknowledgements The authors are thankful to the Natural Sciences and Engineering Research Council of Canada for nancial support (Grants A4984, STR 202047, SCF 19219096 and Canada Research Chair). Li cm n Cc tc gi rt bit n n trung tm Khoa hc T nhin v Hi ng nghin cu ca Canada h tr ti chnh (Ti tr A4984, STR 202047, SCF 192190-96 v ch tch nghin cu Canada). References 1. Abdel-Hameed A (2001) Stirred tank culture of Bacillus thuringiensis H-14 for production of the mosquitocidal d-endotoxin: mathematical modelling and scalingup studies. World J Microbiol Biotechnol 17:857861 2. Aiba S, Humphrey AE, Millis NF (1973) Biochemical engineering, 2nd edn. Academic, New York 3. Anwar A, Saleemuddin M (1997) Alkaline proteases: a review.Bioresour Technol 64:175183 4. APHA, AWWA, WPCF (1989) Standard methods for examination of waters and wastewaters, 17th edn. American PublicHealth Association, Washington DC 5. Aronson A (2002) Sporulation and d-endotoxin synthesis by Bacillus thuringiensis. Cell Mol Life Sci 59:417425

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6. Avignone-Rossa C, Arcas J, Mignone C (1992) Bacillus thuringiensis, sporulation and d-endotoxin production in oxygen limited and non-limited cultures. World J Microbiol Biotechnol 8:301304 7. Beegle CC (1990) Bioassay methods for quantication of Bacillus thuringiensis d-endotoxin. In: Hickle LA, Fitch WL (eds) Analytical chemistry of Bacillus thuringiensis. American Chemical Society, Washington DC, pp 1421 8. Braun S (2000) Bioassays of Bacillus thuringiensis, 1D. In: Navon A, Ascher KRS (eds) Production of Bacillus thuringiensis insecticides for experimental uses. Bioassays of entomopathogenic microbes and nematodes. CAB International, Wallingford, UK, pp 4972 9. Chang SW (1993) Studies of growth conditions and estimations of oxygen uptake rate for Bacillus thuringiensis. Thesis, University of Illinois, p 95 10. Chu IM, Lee C, Li TS (1992) Production and degradation of alkaline protease in batch culture of Bacillus subtilus ATCC 14416. Enzyme Microb Technol 14:755761. 12. Flores EA, Perez F, De La Torre M (1997) Scale-up of Bacillus thuringiensis fermentation based on oxygen transfer. J Ferment Bioeng 83:561564 13. Hsu YL, Wu WT (2002) A novel approach for scaling-up a fermentation system. Biochem Eng J 11:123130 14. Humphrey A (1998) Shake asks to fermentor: what have we learned? Biotechnol Prog 14:37 15. Jin B, Leeuwen J, Doelle HW, Yu Q (1999) The inuence of geometry on hydrodynamic and mass transfer characteristics in an external airlift reactor for the cultivation of lamentous fungi. World J Microbiol Biotechnol 15:737936

16. Ju LK, Chase GG (1992) Improved scale-up strategies of bioreactors. Bioprocess Eng 8:4953 17. Juarez P, Orejas J (2001) Oxygen transfer in a stirred reactor in laboratory scale. Latin Am Appl Res 31:433439 18. Kumar CG, Takagi H (1999) Microbial alkaline proteases: from a bioindustrial viewpoint. Biotechnol Adv 17:561594 19. Kunitz M (1947) Crystalline soybean trypsin inhibitor. J Gen Physiol 30:291 310 20. Lachhab K, Tyagi RD, Valero JR (2001) Production of Bacillus thuringiensis biopesticides using wastewater sludge as a raw material: eect of inoculum and sludge solids concentration. Process Biochem 37:197208 21. Liu CM, Tzeng YM (2000) Characterization study of the sporulation kinetics of Bacillus thuringiensis. Biotechnol Bioeng 68:1117 22. Meunier N (1999) Evaluation du potentiel de production de proteases bacteriennes a partir de boues depuration municipales. Msc Thesis, INRS-Eau, Universite du Quebec 23. Parakulsuksatid P (2000) Utilization of microbubble dispersion to increase oxygen transfer in pilot scale bakers yeast fermentation unit. MSc Thesis, University of Virginia 24. Rowe GE, Margaritis A, Wei N (2003) Specic oxygen uptake rate variations during batch fermentation of Bacillus thuringiensis subspecies kurstaki HD. Biotechnol Prog 19:14391443

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33. Vidyarthi AS, Tyagi RD, Valero JR, Surampalli RY (2002) Studies on the production of Bacillus thuringiensis based bio- pesticides using wastewater sludge as a raw material.Water Res 36:48504860 34. Yamamoto T (1982) Identication of entomocidal toxins of 129:25952603 35. Yang XM, Wang SS (1998) Development of Bacillus thuringiensis fermentation and process control from a practical perspective. Biotechnol Appl Biochem 28:9598 36. Zouari N, Jaoua S (1999) Production and characterization of metalloproteases synthesized concomitantly with d-endotoxin by Bacillus thuringiensis subsp. kurstaki strain grown on gruel-based media. Enzyme Microbial Technol 25:364 371 37. Zouari N, Achour O, Jaoua S, (2002) Production of delta- endotoxin by Bacillus thuringiensis subsp kurstaki and over coming of catabolite repression by using highly concentrated gruel and sh meal media in 2 and 20 dm3 fermentors. J Chem Technol Biotechnol 77:877882. Ti liu tham kho 1. Abdel-Hameed A (2001) khuy b vn ha ca vi khun Bacillus thuringiensis 14-H cho sn xut ca cc mosquitocidal 861 2. Aiba S, Humphrey AE, Millis NF (1973) Sinh hc k thut, ti bn ln 2. Hc tp, New York -Ni c t: m hnh ton hc v cc nghin cu nhn rng. J th gii Microbiol Biotechnol 17:857Bacillus

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4. APHA, AWWA, WPCF (1989) tiu chun phng php kim tra cc vng nc v nc thi, ti bn ln 17. Hip hi Y t cng cng M, Washington DC 5. Aronson Mt hnh thnh bo t (2002) v -Ni c t tng hp ca vi khun

Bacillus thuringiensis. Cell Mol Cuc sng Khoa hc 59:417-425

6. Avignone-Rossa C, Arcas J, Mignone C (1992) Bacillus thuringiensis, hnh thnh bo t v -Ni c t sn xut trong nn vn ha oxy hn ch v khng hn ch. J th gii Microbiol Biotechnol 8:301-304 7. Beegle CC (1990) BioAssay phng php nh lng vi khun Bacillus thuringiensis -Ni c t. Trong: Hickle LA, Fitch WL (bin son) phn tch ha hc ca vi khun Bacillus thuringiensis. Hi Ha hc M, Washington DC, trang 14-21 8. Braun S (2000) sinh trc nghim ca Bacillus thuringiensis, 1D. Trong: Navon A, Ascher KRS (bin son) Sn xut thuc tr su Bacillus thuringiensis s dng th nghim. Cc sinh trc nghim ca cc vi khun entomopathogenic v tuyn trng. CAB International, Wallingford, Anh, trang 49-72 9. Chang SW (1993) nghin cu v iu kin tng trng v c tnh t l hp thu dng kh cho vi khun Bacillus thuringiensis. Lun vn, i hc Illinois, p 95 Chu IM, Lee C, Li TS (1992) Sn xut v suy thoi ca protease kim trong 10. nn vn ha hng lot ca subtilus Bacillus ATCC 14416. Enzyme Microb40

Technol 14:755-761 Doran PM (1995) X L Sinh nguyn tc k thut. Hc tp, San Diego 11. Flores EA, Perez F, De La Torre M (1997) Quy m-up ln men vi khun 12. Bacillus thuringiensis da trn chuyn oxy. J men Bioeng 83:561-564 YL Hsu, Ng WT (2002) Mt phng php mi cho h thng rng mt-up ln 13. men. Biochem Eng J 11:123-130 Humphrey A (1998) Lc bnh ln men: chng ti c nhng g hc? 14. Biotechnol Prog 14:3-7 Jin B, Leeuwen J, Doelle HW, Yu Q (1999) S nh hng ca hnh hc v 15. c im thy ng lc v khi lng chuyn nhng trong mt l phn ng khng vn bn ngoi cho vic trng nm si. J th gii Microbiol Biotechnol 15:73-79 Ju LK, Chase GG (1992) chin lc m rng quy m ci tin ca phn ng 16. sinh hc. X L Sinh Eng 8:49-53 Juarez P, Orejas J (2001) chuyn oxy trong mt khuy ng l phn ng v 17. quy m trong phng th nghim. Am Latin Res Appl 31:433-439 Kumar CG, Takagi H (1999) protease kim vi sinh vt: t mt quan im 18. bioindustrial. Biotechnol ADV 17:561-594 Kunitz M (1947) cht c ch trypsin u nnh tinh th. J tng Physiol 19. 30:291-310 Lachhab K, Tyagi RD, Valero JR (2001) Sn xut biopesticides Bacillus 20. thuringiensis s dng bn thi lm nguyn liu: nh hng ca nng cht rn bn v truyn cht c. Quy trnh Biochem 37:197-208

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Liu CM, Tzeng YM (2000) M t c tnh nghin cu v ng hc hnh thnh 21. bo t ca vi khun Bacillus thuringiensis. Biotechnol Bioeng 68:11-17 Meunier N (1999) nh gi du potentiel de sn de protease bactriennes 22. partir de boues d puration municipales. Lun vn thc s, INRS-Eau, Universit du Qubec Parakulsuksatid P (2000) S dng phn tn microbubble tng chuyn oxy 23. trong bnh quy m th im ln men ca nm men n v. Lun vn thc s, i hc Virginia Rowe GE, Margaritis A, Wei N (2003) cc bin th c th t l hp thu oxy 24. trong qu trnh ln men ca phn loi vi khun Bacillus thuringiensis kurstaki HD. Biotechnol Prog 19:1439-1443 Sachdeva V, Tyagi RD, Valero JR (2000) Sn xut biopesticides nh l mt 25. phng php mi ca vic s dng bn thi / x l. Khoa hc nc Technol 42:211-216 Sachidanadham R, Kumar IA, Krishnan MR, Jayaraman K (1999) M hnh 26. ton hc da trn d ton ca h s chuyn oxy th tch trong sn xut cc enzyme phn gii protein trong Bacillus amyloliquefaciens. X L Sinh Eng 2:319-322 Schell DJ, nng dn J, Hamilton J, Lyons B, McMillan JD, Saez JC, Tholudur 27. A (2001) nh hng ca iu kin hot ng v kch thc tu chuyn oxy trong qu trnh sn xut cellulase. Appl Biochem Biotechnol 91:627-642 Tirado-Montiel ML, Tyagi RD, Valero JR (2001) x l nc thi bn nh mt 28. nguyn liu sn xut vi khun Bacillus thuringiensis biopesticides da. Res nc 35:3807-3816

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Tirado-Montiel ML, Tyagi RD, Valero JR, Surampalli RY (2003) 29. biopesticides sn xut bng cch s dng bn thi lm nguyn liu: tc ng ca qu trnh tham s. Khoa hc nc Technol 48:239-246 Tyagi RD, Foko VS, Barnabe S, Vidyarthi AS, Valero JR (2002) ng thi 30. sn xut, kim protease do Bacillus thuringiensis biopesticide s dng bn thi lm nguyn liu th. Khoa hc nc Technol 46:247-254 Vidyarthi AS, Desrosiers M, Tyagi RD, Valero JR (2000) Foam kim sot 31. trong sn xut biopesticide t bn cng. J Ind Biotechnol Microbiol 25:86-92 Vidyarthi AS, Tyagi RD, Valero JR (2001) nh hng ca cht hot ng b 32. mt v sn xut bng cch s dng bn thi biopesticides nh mt nguyn liu th. Khoa hc nc Technol 44:253-260 Vidyarthi AS, Tyagi RD, Valero JR, Surampalli RY (2002) nghin cu v vi 33. khun Bacillus thuringiensis sn xut da biopesticides s dng bn thi lm nguyn liu. Res nc 36:4850-4860 Yamamoto T (1982) Xc nh cc cht c entomocidal ca Bacillus 34. thuringiensis bi sc k lng hiu nng cao. J tng Microbiol 129:2595-2603 XM Yang, Wang SS (1998) pht trin ca qu trnh ln men Bacillus 35. thuringiensis v kim sot qu trnh t mt quan im thc t. Biotechnol Appl Biochem 28:95-98 Zouari N, Jaoua S (1999) Sn xut v c tnh ca metalloproteases tng hp 36. dng ng thi vi ni c t-d ca Bacillus thuringiensis subsp. Kurstaki ging trng trn da trn phng tin truyn thng cho. Enzyme vi sinh vt Technol 25:364-371 Zouari N, Achour O, Jaoua S, (2002) Sn xut-ng bng ni c t ca vi 37. khun Bacillus thuringiensis subsp kurstaki v khc phc bng cch s dng p

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catabolite tp trung cao v phng tin truyn thng cho bt c trong 2 v dm 20 3 fermentors. J Chem Technol Biotechnol 77:877-882

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