ORIGINAL ARTICLE

Solid-state fermentation of coconut kernel-cake as substratefor the production of lipases by the coconut kernel-associatedfungus Lasiodiplodia theobromae VBE-1

Balaji Venkatesagowda & Ebenezer Ponugupaty &

Aneli M. Barbosa & Robert F. H. Dekker

Received: 14 November 2013 /Accepted: 10 February 2014 /Published online: 13 March 2014# Springer-Verlag Berlin Heidelberg and the University of Milan 2014

Abstract Plant oil-extracted seed-cakes are good fermenta-tion substrates for producing lipases that find application intransesterification of seed oils into biodiesel. This work de-scribes the production of lipases by five lipolytic, oil-seedassociated fungi (Aspergillus niger, Chalaropsis thielavioides,Colletotrichum gloeosporioides, Lasiodiplodia theobromae,and Phoma glomerata) by Solid-State Fermentation (SSF)on eight plant oil-seed cakes. The highest lipase activity wasfrom the Coelomycete Lasiodiplodia theobromae VBE-1grown on coconut kernel-cake, and was selected to optimizelipase production. The effects of supplementing coconutkernel-cake with mineral salts and coconut oil on lipase pro-duction by L. theobromae VBE-1 resulted in enhanced lipaseactivity. The effects of time of growth, moisture content, initialpH, temperature, as well as nutritional factors (carbon, nitro-gen, vegetable oils, surfactants) when added to coconutkernel-cake, on lipase production were examined by a one-factor-at-a-time approach, and identified key variables foroptimization by Response Surface Methodology (RSM). A26 factorial central-composite experimental design with eightstarting points and six replicates at the central point was usedfor lipase optimization. After validating the predicted levels ofthe factors, lipase production rose to 698 U/g Dry Substrate(DS) over un-optimized conditions (450 U/g DS).

Keywords Solid-state fermentation . LasiodiplodiatheobromaeVBE-1 .Lipase .Responsesurfacemethodology .

Coconut kernel-cake

Introduction

Biodiesel from plant oil-seed crops has attracted wide atten-tion globally as an alternative biofuel. An environmentallyfriendly approach to biodiesel is through enzymes such asmicrobial lipases that can transesterify plant seed-oils in thepresence of alcohols into fatty acyl alkyl esters that constitutea diesel substitute. Oil extraction of plant oil-seed yields a by-product called a seed “cake”, a nutritionally rich feedstock thatcan be used for the production of microbial enzymes such aslipases through solid-state fermentation (SSF).

Lipases (triacylglycerol acylhydrolases, EC 3.1.1.3) are dis-tinguished by a phenomenon called interfacial activation, andincrease activity when they act at the lipid-water interface ofmicellar or emulsified lipidic substrates. Lipases catalyze thehydrolysis of triacylglycerols, as well as interesterification andtransesterification reactions. These enzymes are produced on acommercial scale to meet the growing demands for processingfats and oils, and in detergent formulations (Novozymes A/S,Denmark http://www.biotimes.com/EN/Pages/default.aspx).Various microbial sources, particularly fungi, are consideredthe preferred choice for commercial scale lipase production inview of the high yields of the enzymes produced. Fungallipases are endowed with rare characteristics, such as theiractivity to operate under mild conditions, stability in organicsolvents, high substrate specificity, and regio and stereoselectivity. As such, they find applications in food processing,detergent formulations, decomposition and removal ofoleaginous substances, wastewater treatment, and recently inthe production of biodiesel (Romano et al. 2007; Treichel et al.

B. Venkatesagowda : E. PonugupatyCentre for Advanced Studies in Botany, University of Madras,Guindy Campus, Chennai 600 025, Tamil Nadu, India

B. Venkatesagowda (*) :R. F. H. DekkerBiorefining Research Institute, Lakehead University, Thunder Bay,Ontario, Canada P7B 5E1e-mail: kvsbalaj@gmail.com

A. M. BarbosaDepartamento de Quimíca - CCE, Universidade Estadual deLondrina, CEP, Londrina, Paraná 86051-990, Brazil

Ann Microbiol (2015) 65:129–142DOI 10.1007/s13213-014-0844-9

2010; Kumar et al. 2011; Salihu et al. 2012; Hamdy and Abo-Tahon 2012; Sethi et al. 2013).

Microbial lipase production has traditionally been studiedusing plant-derived oils (corn, olive, rapeseed, sunflower) andanimal fats (Domınguez et al. 2003; Papanikolaou et al. 2007).Lately, there has been interest in the industrial microbiologyand biotechnology sectors in using low-valued fatty wastes,and carbon (C) sources such as fatty acids, fats, soapstocks, n-alkanes, grease-containing waste-waters, and waste cookingoils as fermentation feedstocks for the production of value-added products including lipases (Kamzolova et al. 2005;Fickers et al. 2005; Papanikolaou et al. 2007; Darvishi et al.2009; Papanikolaou and Aggelis 2010; Mafakher et al. 2010;Sethi et al. 2013). Low-valued substrates such as beef tallowand poultry fat have also been used as feedstocks by variousstrains of the yeast, Yarrowia lipolytica, to produce lipases(Bednarski et al. 1994; Papanikolaou et al. 2007).

Yarrowia lipolytica, an oleaginous yeast, is of particularcommercial interest because of its unique ability to produce abroad range of organic acids (Krebs cycle intermediates, andesp., citric acid) when cultivated on vegetable oils, and itslipases are known to transform lipids into value-addedoleochemical products (Darvishi et al. 2009; Papanikolaouand Aggelis 2010). Lipase production by species of this yeasthas been studied to increase enzyme titers through chemicalmutation, genetic engineering or mutagenesis approaches, andnew over-producing genetically-engineered strains (Fickerset al. 2005), as well as combinations of biomolecular engi-neering and mutagenesis technologies using an expressionsystem with strains containing multi-copy integration of ex-pression cassettes, have resulted in very high lipase activities(Pignede et al. 2000; Fickers et al. 2005).

The choice of a suitable fermentation technology is crucialfor the economic production of enzymes. Solid-state fermen-tation has increasingly become recognized for large-scaleproduction of industrial enzymes as less-sophisticated andrelatively inexpensive equipment is used compared to that ofconventional fermentation processes. SSF provides a feasiblealternative to producing industrial enzymes at lower costs withthe advantage of using agro-industrial residues as fermenta-tion substrates; many of which are considered cheap as theyconstitute processing waste products (Salihu et al. 2012).Agro-industrial residues studied for lipase production bySSF have included seed oil-cakes from oil-extracted coconut(Benjamin and Pandey 1997), gingelly (sesame) (Kamini et al.1998), rice hulls (Romano et al. 2007), pongamia (Balaji andEbenezer 2008), castorbean (Godoy et al. 2009), mustard seedoil cake (Sethi et al. 2013), and mixed substrates (wheat branand sesame oil-cake) (Mala et al. 2007). Various configura-tions for SSF to produce lipases have included tray-type andpacked-bed bioreactors (Gutarra et al. 2005). Coradi et al.(2013) compared lipase production by SmF and SSF usingagro-industrial residues as cheap substrates.

The industrial demand for new sources of lipases withdifferent and novel enzymatic properties stimulates a continu-ing global interest to screen and select new strains of lipolyticmicroorganisms (Vargas et al. 2004). Towards this goal, mi-crobial lipases have been extensively explored on account oftheir suitability for industrial processes. Lipase production byplant oil seed-associated fungi over the years has receivedrelatively little attention. Recently, we reported five lipolyticfungal isolates producing high lipase titers among 1,279 seedoil-associated fungal isolates that were screened for lipaseactivity (Venkatesagowda et al. 2012). The best producerof lipases within this group was the coconut kernel-associated fungus, Lasiodiplodia theobromae VBE-1, acoelomyceteous fungus. We have extended our studiesfurther by assessing lipase production by SSF by grow-ing the five lipolytic fungal isolates on different oil-seedcakes. L. theobromae VBE-1 produced the highest lipasetiters and was selected for further study. Herein, wereport on the influence of nutritional factors on extracel-lular lipase production by L. theobromae VBE-1 whengrown on coconut kernel-cake by SSF. Optimization oflipase production by a one-factor-at-a-time approachidentified key fermentation variables for further optimi-zation by the Response Surface Method (RSM). Afterexperimental predicted values were validated for lipaseproduction, lipase production was increased 1.55-foldover un-optimized conditions.

Materials and methods

Materials

The plant oil-seed cakes (seed oil-extracted residues) used inthis study were derived from castorbean (Ricinus communis),coconut kernel (Cocos nucifera), cottonseed (Gossypium sp.),mahua (Madhuca indica), neem (Azadirachta indica), peanut(Arachis hypogea), pongamia (Pongamia pinnata), and sesa-me (Sesamum indicum). The chemical composition of thesecakes is summarized in Table 1. The cakes were producedfollowing the pressing of the plant seeds to extract the oils, andwere obtained from Sri Venkateswara Oil Mill Pvt Ltd,Veppanapalli, India. They are regarded as relatively inexpen-sive substrates with prices ranging fromUS $ 0.010 to $ 0.080per kg.

All vegetable oils were of food-grade, and all laboratorychemicals were of analytical grade.

Fungi and nutrient medium

Five lipolytic oil seed-associated fungi: Aspergillus niger,Chalaropsis thielavioides, Lasiodiplodia theobromae VBE-1(GenBank Accession No. EU852567), Phoma glomerata, and

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Colletotrichum gloeosporioides (Venkatesagowda et al. 2012)were evaluated for lipase production by SSF. The fungalisolates were sub-cultured on potato-dextrose-agar and storedat 4 °C. The oil-seed cakes in this study were supplementedwith mineral salts of Czapek-Dox medium (CDM; sodiumnitrate (2.0 g), potassium phosphate (1.0 g), magnesium sul-fate (0.5 g), potassium chloride (0.5 g), ferrous sulfate (0.01 g)and distilled water (1,000 mL), pH adjusted to 6.5; sucrosewas omitted) in the cultivation of the fungal isolates. In SSFeach experiment was performed in triplicate, and the resultsrepresent the mean ± SD.

Determination of fungal biomass

Fungal biomass was determined by an indirect estimationof the chitin content (as N-acetylglucosamine) in the fungalcell wall following the procedure of Elson and Morgan(1933). After the SSF and washing steps to extract thelipases (see below), samples of the washed fungal-colonized material (1 g) were treated with 5 mL of 6 NHCl for 6 h/100 °C. The hydrolyzed samples were thenevaporated to remove excess HCl, and the resulting residue

washed with distilled water and filtered. The volume offiltrate was recorded, and 2 mL aliquots taken for assay ofN-acetylglucosamine by treating the samples with 1 mL offreshly prepared 2 % (v/v) acetylacetone in 0.5 M Na2CO3,and heated for 15 min/100 °C. After cooling, 5 mL of 95 %ethanol and 1 mL of Ehrlich’s reagent (1.33 % (w/v) of p-dimethylaminobenzaldehyde dissolved in 100 mL of 1:1(v/v) ethanol:c.HCl) were added, and the contents thor-oughly mixed. The resulting purple-red color was allowedto develop for 30 min, and then read in a spectrophotometerat A530 against a blank. A calibration N-glucosamine curvewas prepared using a stock solution (250 mg of N-glucosamine/100 mL) from which working standard solu-tions (1–100 μg/mL) were prepared. The N-glucosaminecontent is expressed as mg/g of dry fungal-colonizedmaterial.

Assay of lipase activity

Lipase activity present in the filtrates resulting fromextracting the oil-seed cakes following SSF were assayedagainst p-nitrophenyl palmitate (p-NPP, Sigma) as the

Table 1 Chemical composition of the different plant oil-seed cakes used in solid-state fermentation for fungal lipase production

Composition of oil-seed cakes (%)a Oil-seed cakes

Castorbeanb Coconut kernel Cottonseed Mahua Neemc Peanut Pongamia Sesame

Dry matter 91 88.8 94.3 89 87 92.6 96 83.2

Crude fiber 11.2 10.8 15.7 2 7.2 5.3 1 7.6

Crude protein 28 25.2 40.3 23 0.5 49.5 34.5 35.6

Residual oil 10 15 9 14 12 20 24 18

Ash 11.8 6.0 6.8 7.1 5.3 4.5 9.5 4.8

Fatty acid composition of the residual oils (%)d

Caprylic acid (C8:0) – 6.21 – – – – – –

Capric acid (C10:0) – 6.15 – – – – – –

Lauric acid (C12:0) – 51.02 – – – – – –

Myristic acid (C14:0) – 18.94 0.866 – 1.6 – – –

Palmitic acid (C16:0) 1.07 8.62 24.4 22 14.3 7 5.8 8.9

Stearic acid (C18:0) 0.91 1.94 2.54 24 23.1 5.1 7.7 4.6

Oleic acid (C18:1) 4.48 5.84 18.06 42 49.7 60 63.6 42.5

Ricinoleic acid (C18:1)e 88.30 – – – – – – –

Linoleic acid (C18:2) 4.54 1.28 52.5 9.6 9.8 24.3 18.3 43

Linolenic acid (C18:3) 0.57 – 0.201 – – – – –

Arachidic acid (C20:0) – – 0.294 2.4 1.5 3.6 4.6 1

Behenic acid (C22:0) – – 0.139 – – – – –

Lignoceric acid (C24:0) – – 0.120 – – – – –

a Chemical composition of different plant seed oil-cakes as reported by Ramachandran et al. (2007); a Chempro; http://www.chempro.in/fattyacid.htmbCastorbean oil cake contains the toxin ricin (0.07 %), a naturally occurring lectinc Neem oil cake contains azadirachtin (0.05 %), a compound belonging to the limonoid group of metabolitesd No free fatty acids present in the cakese 12-hydroxy-9-cis-octadecenoic acid, an unsaturated omega-9 fatty acid and a major component of the seed oil from mature castorbeans

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substrate for 15 min at 37 °C as previously reported(Venkatesagowda et al. 2012). One unit (U) of lipaseactivity is defined as the amount of enzyme that liberates1 μmol of p-nitrophenol per min. Lipase activities arisingfrom SSF are expressed as U/g dry substrate (DS).

Lipase production by solid-state fermentation

Eight oil-seed cakes (see Materials) were evaluated as sub-strates for SSF in the production of lipases by five lipolyticfungi. Erlenmeyer flasks (250 mL) containing 20 mL of CDMwith and without added coconut oil (1 %, v/v based on CDM)as lipase inducer, were mixed with 20 g oil-seed cake (ratio ofCDM to cake was 1:1, moisture content 50 %), sterilized(121 °C for 30 min), and inoculated with four 7-mm diameteragar plugs colonized with fungal mycelium. The flasks wereleft under stationary conditions at 25 °C and initial pH (pHi) of6.5 for ten days.

Effect of substrate combinations on lipase productionby Lasiodiplodia theobromae VBE-1 on solid-statefermentation

The influence of different combinations of oil-seed cakes assubstrates on lipase production was evaluated withL. theobromae VBE-1 using coconut kernel-cake (10 g DS)as the base substrate for SSF. Oil-seed cakes (10 g DS;castorbean, cottonseed, mahua, neem, peanut, pongamia, ses-ame), as well as wheat bran and rice bran, were each mixedwith coconut kernel-cake in a ratio of 1:1. CDM (20 mL) wasblended with the cake mixtures in the flasks. The experimentswere performed under stationary conditions over ten days atpHi 6.5, 25 °C and a moisture content of 50 %.

Lipase production by Lasiodiplodia theobromae VBE-1in solid-state fermentation by a one-factor-at-a-time approach

To enhance extracellular lipase production by L. theobromaeVBE-1 grown by SSF, various fermentation variables wereevaluated including carbon and nutrients by a one-factor-at-a-time approach while keeping time of growth (ten days), pHi

(8.0), moisture content (60 %) and temperature (25 °C) con-stant, and were further used in experiments examining theinfluence of nutrient composition on lipase production.

The fungal isolate was grown in a series of 250 mL Erlen-meyer flasks containing CDM (20 mL, pH 8.0) blended withcoconut oil (0.2 mL; 1 %, v/v based on CDM) as lipaseinducer, and then mixed with coconut kernel-cake (20 g).The moisture content was adjusted to 60 % with CDM. Theflasks were inoculated with four 7-mm diameter mycelial-colonized agar plugs and left stationary at 25 °C forten days. Following growth, lipases were extracted from the

fermented solids as described below, and the filtrates assayedfor lipase activity.

In experiments evaluating the composition of the nutrientmedium on lipase production by SSF, the following wereseparately incorporated into CDM at 1 % (w/v) concentration:carbon sources (glucose, fructose, sucrose, maltose, xylose,mannitol, starch, carboxymethylcellulose, glycerol); nitrogensources (inorganic: ammonium nitrate, sodium nitrate, ammo-nium sulfate; organic: urea, yeast extract, peptone, tryptone,casein); lipid substrates (myristyl alcohol, tributyrin, tristearin,oleic acid, cholesterol, and vegetable oils derived from al-mond, castorbean, coconut, mustard, neem, olive, peanut,pongamia, sesame, sunflower); and emulsifying agents(Tweens 20, 40, 60, 80; Tri ton X-100; sodium-dodecylsulfate; sodium deoxycholate; polyvinylpyrrolidone;gum arabic). Each experiment was performed in triplicate, andthe results represent the mean ± SD.

Extraction of lipases from oil-seed cakes following solid-statefermentation

Following SSF, 50 mL of Tris-HCl buffer (50 mM, pH 8.0)was added to each flask and placed on a rotary shaker(200 rpm) for 1 h at 25 °C. The contents of each flask werethen filtered (Whatman #1 filter paper) and the filtrate collect-ed. Next, 25 mL buffer solution was added to the extractedresidue, and the contents mixed for a further 1 h at 25 °C, andfiltered. This step was repeated once more. The resulting twofiltrates were pooled with the first extract (∼95 mL totalvolume) and used as the source of enzyme for assay of lipaseactivity. The expended oil-seed cake residues were used tomeasure fungal biomass as described above.

Optimization of lipase production by solid-state fermentationusing Response Surface Methodology

Based upon the results obtained from a one-factor-at-a-timeapproach, the variables selected were: coconut oil (X1), incu-bation temperature (X2), initial moisture content (X3) andTriton X-100 (X4), and were used to assess lipase productionby SSF using coconut kernel-cake as substrate. The effects ofeach variable on lipase production under defined conditionswere determined by modulating variables according to a full2k factorial design (Leardi 2009). Composite design, whichgives the total combinations of (2k+2k+no), provided 30experimental runs, where k is the number of independentvariables, and no the number of replicates of experiments atthe central point. For the four factors, a 26 factorial central-

2) and six replicates at the central point, resulting in 30experiments (Design Expert 7.0, StatEase, Minneapolis,USA, 2006) was used to optimize the variables. The 2k facto-rial design was performed with four factors at five-coded

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composite experimental design with eight starting-points (α=

levels (–2,–1,0,+1,+2) in duplicate, with central points intriplicate to determine the experimental error (Box et al.1978). The real values and coded values of the independentvariables are given in Table 2. The variables were codedaccording to Eq. (1)

xi ¼ X i−X 0

δXð1Þ

where xi is the coded (dimensionless) value of the variable Xi,Xo the value ofXi at the central point, and δX is the step changeof the variable. The responses of the input variables wereevaluated as a function of lipase production coded by y (U/gDS). The experimental results of the central composite designwere fitted in a second-order polynomial equation (Eq. 2) by amultiple regression technique. The behavior of the systemwasexplained by:

active coefficients, and xi and xj are coded independent vari-ables. The design matrix is shown in Table 2. Lipase activity(U/g DS) was the measured response.

All calculations involved, as well as the drawing of the 2-Dcontour and 3-D surface plots, were performed using DesignExpert 7.0 software. The quality of fit of the second-ordermodel equation was expressed by the coefficient of determi-nation (R2), and its statistical significance determined by theF-test. The significance of the regression coefficients wastested by the t-test. The optimum levels of variables to obtainmaximum lipase production were determined by runningexperiments using the optimum values for variables givenby response optimization (Myers et al. 2009) for confirmationof the predicted value, and lipase production was confirmed.In order to validate the adequacy of the model, a total of threeverification experiments were carried out under optimal fer-mentation conditions within the experimental range.

Results and discussion

Lipase production by five lipolytic fungi grown on differentoil-seed cakes by solid-state fermentation

Table 1 summarizes the composition of the various oil-seedcakes evaluated for lipase production by SSF. The cakescontain high proportions of fiber, protein and residual oils.The fatty acid compositions of the residual oils in the cakes arealso included. Altogether, the oil-seed cakes constitute a nu-tritionally rich feedstock to support fermentation for the pro-duction of value-added chemical products such as enzymes.

Screening for lipolytic activity of 1,279 endophytic fungiisolated from seven different plant oil-bearing seeds resultedin forty fungal isolates that tested positive for extracellularlipase activity (Venkatesagowda et al. 2012). Among thisgroup were five isolates (Aspergillus niger (94 U/mL),Chalaropsis thielavioides (96 U/mL), Lasiodiplodiatheobromae VBE-1 (108 U/mL), Phoma glomerata (95 U/

Table 2 Experimental design matrix defining conditions for optimiza-tion of lipase production by Lasiodiplodia theobromae VBE-1 on solid-state fermentation and the obtained responses

Exp. Variables in coded levels Responses

X1 X2 X3 X4 Lipase activity (U/g DS)

Observed Predicted

1 −1 −1 −1 −1 416.5 396.6167

2 +1 −1 −1 −1 459.0 406.4458

3 −1 +1 −1 −1 379.7 362.9458

4 +1 +1 −1 −1 379.3 376.9500

5 −1 −1 +1 −1 340.1 339.6458

6 +1 −1 +1 −1 337.8 330.7000

7 −1 +1 +1 −1 322.0 340.4500

8 +1 +1 +1 −1 321.2 335.6792

9 −1 −1 −1 +1 378.5 335.5292

10 +1 −1 −1 +1 335.7 319.7833

11 −1 +1 −1 + 501.3 510.9333

12 +1 +1 −1 +1 527.4 499.3625

13 −1 −1 +1 +1 394.5 399.3833

14 +1 −1 +1 +1 376.6 364.8625

15 −1 +1 +1 +1 585.2 609.2625

16 +1 +1 +1 +1 556.5 578.9167

17 −2 0 0 0 346.3 344.8375

18 +2 0 0 0 296.9 324.3208

19 0 −2 0 0 316.4 376.2875

20 0 +2 0 0 590.6 556.6708

21 0 0 −2 0 300.4 371.8375

22 0 0 +2 0 439.9 394.4208

23 0 0 0 −2 331.5 351.6042

24 0 0 0 +2 527.9 533.7542

25 0 0 0 0 514.4 557.7333

26 0 0 0 0 567.9 557.7333

27 0 0 0 0 566.9 557.7333

28 0 0 0 0 562.7 557.7333

29 0 0 0 0 568.5 557.7333

30 0 0 0 0 566.0 557.7333

Factors Real levels

−2 −1 0 1 2

X1, coconut oil (%, v/v) 0 0.5 1 1.5 2.0

X2, temperature (°C) 15 20 25 30 35

X3, moisture content (%) 40 50 60 70 80

X4, Triton X-100 (%, w/v) 0 0.5 1 1.5 2.0

DS dried solids

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y ¼ β0 þX

β ixi þX

β iix2i þ

Xβ iixix j ð2Þ

where y is the predicted response, β the intercept term, βi thelinear coefficients, βii the quadratic coefficients, βij the inter-

mL) and Colletotrichum gloeosporioides (98 U/mL))exhibiting the highest extracellular lipase activity by sub-merged fermentation (SmF). Exceptionally high lipase activ-ity was reported for Yarrowia lipolytica 704 grown on rape-seed oil resulting in enzyme titers of 2,760 U/mL Kamzolovaet al. (2005); lipase activity was assayed by the titrimetrymethod (Stuer et al. 1986). The titrimetry method measuresthe amount of carboxyl groups of free fatty acids releasedfrom the hydrolysis of triacylglycerols (vegetable oils) bytitrating against NaOH, and is known to generally over-inflate lipase titers compared with the highly sensitive spec-trophotometric method employing p-NPP as chromogenicsubstrate, as used in the work reported herein. Likewise, inanother study, Fakas et al. (2010) also reported rather highlevels of lipase (4,100 to 7,300 U/mL; lipase measured bytitrimetry) using newly isolated Penicillium sp. strains culti-vated on olive oil as substrate in shake-flasks. Agro-industrialwastes have been used as alternative substrates for lipaseproduction by Y. lipolytica strains M1 and M2 resulting inlipase titers of 11 and 8.3 U/ml, respectively, on olive oil-containing media (Mafakher et al. 2010).

In extending our work, the five fungal isolates were eval-uated by SSF in attempts to enhance lipase production. Whengrown on eight different plant seed-oil cakes, each fungalisolate showed a preference for a particular, albeit different,oil-seed cake, and this appeared to be inherent in the origin ofeach fungal isolate (Table 3). These isolates had, therefore,adapted to their host plant seed-oil niche where availability ofessential nutrients in the cakes exerted a nutritional effect onlipase production. All five fungal isolates showed high lipaseactivities (range 220–300 U/g DS), thus, confirming an earlierstudy (Venkatesagowda et al. 2012) conducted in SmF thatthese isolates were highly lipolytic.

Nutrient supplementation has been reported to enhance li-pase activity when this was added to the solid substrate in SSF(Pandey 2003; Ramachandran et al. 2007; Balaji and Ebenezer2008; Coradi et al. 2013), and plant seed-oils are known to beexcellent inducers of lipases (Rodriguez et al. 2006; Coradiet al. 2013). As L. theobromaeVBE-1 was previously found toproduce high l ipase t i ters (108 U/mL) by SmF(Venkatesagowda et al. 2012), this isolate was selected toevaluate the influence of adding coconut oil as lipase inducerto seed-oil cakes in attempts to enhance lipase activity. Resultsshown in Table 3 demonstrated that lipase activities could beenhanced several-fold through adding coconut oil (1 %, v/v) tothe various oil-seed cakes, and the trend for lipase productionby the different fungal isolates was similar to that observed inthe isolates grown in the absence of coconut oil. However,there were two exceptions, Chalaropsis thielavioides (isolatedfrom coconut kernel) produced higher lipase activity onpeanut-cake than coconut kernel, and P. glomerata isolatedfrom peanut, preferred castorbean-cake, although its activityon peanut-cake was somewhat similar. Peanut-cake is a

nutrient-rich substrate that supports fungal growth (Pandey2003). Sesame-cake was the best substrate for lipase produc-tion by A. niger. Similarly, Chalaropsis thielavioides producedthe highest lipase activity on peanut-cake; L. theobromaeVBE-1 on coconut kernel-cake, P. glomerata on castorbean-cake and peanut-cake. The pongamia-cake supported thehighest lipase activity for Colletotrichum gloeosporioides (Ta-ble 3). Similar results were obtained with Aspergillus terreusby using mustard seed oil cake (Sethi et al. 2013).

Lipase production by Lasiodiplodia theobromae VBE-1on seed oil-cake combinations by solid-state fermentation

Of the five fungal isolates screened on the different oil-cakes,L. theobromae VBE-1 exhibited the highest lipase activity(450 U/g DS; Table 3) and was chosen for further studies onlipase production by SSF on coconut kernel-cake. In a sepa-rate experiment, the effect of supplementing coconut kernel-cake on SSF by L. theobromae VBE-1 with mineral salts(CDM) was examined and resulted in enhanced lipase levelscompared with water (from 256±7.2 to 328±5.1 U/g DS).Adding coconut oil as lipase enzyme inducer to coconutkernel-cake in the presence of CDM promoted lipase produc-tion to 503±12.3 U/g DS; an overall 1-fold increase.

When each of the different seed-oil cakes were mixed withcoconut kernel-cake (ratio 1:1) and used in SSF,L. theobromaeVBE-1 lipase activity did not increase over coconut kernel-cake alone as substrate (Table 4). The reason for this may beinherent in the nature of the chemical constituents present inthe different seed oil-cakes, which affected fungal growth, asjudged by the amount of fungal biomass produced (Table 4).Castorbean-cake resulted in the lowest lipase activity andfungal growth. Phenolic compounds are present in many plantoil-seeds, and are known to inhibit microbial activity (Akandeet al. 2010). The two cereal brans mixed with coconut kernel-cake resulted in lipase activities higher than that supported bythe other oil-cake mixtures (Table 4). Similar observationshave been reported by other investigators (Benjamin andPandey 1997; Edwinoliver et al. 2010; Sethi et al. 2013).Since coconut kernel-cake facilitated high enzyme yields, thissubstrate was used in further studies. Coconut kernel-cake is awaste residue produced after extracting oil from coconut.

Influence of nutrients and fermentation parameterson enhancing lipase production by Lasiodiplodia theobromaeVBE-1 in solid-state fermentation

Fermentation parameters such as time of growth, moisturecontent of the solid substrate, pHi and temperature influencemicrobial growth and subsequent enzyme production. Maxi-mal lipase production by SSF on coconut kernel-cake byL. theobromae VBE-1 was found to occur at ten days growth,a moisture content of 60 %, pHi of 8.0, and at a temperature of

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25 °C. These parameters were then used in further studiesexamining the influence of various nutrients on lipase produc-tion applying a one-factor-at-a-time approach (see Table 5).Moisture content of the solid substrate is an important param-eter for SSF, and can vary between 60 % and 80 % (Zadraziland Puniya 1995). Sethi et al. (2013) reported maximumlipase production pH of 6.0 in Aspergillus terreus using mus-tard seed oil cake.

The best carbohydrate C source of the sugars evaluated wassucrose (515 U/g DS). Glucose appeared to be a repressor oflipase synthesis (6 U/g DS), and this was also observed forlipase from Botryosphaeria ribis EC-01 grown in SmF(Messias et al. 2009). Maltose, starch and cellulose, by con-trast, which are composed of glucose, resulted in relatively

higher lipase activities but lesser so than sucrose (Table 5).The best N source for lipase production were complex organicN sources; yeast extract (530 U/g DS) and peptone (520 U/gDS). The inorganic N sources examined, with the exception ofNaNO3, resulted in lower lipase activities (Table 5). Ureaproduced 200 U lipase/g DS. By contrast, a 6-fold higherlipase activity on urea than yeast extract was reported forRhizopus homothallicus by SSF on sugarcane bagasse(Rodriguez et al. 2006). Selection and addition of N sourcesplay vital roles in lipase production (Sethi et al. 2013).Montesinos et al. (2003) reported that simultaneous increasesof C and N concentrations enhanced lipase titers in Candidarugosa ATCC, but N-limitation in the nutrient media sup-pressed lipase secretion.

Table 3 Effect of different plant seed oil-cakes on lipase production by five lipolytic fungi on solid-state fermentation

Fungal isolate Lipase activity (U/g DS)

Oil-seed cakes

Neem Peanut Sesame Castorbean Pongamia Mahua Coconut kernel Cottonseed

Absence of inducer

A. nigera 125±3.7 227±5.3 300±1.8 243±5.4 214±4.1 80±3.4 229±4.2 137±5.3

C. thielavioidesb 96±4.3 248±6.3 186±4.2 117±6.3 120±7.3 136±2.8 197±3.7 40±5.7

C. gloeosporioidesc 45±3.6 84±5.0 230±4.3 130±3.7 237±5.8 65±7.5 126±5.3 112±4.5

L. theobromae VBE-1d 48±4.3 156±7.1 160±4.6 136±5.7 246±3.7 174±6.3 206±4.2 148±5.7

P. glomeratae 8±5.4 138±4.2 149±3.8 220±6.3 67±4.8 24±3.5 137±6.3 80±5.3

Presence of inducer (coconut oil, 1 %, v/v)

A. niger 217±7.5 352±12 380±9.0 344±19.0 269±13.0 161±8.4 241±12.4 300±15.4

C. thielavioides 193±7.7 371±13 263±8.0 243±11.6 312±14.0 219±12.2 225±12.8 104±9.0

C. gloeosporioides 147±4 211±10 400±21 217±11.8 408±20.1 132±6.5 371±17.8 267±12.9

L. theobromae VBE-1 114±7.5 202±6 360±8.6 390±19.8 340±17.1 284±14.1 450±20.3 227±11.7

P. glomerata 32±10 375±10 265±8.5 378±19.0 191±9.5 97±5.0 280±7.0 172±9.0

a Isolated from sesame seedb,d Coconut kernelc Pongamia seede Peanut

Table 4 Effect of oil-seed cakecombinations on lipase produc-tion by L. theobromae VBE-1 inSSF

Substrate combinations(1:1, w/w)

Lipase activity (U/g DS) Biomass (N-acetylglucosaminecontent, mg/mL)

Coconut kernel-cake (CKC) 580±14.9 98±0.98

CKC: Neem cake 436±9.2 45±0.45

CKC: Peanut cake 524±11.1 34±0.34

CKC: Sesame cake 447±9.4 66±0.66

CKC: Castor cake 241±5.1 20±0.2

CKC: Pongamia cake 537±13.5 73±0.73

CKC: Mahuca cake 555±11.7 68±0.68

CKC: Cottonseed cake 561±11.9 61±0.61

CKC: Wheat bran 566±14.8 84±0.84

CKC: Rice bran 524±14.5 55±0.55

Ann Microbiol (2015) 65:129–142 135

Table 5 Optimization of lipase production by Lasiodiplodia theobromae VBE-1 on solid-state fermentation with coconut kernel-cake applying a one-factor-at-a-time approach

Experimental conditionsa Variables Lipase activity (U/g DS) Biomass (N-acetylglucosamine content, mg/mL)

Time of growth (d)b 1 15±0.2 2±0.2

2 80±1.5 3+0.7

3 159±3.1 15±0.3

4 264+5.2 23±0.4

5 385±7.6 42±0.9

6 466±9.2 52±0.1

7 482±11.5 62±0.7

8 531±12.4 75±0.3

9 540±12.6 78±0.7

10 554±14.9 80±0.4

11 550±14.8 80±0.2

Moisture content (%)c 40 66±1.3 34±0.1

50 326±6.4 73±0.5

60 461±9.2 82±0.9

70 286±5.6 41±0.3

80 211±4.1 35±0.8

pHid 1 2±0.03 22±0.04

2 30±0.5 32±0.9

3 133.6±2.6 44±0.23

4 237.1±4.6 51±0.54

5 340.9±6.7 60±0.89

6 542.5±10.7 71±0.03

7 548±14.8 120±0.5

8 576±8.4 127±0.7

9 560±11.0 110±0.8

10 224.8±1.4 88±0.9

Temperature (°C)e 10 199±3.9 27±0.43

15 216±4.2 30±0.12

20 353±6.9 51±0.79

25 476±9.4 98±0.0

30 414±8.1 88±0.2

35 410±8.1 75±0.05

40 112±2.2 46±0.08

45 103±2.0 42±0.1

50 80±1.5 35±0.9

Carbon sourcesf Glucose 6±0.1 21±0.2

Fructose 422±8.3 13±0.4

Xylose 200±3.9 15±0.1

Sucrose 515±12.1 110±0.05

Maltose 260±5.1 12±0.5

Mannitol 312±6.1 110±0.3

Starch 360±7.1 90±0.08

Cellulose 448±8.8 68±0.8

Glycerol 232±4.5 38±0.56

Lipidsf Tristerin 228±4.5 34±0.6

Tributyrin 416±8.2 70±0.12

Oleic acid 380±7.5 42±0.62

Cholesterol 158±7.31 21±0.42

136 Ann Microbiol (2015) 65:129–142

Various lipids ranging from triacylglycerols, vegetable oils,surfactants, and cholesterol were examined for their influenceon lipase production by L. theobromae VBE-1 grown oncoconut kernel-cake. The results are shown in Table 5, andthe highest lipase activity resulted for coconut oil (580 U/gDS), Tween 60 and Triton X-100 (each 512 U/g DS). Lowlipase activity was observed with oleic acid (380U/g DS). Thereason for this may be explained from an observation reportedby Barth and Gaillardin (1997) with Y. lipolytica in thatextracellular lipase activity required oleic acid as a stabilizer/activator, whereas the cell-bound lipase did not. As for theeffect of fatty acids on lipase production, experimental data inthe literature is contradictory. With respect to Candidalipolytica, fatty acids (viz., capric, lauric, and oleic acids) werereported to inhibit lipase production (Zvyagintseva 1971),whereas others reported that oleic acid as well as rapeseedoil stimulated lipase production (Ota et al. 1982; Pignede et al.2000).

Lasiodiplodia theobromae VBE-1 grew relatively well(38 mg/mL) when cultivated on medium containing ARgrade glycerol producing lipase titers of 232 U/g DS(Table 5). In Y. lipolytica cultivated on medium

containing glycerol; however, it was noted that lipasewas only produced when the glycerol concentration inthe medium fell below 4.0 g/L (Kamzolova et al. 2011).

Of the oils tested, lipase production was lowest with al-mond oil (116 U/g DS). Lipase production by Y. lipolytica onolive oil medium resulted in lipase activity of 34.6 U/mL, andinorganic N sources showed no significant influence on lipaseproduction (Darvishi et al. 2009). Of the five strains ofAspergillus sp. and Penicillium expansum cultivated on wastecooking olive oil, the highest lipase activity of 645 U/mL(titrimetry assay) occurred with A. niger NRRL 363, andenzyme activity was fungal strain and time dependent(Papanikolaou et al. 2011).

Surfactants are surface-active amphiphilic molecules thatwhen added to microbial cultures increase cell membranepermeability facilitating enzyme export into the culture medi-um that can result in marked increases in enzyme titers. Theyare also known to activate and inhibit lipase activity, and in thecase of lipophilic fungi, surfactants can act as a C source forlipase synthesis (Messias et al. 2009). Of the surfactantsevaluated, high lipase activity resulted from Triton X-100(512 U/g DS). A similar observation was reported for

Table 5 (continued)

Experimental conditionsa Variables Lipase activity (U/g DS) Biomass (N-acetylglucosamine content, mg/mL)

Coconut oil 580±11.4 86±0.4

Castor oil 280±5.5 20±1.2

Sunflower oil 418±8.2 40±0.8

Peanut oil 350±6.9 35±0.5

Pongamia oil 520±10.2 78±0.5

Sesame oil 515±10.1 65±0.76

Neem oil 422±8.3 43±0.11

Olive oil 490±9.7 30±0.9

Almond oil 116±2.2 72±1.4

Mustard oil 210±4.1 17±0.3

Surfactantsf Tween 20 92±9.7 30±0.9

Tween 40 208±4.1 18±0.3

Tween 60 512±7.2 45±0.8

Tween 80 368±7.2 15±0.1

Triton X-100 512±10.1 62±0.98

SDSg 416±8.2 160±1.5

Polyvinylpyrrolidone 157±3.1 70±1.1

Gum arabic 300±5.9 105±0.67

a All experiments were conducted on coconut kernel-cake and CDM (containing 1 %, v/v coconut oil) in a ratio of 1:1 under static conditionsb pHi 6.5, 25 °C, moisture content of 50 %c pHi 6.5, 25 °C, 10 dayd 25 °C, 10 day, 60 % moisture contente pHi 8.0, 10 day, 60 % moisturef pHi 8.0, 25 °C, 10 day, 60 % moisture contentg Sodium dodecylsulfate

Ann Microbiol (2015) 65:129–142 137

Colletotrichum gloeosporioides (Balaji and Ebenezer 2008).Surfactants like Triton X-100 and Tweens (20, 40) have beenreported to influence lipase production in Botryosphaeriaribis EC-01 (Messias et al. 2009). Mahadik et al. (2004)reported maximum lipase activity by a mutant A. niger UV-100 strain cultivated on nutrient media based on olive oil towhich was added Triton X-100.Whereas lipase production bywild strains of Y. lipolytica was critically affected by thepresence of surfactants, as well as the degree of aeration andagitation (Kamzolova et al. 2005).

Various metal cations were also examined on lipase produc-tion, and divalent cations, Ca++ (520U/g DS) and Cu++ (480 U/g DS), resulted in high lipase activities. The chelating agentEDTA resulted in low lipase activity (92 U/g DS) indicatingthat metal cations (e.g., Ca++ and Cu++) may be necessary forlipase synthesis by L. theobromaeVBE-1. High lipase produc-tion was also reported with Ca2+ and Mg2+ in Aspergillusterreus using mustard seed oil cake (Sethi et al. 2013).

The environmental and nutritional factors influencinglipase production by L. theobromae VBE-1 in SSF werepHi 8.0, incubation temperature 25 °C, sucrose as Csource, yeast extract as N source, and coconut oil asthe vegetable oil. The surfactants Tween-60 or TritonX-100 could also act as a source of lipid for inducinglipase activity.

Optimization of lipase production by Lasiodiplodiatheobromae by solid-state fermentation using responsesurface methodology

Response surface methodology, a combination of statisticaland mathematical algorithms for developing, improving andoptimizing processes and products (Myers et al. 2009), isadvantageous over conventional methods of product optimi-zation as it involves less experimental trials, and also becauseof its suitability for multiple factor experiments (Chang et al.2006).

Based on the results obtained from a one-at-a-time factorapproach, four factors were chosen using RSM: coconut oil(X1), temperature (X2), moisture content (X3) and TritonX-100 (X4) to optimize lipase production by L. theobromaeVBE-1 by SSF on coconut kernel-cake. The response-surfaceoptimization for lipase production was performed using acentral-composite design involving 30 experimentalruns, and results for the four-factor experimental designare presented in Table 2. Highest lipase production(590.6 U/g DS) was observed with experimental run20. Applying multiple regression analysis to the exper-imental data, the response variable (Y) and the testvariables were related to the second-order polynomialequation:

Y lipase production;U=g DSð Þ ¼ 557:7333 − 5:12917 X 1 þ 45:09583 X 2 þ 5:645833 X 3 þ 45:5375 X 4þ1:04375X 1X 2−4:69375X 1X 3−6:39375X 1X 4 þ 8:61875X 2X 3 þ 52:26875X 2X 4 þ 30:20625X 3X 4−55:7885X 2

1−22:8135 X 2

2 − 43:651 X 23 − 28:7635 2

4

ð3Þ

Analysis of variance (ANOVA) of the quadratic re-gression model (Table 6) demonstrated that the modelwith Fisher’s test (F model, mean square regression/mean square residual=13.19) yielded a low probabilityvalue [(P model>F)=0.0001] and implied that the mod-el was statistically significant. The coefficient of deter-mination (R2 0.9249) indicated that the model couldexplain 92.49 % of the variability with 7.51 % of the

total variations unexplained indicating the model’sapplicability.

The value for the adjusted coefficient of determina-tion (adj.R2 0.8548) was sufficiently high to advocatehigh significance of the model and good reliability ofthe experimental values (Khuri and Cornell 1987). Thecoefficients of the regression equation (Eq. 3) weredetermined using Design Expert software. The result

Table 6 Statistical data from RSM using a central-compost design

Source Sum of squares df Mean square F-Value p-value Prob>F

Model 291,214.19 14 20,801.01 13.19 <0.0001 significant

Lack of fit 21,367.90 10 2,136.79 4.69 0.0508 Not significant

Pure error 2,274.09 5 454.81

Correlation total 314,856.18 29

Source Standard deviation R-Squared Adjusted R-Squared Predicted R-Squared PRESS

Quadratic 39.70 0.92 0.85 0.59 126,353.8 Suggested

Cubic 27.40 0.98 0.93 −0.37 432,978.7 Aliased

138 Ann Microbiol (2015) 65:129–142

revealed that the linear coefficients (X1, X2, X3, X4), aquadratic term coefficient, and cross-product coefficientswere significant, having very low P values (p<0.0001)(Table 7).

From the analysis of variance (ANOVA) of all factorsemployed to ensure maximum enzyme production, the modelpredicted the highest lipase activity of 609.86 U/g DS, whilethe experimental trials yielded 590.6 U/g DS. After solvingthe regression equation (3), the highest lipase productionpredicted by the model using the optimum values of the testvariables in real units were: coconut oil (0.985 %, v/v), tem-perature (30 °C), moisture content (66.4 %) and Triton X-100(1.5 % v/v). All the factor values for the four variables werefound to be present within the design space. The 2-D contourplots and the 3-D response-surface plots were generated fromthe regression equation by keeping the two variables at zeroand changing the other two variables in different combinations(Fig. 1). The validation experiments conducted with the RSM-based predicted values of the nutrient media componentsshowed a significant enhancement of lipase production byL. theobromae VBE1. Lipase production was 568.5 U/g DSin the original nutrient medium with the four components attheir central-point levels. Design Expert software predicted amaximum lipase activity of 609.86 U/g DS in optimizedmedium involving the four factors. After validating the pre-dicted levels of the components, lipase production rose to698.1 U/g DS as against 450 U/g DS obtained before optimi-zation. A lipase activity of 698.1 U/g DS by L. theobromae

Table 7 ANOVA analysis for a response surface quadratic model

Model term Coefficient estimate Standard error P-value

Intercept 557.73 16.20

X1 −5.12 8.10 0.5363

X2 45.09 8.10 <0.0001

X3 5.64 8.10 0.4967

X4 45.53 8.10 <0.0001

X12 −55.78 7.58 <0.0001

X22 −22.81 7.58 0.0088

X32 −43.65 7.58 <0.0001

X42 −28.76 7.58 0.0018

X1X2 1.04 9.92 0.9176

X1X3 −4.69 9.92 0.6431

X1X4 −6.39 9.92 0.5292

X2X3 8.61 9.92 0.3989

X2X4 52.26 9.92 <0.0001

X3X4 30.20 9.92 0.0082

Fig. 1 Contour and 3-Dresponse-surface curves showingthe effect of a temperature andcoconut oil; b moisture contentand coconut oil; c Triton X-100and coconut oil; d moisturecontent and temperature; e TritonX-100 and temperature; f TritonX-100 and moisture content; onlipase production (U/g DS or U/gDM (dry matter)) byLasiodiplodia theobromae VBE-1 by solid-state fermentation oncoconut-kernel cake

Ann Microbiol (2015) 65:129–142 139

VBE-1 represents one of the highest reported levels of lipaseactivity reported for lipase assayed against p-NPP as substrate.

Other investigators also emphasized the advantages ofRSM for optimizing lipase production by SSF in other fungi(Di Luccio et al. 2004; Kempka et al. 2008; Kumar et al.2011). Lipase production by Aspergillus carneus by SFFusing RSM with five experimental variables resulted in a 1.8fold increase in enzyme output as against a meager lipaseincrease from a one-at-a-time factor approach (Kaushik et al.2006).

According to the literature, the maximum lipase productionreported by SmFwas 316 U/mL, and occurred for B. ribis EC-01 (Messias et al. 2009). In SSF, high lipase titers have beenreported for Colletotrichum gloeosporioides (983 U/g DS;Balaji and Ebenezer 2008; and A. niger 620.8 U/g DS;Edwinoliver et al. 2010), but in both cases, lipase was assayedby the titrimetrymethod, which generally gives higher activityvalues than the p-NPP assay procedure.

Scale-up of solid-state fermentation at different levelsfor lipase production by Lasiodiplodia theobromae VBE-1

Large-scale production of lipases by SSF with L. theobromaeVBE-1 was conducted on coconut kernel-cake in attempts todetermine if scale-up could significantly increase lipase titers.Scale-up to different levels of coconut kernel-cake batches of100, 500 and 1,000 g in flasks, 2 kg in a tray, and 10 kg inautoclavable polyethylene bags, produced lipase activities of614-762 U/g DS (average of 667±7.89 U/g D,S) see Table 8.This trend occurred for times sampled at two to ten days ofcultivation. Similar results on scaled-up levels were reported

for A. niger (Kamini et al. 1998; Edwinoliver et al. 2010) withlipase yields varying somewhat from the 10 g-level scale, asalso observed in the work presented here. At scaled-up levels,some lipase activity was lost during the extraction steps, and,thus, less enzyme activity resulted. Coconut kernel-cake isconsidered to be a relatively cheap substrate for producinglipase by L. theobromae VBE-1 at scaled-up levels (614 U/gDS at the 10 kg scale, and represents 83 % of the lipase yieldobtained with 10 g) by SSF.

Conclusion

Plant oil seed-associated fungi (A. niger, Chalaropsisthielavioides, L. theobromae, P. glomerata, Colletotrichumgloeosporioides) were demonstrated to have a potential tosecrete industrially important lipases. L. theobromae VBE-1when cultivated by SSF on coconut kernel-cake and opti-mized by RSM, resulted in high lipase production. Factorssuch as coconut oil, temperature, moisture content and surfac-tant enhanced production of lipase by the RSM model devel-oped. After validating the predicted values using RSM, lipaseproduction by L. theobromae VBE-1 increased to 698.1 U/gDS. This study is the first evidence of lipase production bySSF using coelomyceteous fungi.

Acknowledgments The University Grants Commission - Major Re-search Project (F.30-26/2004; SR 29-10-2004) at the University of Ma-dras, INDIA, is acknowledged for financial support. The authors aregrateful to the Biorefining Research Institute at Lakehead University(Canada) for support during the writing of the manuscript.

Table 8 Scale-up of solid-state fermentation at different levels for the production of lipases by Lasiodiplodia theobromae VBE-1

Scale of fermentationa Lipase activity (U/g DS)

Time of growth (d)

2 4 6 8 10

10 gb,c 78±1.5 254±5.2 453±9.2 628±12.4 744±14.9

100 gb,c 92±2.4 316±3.1 536±4.4 580±6.2 644±7.3

500 gb,d 86±3.2 295±4.6 422±2.6 543±8.4 618±5.3

1,000 gb,d 120±2.3 310±3.3 480±5.6 633±4.2 762±12.3

2 kg (tray level)d,e 72±1.3 280±4.5 449±1.4 568±3.1 621±5.7

10 kg (autoclavable polyethylene bag level)d 83±2.1 192±8.5 360±2.3 496±2.2 614±1.8

a Cultivated on coconut kernel-cake - CDM (ratio of 1:1) under optimized conditions after validation of the RSMmodel (pHi 8.0, 30 °C, 66.4%moisture,coconut oil (0.985 %, v/v), 1.5 % Triton X-100) for maximal lipase productionb Erlenmeyer flasks usedcWhole sample extracted for enzyme (100 g of fermented solids was extracted with 50 mM Tris-HCl buffer (pH 8.0); firstly with 500 mL, and then twofurther extractions each of 250 mL, and the three extracts combined)d Aliquot sample of 100 g removed and used to extract enzyme as described by the above protocole Tray dimensions: 40×25×5 cm (coconut kernel-cake level spread out to a height of 2 cm)

140 Ann Microbiol (2015) 65:129–142

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