Process Biochemisuy 98 (2020) 224-232 ELSEVIER Content ists available at Seencedirect, Process Biochemistry journal homepage: ww. e\sevier.comilocate/procbio Review Review on biopesticide production by Bacillus thuringiensis subsp. kurstaki since 1990: Focus on bioprocess parameters Wafa Jallouli’, Fatma Driss*’, Luc Fillaudeau, Souad Rouis’ ‘ahr of penis, Co of Mehl of Sx, Ut of fa, RO. Ba “117% OT, Sf, Tata ounce inte Bo & Chena! mgnering Une of Toe CNBES S54 INEAK 792 NSA, TB. INSA Teo, 135 eee de Regu ARTICLE INFO ABSTRACT Koren irepectve we of chem pnices ase overeat desde, several problems sich a, wate and a ete {cod sources palon, and peeration of 6 sletive pressure causing the emergence of pest restr. erat nse, reenchr ave ben fing more on he we flog cota ara erate eee. Brees ‘ects rhe (2 i one of the tox ely sued acer in instal technology and comme elaecde Giada an environmental stainable bop, There, ge iter hs ben allocated fore ‘each on hs acter an several seis have Den pubis on he nts ee, We ed tcevauate he sete poccton over the ia thirty years forte fst tin, nto of on and eo ‘Paphiclosn sng prclry on Brags vat (A) is worth emphasing ht the Be rote enanerng inven actos ing growth, sordation ad xin rman sty thas teen {ity tvetignedn previous evens To hs ed tesco set of is eview pote an psa are Stout thee colon such at utionalequtnent elie ea ad fer enaintecoolgien Relea infomation was oletdin compare ables ha coud b eye or thee commun need ini hoe Moers 1. Introduction 90 % ofthe world’s blopestcide matt, While the Bt products are Since the 19605, pest management in industrialized countries has been based on the intensive use of synthetic chemical pesticides. Undeniably, these pesticides have contributed to increasing crop yields by nearly 709% in Europe and 100 inthe USA [1]. However, the use fof synthetic pesticides has significantly become debatable due to a number of interacting factors, especially the fact that all major Insect pests ate developing resistance to the various classes of chemical in secticides used against them worldwide. Over 500 species of arthropod pests have resistance to one or more Insecticides [21, while herbicide. resistant weeds count about 200 species (°]. Biopesticides are used as ‘an integral part of Integrated Pest Management (IPM) and they ae Classified into three groups according to ther orgin (microbial, plant ioetemical) or animal (semiochemieaD), and can be used both in conventional nd in organic farming (4) ‘Most commercial biopestiides are of microbial origin and are pri marily based on the Boclus thuringiensis (Bt) microorganism [5]. Bt based biopesticides are of overarching importance and represent almost “Cowesponding author at: Laboratory of Biopesticides, Cente of Botchnology of Sax, Unversity of Sax, P.O. Box. Widely available in North America and represent $5 % of the bioin: secticide marke, they are less popular in Europe representing only 8% ofthe same market. The low level of Bc products inthe European Union (EU) is mainly due to the greater complenity of EU-based biopesticide regulations [5]. Nevertheless, marketshare growth of blopestiides Is predicted to outpace that of chemical ones, with an annual growth rate (9 15% [7]. In fact, they are expected to increase from about 29% of the flobal pesticide market in 2003 to about 8% (estimated to exceed 82 billion USD) in 2020. Br spores and erystals have been commercialized to control a range of diferent inset orders during thelr larval stages, such as Diptera, Coleoptera, Hymenoptera, Mallophaga among others [8]. The major Beased bioinscticides targets are herbivorous lei dopteran larvae like cabbageworms, cabbage loopers, hornorms, European com borers, cutworms, some armyworms, diamondback moths, tent caterpillars and Indianmeal moth larvae in stored grain ‘This might explain the fact of relying on kurstakl serotype strains, especially that of Buk HDI [9,10]. Bris known to be safe fr vertebrates and a good numberof reviews has concluded tht itis one ofthe safest 177", 3016, Sl, Tunisia malades slea20yaheo (Wl, eis tas een (Fel se adenine tolose. (Lille, sound rouseebsravcin (8. Rous. heps/diorg/10.1016/ procbio.2020.07.023 Received 9 Match 202; Received! in reisd form 7 July 2020; Aceptd 29 Fay 2020 ‘Available onfne 11 August 2020 1359-51127 © 2020 Esevier IA rights reservedW, Jala ea products avatlable in tems of impacts on non-target insects (1,12). Consequently, the use of this bacterium Is an important” en- Vironmentally fiendly prt of pest management toolkit. Be Is a ubiquitous Gram-positive bacterium. It ean be found in a ‘reat diversity of ecosystems including soil, water, dust from grain ‘storage, dead insects, leaves from deciduous tres and diverse conifers [05]. 10s @ spore forming bacterium producing crystalline inclusions consisting of one or more insecticidal proteins known as endotoxins and commonly called Cry protein (14. When 8endotoxins (or Cry toxins) are ingested by a susceptible insect, the crystal proteins are solubilized by the extreme alkaline pH of the insect midgut and pro- teolyticaly activated by midgut proteases. Then, the activated toxins ‘ind to specific receptors located inthe insect cell membrane leading to ‘the destruction ofthe epithelia cells lining the insect gut. ts general ‘believed that these toxins at by creating pores in the cell membrane (15). Although the bacterium contributes tothe death ofthe insect, the Seendotoxins are capable of kiling some species on their own if pro: ‘duced at sufficient high doses C16). Whether i i through government grants or companies doing re search and development or even via non-profit foundations funding, a ‘considerable investment was made in the Fld of Bi-basebiopestiides Which have gained more and more interest. For instance, the European ‘commission has been funding more than twenty projects dealing with the ability of Br to be used as biopeticides from 1986 to the present date, Among these projects, we ca cite the ongoing project coordinated. in France, entitled “IPM-4-Cirus, Gtrus disease Integrated Pest Management: from Research to Market” (ID: 734921; Period: From 2017 04:01 to 2021 03:31; Total cost: 801 000 EUR) [17]. IPN: CITRUS focuses on two promising and newly identified strains (Bik BLBI and Lip). These strains were shown tobe more efficient than the ‘commercial strain (Bek HDI) against Lepldopteran pests both in terms ‘of aetivity and production yield [18,12]. The ultimate objectives ofthe project are: optimization ofthe bioproduetion processes, increase of the ‘rains Intrinsic toxlity and generation of high added-value biopro- ducts ‘Within the framework ofthis project, i is expected to define the ‘nutritional requirements for Bt cell growth, endotoxin production and. ‘entomotoxiety and to Improve the productivity beyond the current state, To this end, an exhaustive state of the art reporting the previous ‘conducted research on Be boinsecticide production with Bk HDI, BLBY and Lip would be very useful. Over the last thirty years, several sc: ‘entific publications dealing with Br biopesticide have appeared thanks to the important development in the understanding of Bt and its mode ‘of action (20). Reviews reporting this Literature focused on gene dis ‘covery, toxin mode of action and resistance evolution, with less interest to Be production. In parallel, most ofthe work conducted by the private ‘companies is either unpublished or patented. ‘The present review provides the readers an overview on the aval: able publications about Buk biopesticde production, during the last thirty years, and addresses this bacterlam culture media and culture ‘conditions emphasing on the fermenter operation modes for biopes Ueide production 2. Scientific literature related to Bt biopestici le production Bibliographic research using the database in engineering sciences (web of science (WOS); Thomson Reuters) for the timespan 1990-2000 (done the 10th of June 2020), reveals 4998 publications quoting “ ‘thuringiensis’ inthe tite (profile 1). This number includes publications dealing with biopesticide production ftom diferent Br subspecies such as subspecies dvulensis (21), alzawal (22), tenebrions (2°) ete. AS stated above, inthis review, we focused on the literature dealing with ‘the subspecies kurta Pron Mchenry 98 (2020) 26-252 ‘Tablet [amber of centie publi tons pee profile. Timespan: 1990 to 2020. Tie “TilerReyworsy role t_ hngie “8 Prosle2 5 Marat Sava iss Prosle3 iwi Tare endanin 8 Prosle 4 turagne Sartal/sporiaion 73 Profle tarlost Sarl fermentation 62 Profle turmge Sura Knees 9 Profle? tarmgne Sarak Bopoces 8 2.1. Quantitative analysts of publication on Bik ‘The first section of ths review highlighted the scentfe publica tions quoting *B. thuringiensis” inthe tte associated with *kurstak in the ttlevheywords/abstract concomitant with five topies viz, “en dotoxin’, “sporulation”, “fermentation”, “kinetics” and “bioproces”. ‘Thus, profiles from 2 to 7 are proposed (able 1). The bibliographic research was basically conducted using WOS. The obtained results were enriched by those collected from SpringerLink and. ScienceDirect. However, several data are missing either because they are reported in unavallable papers or unpublished. Table 1 states the total number of publications per profile between 1990 and 2020. Further information about the annual and the cumulative publication numbers per profile were given in (Fis. 1). The obtained results showed thatthe cumulative number of publications about Buk associated with the above cited topics Increased continuously, since 1990, to reach 1159, 398,73, 62, 9, and 8 in profiles 2-7, respectively. The corresponding curves have the same shape bur wlth ferent starting points. Indeed, publications about endotoxin” (profile 3) and “sporulation” (profile 4) started to appear in 1090 just lke those dealing with Bok (profile 2, However, publications started 10 use the terms “fermentation” (profile), “kinetics” (profile 6) and “bioprocess" (profile 7) in 1991, 1999 and 2004, respectively (ig. Taking these data into aceount allows us to conclude thatthe fist studies conducted on Bk mainlyfocused on the exploration of the bacterium especially its life eycle and the metabolites responsible forts entomopathogenic activity. The number ofthe corresponding publica tions peaked around the yeas 1994 and 2007 for profile 3 and around the years 2007-2008 for profile 4. However, papers related to Bt large- scale exploitation began to appear later, in the beginning of the 21st century, with the appearance of publications dealing with “bioprocess” In 2004 (profile 7). Indeed, as reported by Sanchis [2], two main facts have orlented the scientific research In relation to commercial interest In Be the fists the discovery of HDI strain whichis 2-200 times more toxleagalnst key agricultural pests [25] and designated for the first time as @ Austak! variety the second main fact Is when scientists and environmentalists recognized that chemical pesticides were harmful 10 the environment and could be replaced by Br-based products 22. Geographical origin of Bk scenafcIteraure ‘This review was writen in the context of the above cited project Involving six countries viz, France, Tunisia, Lebanon, Italy, Turkey and Germany. Thus, it investigated these countries positioning inthe sc entific production among those interested in research on Btkbiopest cide. It mainly focused on the scientific production related to the bio- process parameters revealed by profiles 5-7 dealing with the topics of “fermentation”, “Kinetics” and “bloprocess, respectively (Table 2). A quick glance at these profiles allows us to deduce that the exploration of| the Issue Is much more coneentrated in America (USA, Canada, Mexico and Brau). Fewer works originated in Asla witha Chinese and Indian Predominance, Africa and Europe rank third and fourth, respectivelyW, Jala ea = i. 234 i ib i UPPPEEECEEEEGUETEL Publation Pron Mchenry 98 (2020) 26-252 gl = ang “3 “ae = 32 of «s- ; we RERRGEE == i 5S ef 2 at a year nag at “a5 o3f od 5 Publication year Fig. 1. Annual (APN) and cumulative numbers of pulleatlon (CPN) fr A) peoles 2 and 3 B) 4, 5,6 and 7. Profle are defined as explained in ble 1. Tmespan 1990 io 2020 Interestingly, the data showed that Tunisia and France play an im- portant role in their respective continents forthe publication of such studies (5 for Tunisia and 3 for France). Fewer publ ‘duced in taly 2 publications) and Lebanon (1 publi tifle production was recorded for Germany and Turkey. These ob: servations may indicate that the Brbased biopestcide industrial production is entaled by the evolution of the geographieal distribution of scientific production. 3. Btk biopesticde production Unlike profiles 2,3 and 4 which mostly focused on Hk culture ‘media and nutritional requirements, profiles 5-7 dealt with Bek bio- process parameters. Occasionally, the provided information Is com: pared to those of other subspecies allowing the Bek study ina broader Bt Context. In the literature included in profiles S-7, the bioprocess i in vestigated from different stand points. For instance, Rowe and Margaritis [2°] studied the economic side of the bioprocess, while Mounset et al. [27) considered the technological feature which is our ‘Table ‘Geographical origin and numberof siete publications of profs 5,6 and 7. Timespan: 1990 to 2020. County __Namberof puication Rae (4) Coty awe) Coury Number of pbiaione Rate (4) cmd a Meso wo Mako ws Inc é ta igi a eee tas TinidaS. fa ral foo mt nas ete 2 ao Gommbia as Syrn : 3 fou Roven 1 ia All counties are counted for publications issued fom more than one county.W, Jala ea main concern inthis review. Relevant information about all these ksues Is detailed below. 31. Nutridonal requirements {A good number ofthe consulted works foused on factors affecting ‘growth, sporulation and toxin formation. These factors are mainly re lated to the Be nutritional requirements suchas, potassium, metal ions and carbor/ltrogen sources. Indeod, the majority of Br strains are able to ferment a variety of carbohydrates including glucose, fructose, starch, maltose, trehalose and ribose [25]. Recently, newly isolated Be strains have been demonstrated to be able to ferment cellulose and xylan [29]. The most used carbon source to produce Be &-endotoxins is ‘lucose which was proven to stimulate the growth and trigger Sen- ddotoxin formation in Br MPK13 [30]. The increase of glucose con }, barley four [59], bird feathers and deolled rice bran [50], glycerol from the biodiesel industry [61], brewer's yeast extract [62], molases with comm steep liquor (22,69) and broller litter extract (6) CIsble #)- Ghribt etal [57] demonstrated that by using soya bean meal and stare for Bek production, -endotoxin production and spore count reached 2.71 gl." and 38 x 10° spores mL, respectively. A similar B-endotoxin con- centration was obtained by Mounsef eta. (55) using 6% wheat bran (24g L-) However, using a combination of gruel and fish meal, Zouat etal. (56) showed that a highest 8-endotoxin production could be reached with Bek strains (3-33 L~"), but the lowest production was reached with Be strains (.24-1.99 gL"). Only 1gL~* and 0.753 LE" of Bendotoxin were obtained by culturing Bek in gruel hydrolysate and 24% molasses based medium, respectively [05]. Moreover, Alves et al. [55] showed that, using diferent combinations of agroindustial residues and by-products, the spore count varied between 5.5 and 21.6 x 10° spores mL". A comparative spore count of 15 x 10* spores mL! was obtained when using brewer's yeast extract as the main ni twogen source [62]. Interestingly, the highest spore count (480 = 10° ‘Table 4 {Complex media used for Be euvation. eal ire tion ‘Sains) Spa and Sends bopoduton a ener 50m) Gril 425 ah eal 29g EK BE BANDE 300mg. "595% 10F pore ma® LS Bhi Te eloangh-! 34» 10 spre Elemeyer 250ml) Che whey’ 50% sya bean mi 109% and lacs 05 6 ae B16 10 ores mL* a Eesmeyer (250ml) Soy ban pain mola 5. Be Res 1 gper at ts Exner (250ml) Grou Bony na pupae 5S, ats 0.5 e105 10 pares tt Elenmejer(12) Gre hdrelsel5g--enmmiomsueS4g1yeatenract sg BRBNSS gL: 34 1 spre! ts lemeyer(SOml) — Hydoaed ade of ck Late BR MDL 0125108 pare mi! wa Ban (1001) Soy Dour 2594" Wslion of MgC, 203g" Cals 102311; 480 1 sors at ba cls 108 ae) Brey ft 2586 (o/s ny Baur; a sleon gl, 20331; AAMDL 958mg 10" apres; 031 x 10Spure a? 8) (ici 10g tach tg) Euler (NA) Bd fetes, died ce an me sagt! to fuer oD Say bean meal 2581 staeh 30 L* Bk 27K meh 8 10 pore“? to Erermeer (11) Wheat bean 65 BELip 18080 cpsal mit and LAOREET seh 201) rowers eet 1% ioe 25 ammonia site 02% HDL 15% 1" epee =? bo fen Notas com tp hor 3, 0s 0.08 DEKH §25 108 sree 2: 750m to Bates 51 Stsch eds waste [BMDL 1.2% 10h spores mL! 1060 mg" ra Erlenmeyer (500m) Bre ter ext Ben 246% 10 pes l= tat Exeemeyer (250ml) Milky eluent 745, tear 264 DeBoer 4 eyelid NA: Not avaiableW, Jala ea spores ml.~*) was generated shen wsing a medium based on soya bean flour with salt solution [5°] and the lowest one was reached when using 2 medium based on barley four (59). For a low-cost Be biopestctde ‘Production, the most used raw-material Is the wastewater sludge [85,00-71], When used at different preparations, the highest viable cell (0.14 x 10° spores mL") and spore count (0.12 > 10" spores ml.~ were obtained when Btk was grown in hydrolysed Black Lake sludge [67] Cable 4). Lachhab et al (55} demonstrated also that using the same medium (wastewater sludge) for inoculum preparation, higher sporulation (17 x 10® spores ml.) and toxicity (12 300 international units (U) mL?) values were obtained. They also reported that the ‘optimum sludge solid concentration was 26. 1.~", which resulted in an Improved potency and high spore count achieving 42 x 10* spores mL? and 12.9701U ml, respectively. Ina later study, Vidyarthia, etal. [1] compared the growth and 8 endotoxin production by Buk ina ‘uyptie soya yeast extract (TSY) medium, soybean based. commercial medium and wastewater sludge medium. They found thatthe highest toxicity was obtained ina sludge medium and was comparable to that, ‘ofthe concentrated commercial Be formulation available on the macket (Foray 488). Among the used wastewaters for Be cultivation, starch Industry wastewater (IW) is the most used one [72-74], Compared to wastewater sluge, Bt fermented SIW showed a low spore count (1.2 x 10" spores mL") but high entomotoxicty (18.4 x 10" spruce bbudworm units (BU) L~"). Higher spore concentration (2.9 x 10" spores mi~*) was obtained using a combination of milky effluent and beer wastewater {75]. These studies indicate that the ehoice of an adequate production medium triggers both spore and 8-endotoxin production ensuring the effectiveness of Basa biological agent. In all the presented cases, it is not possible to make comparative study 10 select the appropriate medium for Be cultivation due to the diferences Inthe ways in which biomass, Bendotoxin and toxety are measuted and expressed, 3.3. Culture conditions Studies conducted by different researchers have shown that process conditions can significantly influence Be erystal-spore complex pro- ‘duction, The fermentation parameters that play an important role in the ‘Bx production are pH, temperature, dissolved oxygen concentration and inoculum preparation, ‘Oxygen supply is a decisive parameter in Be fermentation. Oxygen transfer is a function of the volumetric oxygen transfer coefficient (X12). Its also one ofthe most important factors for the process sale up (76. In practice, the measurement of this coefficient expresses the ‘oxygenation capacity of the medium contained in the bioreactor. Its ‘commonly used to measure the oxygen transfer rate (OTR) and the ‘oxygen consumption rate (OUR) [77]. Several papers have discussed ‘the effet of aeration on growth, sporulation and crystal production for different Be strains [23,27,78,791. Most Br submerged fermentations were earsied out using an aeration rate of one alr volume per medium volume per minute. However, other studies used a higher aeration level (of .4 a volume per medium volume per minute [80]- Sarrafzadeh and Navarro [75] have reported that, using diferent oxygen tansfer rates ‘0, 20, 100 and 250 mmol L~" hin fed-batch cultures, the spore ‘counts were of 100, 93, 84 and 48 %, respectively. So, the highest sporulation rate (1009) was observed in the absence of oxygen and the mature spores were the only population present under this condition at the end of fermentation, We can even observe that sporulation in a large proportion of cells failed under saturated oxygenation [1]. At 100mmol Lh, eels in different physiological states could be ob- served. Furthermore, keeping the dissolved oxygen (DO) concentration ‘at 50% during the vegetative and transition phases then raising the DO {0 100 9 of saturation throughout sporulation allowed @ higher Bat toxicity (PS.82) [21]. However, Ghrbi et al. {79} noted a lower spore ‘let but a higher 8 endotoxin synthesls by a Bk strain BNSS when 60 ‘and 70 % oxygen saturation were ensuted during the fist six hous, Pron Mchenry 98 (2020) 26-252 then decreased t0 40% up to the end of fermentation. Likewise, Bhowmik et al. [32] demonstrated that the fermentation carried out with 30 % DO and an aeration fixed atone vv allowed an increase of 1.67 fold of 8-endoroxin production by Buk strain HD 73 compared toa ron-contolled culture. Moreover, in semi-continuous processes com- bined with batch processes for sporulation, there isan a 53.69% increase In sporulation of Bel IPS 82 under aerated conditions, but toxicity Is about fur times higher under non aerated conditions (J. Considering these Findings together, it is proved that the optimal conultions for spore and 8-endotoxin yields are not the same, eventhough sporulation tnd S-endotoxin formstion occur simultaneously during the ferment tion proces. ‘pH and temperature are major factors influencing Be biopesticide production. Indeed, Br growth occurs in the pH range of 55-85 {28,(6,(8]. The usual intial pH s 6.8-7.2; decreasing to 5.8 as acetate Is released, then Ineteasing to 7.5-8as It Is consumed. Sodium acetate was found to be the best pH contol agen fr Be entomotoxiety and 5- endotoxin produetion [72]. Ndao (4 stated thatthe maximum spor- lation and toxicity were obtained ina medium buffered at an intial pH Of 7.5 and thatthe optimal temperature for growth and toxin produc: tion is 30°C. However, Szkan etal. [46] stated thatthe optimal tem: perature for toxin production depends on which toxin is produced by the bacterium. They found that Cry4Ba synthesis by Bei HDS0O was the best when the microorganism was grown at 25°C, whereas Cry11Aa synthesis was optimal at 20°C, Culture synchrony i also considered an important parameter during Bt biopesticide production since the max- mal efficiency of the final product is achieved when fermentation Is clove to 100 9%, In this context, different inoculum preparation state- Bis such asthe use of heat preteated spores at 60°C for 1S min [40] fand the use of exponential growing cells (cells aged of 6~ 108) [5°] were applied to generate synchronized Br cultures, ‘4. Biokinetics and bioperformances The production of Be cells and spores depends on the specific growth rate () of the micro-organism, which, in turn, depends on the used strain, the concentration of available nuteents, emperatute, pH and dissolved oxygen as well as the metabolic state. Research studies ear ried out on Br production have demonstrated that the maximum Be growth rate varies from 0.4 to 1.9h~* (85). Anderson and Jayaraman [85] have suggested that a high specific growth rate does not ne- cessarily promote sporulation and toxin synthesis. Indeed, a negative correlation between the growth rate and Be produetion parameters ot: curred by varying glucose and yeast extract concentrations. In general, Be growth is characterized by an exponential vegetative growth fl lowed by a stationary phase due to substrate depletion. Spores and crystals formation causes a deerease In the growth rate due to the de- crease in the energy needed to perform the binary fission, which con tebutes to the fnal biomass reduction (9). ‘The specific sporulation rate is another parameter measured during Be kinetics. This rate varies with the medium composition and culture conditions like pH and dissolved oxygen (DO) concentration, etc Indeed, an optimization of culture medium composition is critical 10 control Bt sporulation, sine high growth-fvoring media of vegetative cells may not be adequate for sporulation (25). Vidyarthi et al, 2002 [Ws] reported that comparative low specific sporulation rate values (0.05h-') were obtained in TSY and soya bean based medium. How- ever, the highest value of 0.12h~" was obtained by using wastewater sludge. This high specific sporulation rate leads tothe highest Btk en tomotoxicity. The same authors reported that a linear relationship ex Ists between the specific sporulation rate and that entomotoxicity. The ‘optimum value of specific sporulation rate was of 0.55 ~" ‘In i, sporulation and S endotoxins synthesis are greatly dependent ‘on oxygen supply swhich favored the cellular respeation and metabo- lism leading to higher viable cell and spore counts and S-endotoxin concentration [87]. Interestingly, at 12h of fermentation, the OUR,W, Jala ea which is the thd parameter followed duting Be fermentation was the highest (0.25 mmol L~* h) due tothe ineease in cell eoncentation (growth) and metabolic activities (enzymate synthesis) (87), However, Its worth noting that the OUR peak depends on several parameters seh asthe microorganism requirements and pH regulation. Commonly, at 12h Br sporulation is triggered and transition to sporulation phase begins. For this reason, a ter-hour incubation period was chosen by numerous researchers to perform the substrate feeding achieving high levels of biomass, sporulation and toxicity [0,85,99]. Moreover, Rowe at al. [90], demonstrated thatthe specific OUR of Bik HDI decreased from 8 to 10mmol gh? at one hour after inoculation to less than 2mmol gh" by the growth end. Additionally, Mounset etal. [27] showed thatthe Be oxygen demand for growth and sporulation i not Identical to tha for optimal toxin synthesis. Besides, they found that a linear correlation exists between the amount of consumed oxygen and ‘the maximum cell concentration obtained at different cereal miling Dyproduet (CMB) ratios In the culture medium, with a correlation coefficient of 0.99. In general, OUR increases atthe exponential growth phase, Indeed, at this step, & high substrate consumption rate takes place ater which it decreases withthe cell metabolic activity decline pn. 45. Culture technologies For ft practical application as a biological insecticide, high 8-en otoxin production titers are required. For this reason, several works hhave been carried out on the Br bioinsecticide production using batch fermentation. Boniolo et a. [21] demonstrated thatthe use of batch fermentation allowed a higher biomass concentration, cel productivity ‘and cell yleld. All this closely depended on the amount of $0 % DO applied throughout the fermentation period. In addition, this con ‘entratlon resulted ina higher spore count and markedly Improved the toxic activity ofthe fermentation broth compared to that achieved at low DO concentration (5%). Moreover, the application of various DO profiles during batch fermentation proved thatthe best profile core sponds to 60% and 70 % of oxygen saturation during the first 6-h fermentation period [79]. Then, 40 % of oxygen saturation should be ‘ensured until the end of fermentation, independently of the carbon source origin. Furthermore, Vu etal. [39] demonstrated an improve- ‘ment of S-endotoxin concentration using a batch process performed, with SIW. Using the same fermentation technology, Jowzant et al. (6°), developed a low-cost bioprocess based on agriculture wastes. The ‘oxygen demand Inereased with the fermentation time. Besides, the &- fendotoxin production as well az the bacterial growth increased by raising the oxygen concentration up to 70%. ed-batch culture ha also been widely used for the produetion of Be bloinsectiide. Indeed, an extended dynamic model for Bxk growth and sporulation using an intermittent fed-batch culture each 3.28h with total cell retention in glucose based medium was proposed by Avehortda et al. (92) Using this same strategy, but with two Inter mittent feeds of SIW (at 10 and 20h) during the 72-h fermentation period (8), demonstrated thatthe Sendotoxin concentration and en Tomotoxicity were significantly higher than those obtained by applying the batch process. Indeed, S-endotoxin concentration. and en: tomotoxicity reached 1672.6mg L”* and 18.59 x 10° SBU mL, using tne intermittent feds of SIW compared 10 511.0 mgL." and 15.8 x 10" (SBU) mL, respectively, obtained using the batch fermentation in SIW. However, a fed-batch fermentation carried out with three SIW intermittent feeds at 10, 20 and 24h of fermentation, resulted in the formation of asporogenots variants which decreased the endotoxin concentration and consequently the entomotoxicity value [5°]. More ‘over, Rojas ta [0] demonstrated that a pulse fed-batch process and ‘one-pot combination processes performed a different sales and erried ‘out by the addition of glycerol at 10h of incubation, significantly In- ‘creased the blomass production, spore count and toxiclty compared 10 ‘atch fermentation. Pron Mchenry 98 (2020) 26-252 Despite the relative improvement of 8 endotoxin production by fe batch fermentation when compared to batch fementation, both blor- actor productivity and toxin yield were markedly low due to an In complete consumption of the add substrate For this reason, a two- stage exponentially fed-batch fermentation process involving an inital stage for vegetative growth followed by a second stage for sporulation and toxin production was applied. The best condition corresponds to a fermentation supplied with 190g glucose in 1500ml. At this condition, up to 20.1 of bacterial insecticides per lite were recovered from the fermentation broth with glucose to toxin conversion yield of 0.1593 Pane IIs worth noting that less information was glven about the Bk based bioinsectcies production from an economie stand point. Among the few authors dealing with this Issue, Rowe and Margartis [2°] compared fermentation broths obtained from different fermentation technologies: 1) bateh, I) low density fed-batch (LDPB) and i) high density fed-batch (HDFB). Be based bioinsectcides cost was higher fora batch fermentation than fora LDPB. However, the HDEB has relatively lite adtional cost benefit Although diferent fermentation technologies have been described, probably others are yet t0 be studied to increase the potential of Bt sporescrystal complex which varies according to the medium compo- sition, the strain used and the production conditions. 4, Conclusion (Over the last thirty years the number of scientific publications dealing with Bebased bioinsecticides showed a gradual and steady in crease. The highest number of publications in relation with “sporula tion” and “fermentation” is recorded by Ameria followed by Asia, [Aitieaand Europe. Fewer publications in relation to "bioproces” follow the same geographical disuibution. This distribution i correlated with the fe biopesicde market whichis mostly developed in America. The development of Be biopesticide in the other continents seems to be dependent on many factors such as consumer demand and government policies regarding the use of Bin agriculture. Therefore, itis necessary to strengthen the collaboration between research and industrial in stitutions and accelerate the practical application of research reslts to faeltate Largescale industrial development, mainly in developing counties. ‘Moreover, Its worth noting that reports providing information about kinetics and fermentation technologies are scare and incomplete. Besides, there are diferences in the way of measuring the parameters and expressing the results, which might hamper carrying out com: parative studies. Therefore, standardized measurement methods should be established to enhance the whole field. Declaration of Competing Interest "The authors declare that they have no known competing financial Interests or personal relationships that could have appeared to inl ence the work reported in this paper. Acknowledgments ‘This work was conducted under the IPM-4-Citus projec, funded by the European Commission (1D: 734921; Period: From 2017-04-01 10 2021-0331; _hitps//eordiseuropa.eu/project/ren/207633/foetsheet/ 1). This work was also funded In part by the Tunisian "Ministée de [Enseignement Supérieur et de la Recherche Scientifique” (Program- contract 2019-2023), Appendix A. Supplementary data ‘Supplementary material related to this article ean be found, in theW, Jala ea ‘online version, at dott: //o}.org/10.1016/.proeblo.2020.07.02%, References 1a) 5. rey, agictral ssi concepts, ences nd idence, Ps Tras Soe Land, at Se 3 (149) Cg) 47-465. (2) A taj Natal Eee an rodeo Boia antl, Ann Hee entries Pres, Cami, UK. 200 p 75 IBN 052 6595 (Garda) ISH 521 52585 (perch) et Man 614) 2005) $22 (8) Ha, The nertin Suey fH Rs Wed, 200 ip: sonnets (41S Donel tami FC Fir Vn Hse, Baht, Soude ‘Suny rth bacon a tc dy iy, Ap Ml trehalose, (81 Na Rar cuc, eps prodactn fom atlas area metals act i 3 0) 0) 28 (61 tg Hara Londen, erect pegs he ‘Moog S72 (01) GoW” saps, 171 Roane, the Met tential Bogie, peti Sn he ‘et nd Foe Opearien AES abet 214 fp. 5-28 (a) AL a Ut Sta GX Sie eal Sg of tn ‘Stet mw planes vo, Cat Re Bre 36 GDN) 191 LG copa. The Bape Man Wold Capen, Bs cop pos (10) Ni Horde Haber, Don fas igen ae 09 ‘abhgs or han espn, PEWS hel Let B70 lo) 106-1 tan) cla Och Rata trees ses sk ay 00 UD Sch ttt a gen ape (48) RA Marin RS Towers, Wie aun ad btn Bc ‘tinh on Mra 8 (1) (0s) 07 Se {14 f'bop core va techs tam) eon Bg, DAE bo, Bastar rg adr eile rons Mol Ha Ba nor 2) ce) a (18) Aco, ll e Stuo, Mae of aon fas gia ad ‘Str wh pa or et ot Foon 1 (0 07) 8-8 {861 yuna Saeaon © Neen, ech Soe, Rasa ‘arc ipo ptiogn Td crs 18) 40) 109-15 107 PACE rw Gis dene etd pest magento Fac mth tae Eada Re hoy une ene argos ‘vig ih ele acy ad cds i Ads cr 19) (Guo ate (19) Seth, Anos chia, N Ab 3 Chpie SK Ad Irion nd chara fe Bact drt sip hatoig ‘ew cyt se hight ep ae Lepore) a, ‘eh thaw 96 aon ae (20) Sanne, aa ingens Dhl, Sine, 2012. {all Fs. ilo Rage AP Mae ai, ie, ‘ch erring Omg pln rhage errno gn tev nga on te pet oration sd rsdn pon Sp ‘icetao mowabwa 90) ova eas 38 t221 ON Mori toner fanaa, 3. Di, fet fake cond Sonsom precy yl not fr ge oe {ints vera Pat 6”) Co) 30-138 tan Wl me, Syrup ncn es Beer bea nd stress enn Soper ped son el peptone ‘clade pc nae ec: Pe (3 0 13.10 tae Sci em ico ps oct eset een at: tery of ‘hepsi lags, Asoo, See Bt) ONT) (251 aia, cil avy of HD ewe at dig ter Ala inet at 1S 2) G97 S82 (26) SE Rowe, A ruin, roc ge concn afr he coe ‘nec prodton a eee try ec om ac Ingo aa Basho eg 86 009 77a 2m Saunt, alah da, adam eh ft of ein Sendo hrc! by one ac nr sti Kee ‘Eemnaon osc a as ngs rl, 2001) {2m Fal A. Kern, Machi tel gon ad pein han 1 ney cba et age of emi Balr mera ‘poy a Cham 3) 70 S07 368 tao gn pan SP Sn, aon an hacer of ‘tue pg sires lr topal seve ‘Sts ion, tar Gna ee (20) Semin Rona, Rec ing A et facie ao ‘owt run rand enon ans of ace raga Ae Sta ao cna ee sea tan Pricer, Lay, Brampton of deta ent by Bas (sa (sa (991 0) tar er cer wa 31 a wa ea 91 ‘su (sa (sa (sa (sa (9) Pron Mchenry 98 (2020) 26-252 Faun tina jan nana ee ripen igienlerclgre ey IFES tm An cm ra ts od hn eet ele eer ere or tirdgent ty creeeuca Sa ee ct manag yeep se iy panes ee Si aigiuiel cpl niga poe Lape fare ae enema ‘hurigenss va. goers growth high glacose cocentrations, 3. In Seen meeeree roe aes Caen ean eae ea ee Ce a et durigins, Faulty of Scene, Cairo University, Fay, 2000 eee ee ceria potter ig ton cle n ee ee eee f ets Boring and Yield of Bioinsecticidal Crysal Protein, University of es ee ee eee eee eee ae eet acne pc of me a a eae aie eee fee cee oe eee trees 1 Bec aces oe ee ee he eee rman ee eee Beene ee eee eee ener eee eee eee ee ere Seema ane Pale tors ‘sold-atte fermentation fr the enhanced peduction of Bectas duringien-toxin, teeroer set ae Sie al onsen setae Segre acter eras Tamassia econ (uuu aaa ee eee eee sithitnctae acer eaaa fcr eee cee eae ee Cee ae Ds ee ae ae ae pe ere ee ee sae rnp ter CoS aca analabluann Fa iru edie 1 Tbs Depa Popcorn peepee containedWalid ea (ot) 5 Popa Ath, A med othe pot f opens at ‘pt cr gt pant nota om {ot € har, A teas Me Baer Mal, F. Bono A Ros Prats Growth Seas dings towels oer ive fm bt ny timing he poutine lec, New cho 35 (208) S22 S227. {ay £'Stsncha 8 sopnamhara Verdun, Appeatn of ample Yan erat from apent newer en for grow spon of acs rigs ap amas pyocatyWon1 Ae 17) 2000) 907-6 {sa Cs Joust 6 Abbie, MP Maal Mona Deeopmen of com ‘Seine bproce or proton of nanan nt coer ata ar mgt srt, age Sel Tock 17 (8) (201) 1163-1058, (oa) Ti Aan A tern Ada Bate har se. atc! pre [taone Tena 67) (99) a7. {681 Nowa, A Dhow. lous, Saou, Noon escemet of rn of Decitabine feo hy fore orton 38 ‘ew mem fr Sceenin prddcion, Ap, Basher Bie 6 (Go9n) 4182 (00) K tach RD. Tyah ARV, Prod of Bat res oper Ching wastentr hg a a mateo hes td gels, nent, rte Bache 97 (200) 197-28 {67 NGL Mont yop aero, Wastewater eaten ge a mater fe the produto of ai dros bed opi, Water e310) {on} Sk brn Merms, RD. hog 38. Valeo, RY. Sump, Eom, Decne tn chine neyo Baa ring rented wastes Sedge Sra high secon, orcowr, Techn 100 (19) SW) fab, [ooy 2nuang 8 au, VQ. Wang, 2 Ls, AX Hy Conceive pod of ‘tps ec a en tl arcu Teche 08) CH) a 170) 6 Gong Se Mou NL AN nance act dingo pri om ‘evge de with sali od uirwonc pretence, sr A Sod Po Woda} 07 7588, (70 GSsowr A AIA A Hie, of ac rng cate in ‘rnonser dg snot epi tbe nth peop: Cai), Enum Gen (2) 2016) 6-1. t7ay Kb. That. Vl RS np of ferent pt onal agen on [optical wey ar urges ring the fermentation of tr ie ise senate, Bhp Bos En. 82 (6) (2009) 51-516. 173) KD Ws, RD-Tyat RY Surampall JK. Valeo, Maratea lant be {een spe coment, erento ve dems o Baia Src pert poe ie een medi ee Selo 128 coro 90>ne (74) Gang 56 Zhou a, NL Sach posing waster anew ei {he rstton ot ac ringers, Won to BB) (28) (751 GA Morin, A, Mild Bai HC. Cokes Opinion a he ‘inn ib sn tn wa (oo 0-53 1761 E40 Hoes, Pee, M, Delonte, Salo of Mahara ementtion om ss (ea (ss Pron Mchenry 98 (2020) 26-252 beset on oa fr emt Ble 83 () (197 561-50, ‘Sean RB. Tyeg Val RY. rapa eto far dr ‘naar Soper n th nd bath ese ng woe ‘Sige aa ow moter Chem: err Bates B03) (os St isan at Nao at of xge on he porn, dhroxn hs and oy Bar Surge Wo. Mi ion 2) on) 905 Dre Nou Ta Ja, Inroen of Bars rages ‘bene pridcion by oeconeo eon cate een oe ‘Seqon cote o arate tntyne Mies Tew (0) mM) he Sporeton G3 ADE Stabe § choral, a HM 8, Artin ees 0 meta. tc nes rng praia Baas Sarge) acl 2) OID) ‘Aoi M. Mousa MA Shih, Khe, MH Deepen of a ‘eve mein or eohned pctono Bai Ouran inde. Mc 32 2) | [cD Sha, Fargo kr, $a, O Sou, Padma bosetide [attra va radon sect peso cone with ‘ich preceenf sprlton Bat. Arch Bi Teh 94) GDI) A [XNésn Mic be den producto bape Brae come tat UR oy Meta Tr ofthe tons on he nase of ‘Beiter so nition of is concn edjse IRE odo: Syma Wan 1 Mo, UN Sar, ah 2a onc ints of mca ayn pl seer dng the ohare ent pf apt hn rede y Bla rp Ea wince ty eet ‘Eta mata. Evi Sr esi 82 (@) 01?) 62-600, Shien Kedher 3. nom Zur Overcome of eron cate rere ‘etme poco by apn Ba arteas roa ada ‘Sennen choy Boxes Res 2008 Ga) ‘ir renin a pry set emer me ‘apace Bowe En 33 0) 6-70, GE howe, Aarts XW, Seti open wpa te ios dine ‘eich met of elo argo pees art HD, ate Frog 19 Co) 14 enc Oc Gamer, VE Sos. Merck On uptake en ‘rbial rcect a overview, chen, E 1 (9) (010 289-07 Sargent nese feats wel ne epee ise tp 0 1 00 ere Reece macy ar go) Ma