Get 20M+ Full-Text Papers For Less Than $1.50/day. Start a 14-Day Trial for You or Your Team.

Learn More →

Orchestration an extracellular lipase production from Aspergillus niger MYA 135: biomass morphology and fungal physiology

Orchestration an extracellular lipase production from Aspergillus niger MYA 135: biomass... The impact of biomass morphology and culture conditions on fungal fermentation was widely reviewed in the literature. In this work, we presented three independent experiments in order to evaluate the influence of some of those input factors on a lipase production separately by using the Aspergillus niger MYA 135 and the two-stage fer- mentation technique. Regarding the culture modality, the biomass was pre-grown in a first reactor. Then, the washed mycelium was transferred to a second reactor to continue the study. Firstly, linear effects of fungal morphology and several physiological parameters on a lipase production were explored using the Plackett–Burman design. The dispersed fungal morphology was confirmed as a proper quality characteristic for producing an extracellular lipase activity. Concerning the impact of the carbon source on the biomass pre-growth, the sucrose (E = 9.923, p < 0.001) and the l -arabinose (E = 4.198, p = 0.009) presented positive and significant effects on the enzyme production. On the contrary, the supplementation of 0.05 g/L CaCl displayed a highly negative and significant effect on this process (E = − 7.390, p < 0.001). Secondly, the relationship between the enzyme production and the input variables N:C ratio, FeCl and olive oil was explored applying the central composite design. Among the model terms, the N:C ratio of the production medium had the most negative and significant influence on the enzyme synthesis. Thus, it was concluded that a low N:C ratio was preferable to increase its production. In addition, the bifunctional role of F eCl on this fungus was presented. Thirdly, a prove of concept assay was also discussed. Keywords: Lipase production, Aspergillus niger, Biomass morphology, Fungal physiology Introduction documents related to pharmaceutical industries and Lipases (EC 3.1.1.3) are versatile catalysts that have been medical diagnostic. The oleochemical and waste-related used in hydrolytic and synthetic reactions (Verma et  al. lipase applications exhibited an accelerated growing dur- 2021). As shown in Table  1, a search made by our team ing the last 10 years. In addition, the biosensor field was covering academic and invention patent documents dis- detected as an emerging category involving the utiliza- played an increasing interest of this enzyme in several tion of this enzyme. industrial applications, with a significant growth over Filamentous fungi as enzyme sources are widely used the last decade. Food and flavour industries were the because they are able to produce a large amount of pro- most relevant sectors for products using lipases. In the teins. Native or recombinant biocatalysts from Aspergil- second place, it can mention the number of total patent lus niger, A. oryzae and Trichoderma reesei have been frequently reported. As an example, a lipase from A. niger was immobilized onto a novel macroporous acrylic resin getting a low-cost, stable and recyclable biocatalyst for *Correspondence: lymb@arnet.com.ar Morphogenesis and Fermentation Lab, PROIMI-CONICET, T4001 deacidification of high-acid soy sauce residue oil (Feng MVB San Miguel de Tucumán, Argentina et  al. 2020). However, fermentations involving these Full list of author information is available at the end of the article © The Author(s) 2021. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. Salvatierra et al. AMB Expr (2021) 11:42 Page 2 of 11 Table 1 General interest of lipase-related applications during the last four decades Search methodology (field combinations) Database PubMed Patentscope Lipase Decade Total Decade Total AND (lipase application combined with OR) 1980–1989 1990–1999 2000–2009 2010–2019 1980–2020 1980–1989 1990–1999 2000–2009 2010–2019 1980–2020 AND Date of publication Lipase applications Number of documents Pharmaceutical, Medical diagnostic 2 8 50 170 240 2 76 211 302 602 Oleochemical, Bio/fuel 0 1 15 346 369 0 1 22 84 105 Agrochemical 0 0 0 4 5 0 4 1 3 8 Food, Flavour 233 289 616 1,299 2509 13 100 289 584 1035 Detergent 49 65 65 134 322 32 179 100 211 535 Biosensor 0 0 8 46 56 0 4 2 6 12 Waste, Valorization, Waste management, 2 20 127 354 529 2 18 58 153 233 Bioremediation Cosmetic 0 4 28 117 156 0 26 99 49 171 To represent the scope of this article, the keyword biofuel was more appropriated in PubMed database; while, the keyword fuel was more appropriated in Patentscope database S alvatierra et al. AMB Expr (2021) 11:42 Page 3 of 11 complexes microorganisms are affected by the interrela - Material and methods tion of several parameters, including biomass morphol- Microorganism and culture conditions ogy and culture conditions. In submerged fermentation, The native microorganism Aspergillus niger ATCC MYA filamentous fungi displayed a dispersed (freely hyphae 135, formerly known as A. niger 419 from our culture col- or lax clumps) or a pelletized (spherical agglomerates of lection (PROIMI-CONICET), was used throughout this hyphae) morphology (Quintanilla et  al. 2015). Besides, work. The strain was maintained by monthly transfer to quantitatively measure the effect of culture conditions onto glucose potato agar slants, incubated at 30  °C and on fungal growth, particle parameters such as projected stored at 4 °C. Submerged fermentation was also carried area, circularity, aspect ratio and surface roughness have out at 30  °C on an orbital shaker (INFORS) at 200  rpm. been described to generate a dimensionless morphology Flasks were inoculated with a conidial suspension to get a number. Thus, both fructofuranosidase and glucoamyl - final concentration of about 10 conidia m/L. ase production by A. niger has been negatively correlated with an increasing the morphological number; while, Biomass determination a hypothetical correlation between the morphological The biomass was estimated by drying washed mycelia at number and productivity is proposed for citric acid and 105 °C until constant weight (Colin et al. 2013). secondary metabolites (Cairns et  al. 2019). In the same way, the enzyme productions are influenced by physico- Protein determination and native PAGE chemical factors that should be optimized to reach a Protein concentration was determined according to maximum yield (Geoffry and Achur 2018a). Bradford (1976). Additionally, proteins were separated by In other to control fungal morphology, several strate- native-PAGE using 10% (by mass per volume) polyacryla- gies have been reported. The conventional procedure mide gel. The presence of a lipolytic band was detected is based on the adjustments of chemical, physical and using 1.3 mM of α-naphtyl acetate as substrate. Released biological parameters such as conidia concentration, naphthol was coupled with 1  mM Fast Blue to give a medium composition, temperature, pH, supplementation coloured product. Reactions were carried out at 37  °C of glass beads, agitation systems, fermenter geometry, in shaken plates containing 100  mM phosphate buffer etc. (Papagianni et  al. 2004). Furthermore, Wucherp- (pH = 7.0). fennig et  al. (2011) reported that the culture osmolality also affects fungal morphology and process productiv - Lipase determination ity. In another approach, the macroscopic fungal mor- The lipase activity was measured according to Winkler phology was tailoring by altering the hyphal morphology and Stuckman (1979). One unit of enzyme activity was and the conidia adhesion capacity (Colin et  al. 2013). defined as the amount of enzyme that released 1 μmol of More recently, Karahalil et  al. (2019) reviewed several p-nitro phenol per min. Lipase production was expressed aspects of an interesting technique named microparticle either as a volumetric activity (units per liter of culture enhanced cultivation. That method allows the control of supernatant, U/L) or a specific activity (units per gram of growth physically in submerged fermentation by block- biomass dry weight, U/g ; units per milligram of dry weight ing the aggregation of filamentous microorganisms using protein, U/mg ). protein microparticles such as talc, aluminium oxide, titanium silicom oxide, iron (II, III) and forsterite. Scanning electron microscopy u Th s, as the biomass morphology and the culture con - For observation with scanning electron microscope ditions can affect the process productivity, the influence (SEM), at the end of fermentation, the mycelium was of those variables should be study separately. To do that, collected by filtration, washed with 0.1  mM phosphate the two-stage fermentation strategy can be used being buffer (pH 7), fixed with 2.5% glutaraldehyde and post - the replacement technique one of this culture modal- fixed with 1% OsO . The mycelium was dehydrated in ity (Sternberg and Mandels 1979). Briefly, the biomass is acetone, dried in a critical point apparatus, coated with pre-grown in a first reactor. Then, the washed mycelium gold and observed by using a Zeiss Supra 55VP (Carl is transferred to a second reactor to continue the study. Zeiss, Oberkochen, Germany). For instance, this approach was reported to analyse the transcriptional regulation of xyn2 in Hypocrea jecorina Two‑stage lipase production: replacement technique (Würleitner et al. 2003). Assays were conducted in 50  mL conical flasks contain - In this work, the main objective was to study the effect ing 10  mL of culture medium. The use of this work - of biomass morphology and physiological factors on an ing volume was previously reported for an extracellular extracellular lipase production from Aspergillus niger lipase production by A. niger MYA 135 (Colin et al. 2010). MYA 135 by using a submerged two-stage fermentation. For evaluate both biomass morphology and physiological Salvatierra et al. AMB Expr (2021) 11:42 Page 4 of 11 effectors on the lipase production, the replacement 1994; Gordillo et  al. 1995; Colin et  al. 2010; Griebeler technique reported by Sternberg and Mandels (1979) et  al. 2011; Lanka et  al. 2015). The experimental error was used. Briefly, mycelia were pre-grown during 24  h, was determined by replication of the entire experimental at initial pH 5 and in the presence of 2  g/L NH NO . In matrix. 4 3 the case of Plackett–Burman experimental design, the pre-growth fungal stage was conducted using morpho- Two‑stage lipase production 2: central composite design logical inducers and carbon sources detailed in Table  2. The relationship between the three input variables N:C To induce pelleted or dispersed fungal morphology ratio, FeCl and olive oil, and the enzyme production the corresponding culture medium was supplemented 3 expressed as lipase units per liter of culture supernatant with 0.5  g/L C aCl or 1.0  g/L FeCl , respectively (Colin 2 3 (U/L) were evaluated after 96 h of cultivation by using the et  al. 2013). In the case of central composite design, the central composite design (Myers and Montgomery 2002). pre-growth fungal stage was conducted under a unique This is an experimental design for building a second condition using 1  g/L F eCl and 10  g/L sucrose as mor- order polynomial for the response variable. It involves phological inducer and carbon source, respectively. Bio- three types of trials: 2 k factorial trials, 2 k axial trials and masses were collected and washed by vacuum filtration; nc center point trials, where k is number of factors stud- then, they were weighted and added to different lipase ied in the assay. Values at center point provide informa- production culture media according to the experimental tion about the existence of curvature in the response; that design (Plackett–Burman experimental design or cen- is, they contribute to the estimation of quadratic terms. tral composite design). The initial biomass concentration Axial points are also used to estimate quadratic terms, was 8 and 15 g /L for Plackett–Burman and cen- wet weight while factorial points contribute to the estimation of lin- tral composite experimental designs, respectively. Flasks ear and interaction terms. Each factor was studied at five were further incubated for 72  h under the same condi- different levels (− α, − 1,0 , + 1, + α) (Table  3). Variable tions. Both biomass pre-growth media and lipase pro- settings that represent the axial point of the design were duction media contained the same inorganic supplement decided based on either our own experience or litera- (in g/L): KH PO 1.0, MgSO 7H O 0.2, CuSO 5H O 0.06. 2 4 4 2 4 2 ture reports (Pokorny et al. 1994; Colin et al. 2010; Salihu The nitrogen:carbon (N:C) ratio was studied in produc - et  al. 2011). The experimental error was determined by tion media by varying the concentration of NH NO 4 3 replication of the entire experimental matrix. keeping the concentration of sucrose at 5 g/L.The super - natant was obtained by vacuum filtration and used as enzyme source. Two‑stage lipase production 3: a prove of concept The assay was conducted in 500  mL conical flasks con - Two‑stage lipase production 1: Plackett–Burman taining 100  mL of culture medium. The fermentation experimental design medium comprised (in g/L): sucrose 10.0, KH PO 1.0, 2 4 The effect of 11 environmental factors on the enzyme NH NO 2.0, M gSO 7H O 2.0, C uSO 0.06 and F eCl 4 3 4 2 4 3 production expressed as lipase units per gram of bio- 1.0. The initial pH was adjusted to 7.0 with NaOH. After mass dry weight (U/g ) were evaluated after 96  h dry weight 24 h of incubation, the culture was transferred to another of cultivation by using the Plackett–Burman experimen- 500  mL flask containing 50  mL of 3% (by volume) and tal design (Plackett and Burman 1946) (Table  2). This is was further incubated for 168  h under the same condi- a two-level fractional factorial design for studying n-1 tions. The lipase production was monitored during fer - input variables (factors) using n runs, where n is a mul- mentation using as response variables either the enzyme tiple of 4 (n is the number of experiments). Each fac- activity expressed as lipase units per liter of culture tor is represented at two levels, high and low, which are supernatant (U/L) or the specific lipase activity expressed denoted by (+) and (−), respectively. The effect for a fac - as enzyme units per milligram of protein (U/mg ). protein tor is always described as the change in the response in going from the low level of that factor to the high level. A negative sign means that going from low level to high Statistical analysis level for a factor decreases the response. A positive sign All statistical analysis was performed using Minitab soft- means that going from the low level to the high level ware (Minitab Inc., State College, PA, USA). Data were increases the response. The effect (E) of each variable on expressed as means ± standard deviation. Differences the response was determined by subtracting the average were accepted as significant when p < 0.05. The fitness of response of the low level from that of the high level. Lev- models was checked by both the determination coeffi - els of each input variable were decided based on either cient (R ) and the adjusted determination coefficient (adj our own experience or literature reports (Pokorny et  al. R ). S alvatierra et al. AMB Expr (2021) 11:42 Page 5 of 11 Table 2 Plackett–Burman design: experimental procedure, variables and statistical analysis Two-stage Pre-growth stage (24 h) Lipase production stage (72 h) Response variable Fermentation Initial pH 5 Initial biomass concentration: 8 g /L wet weight Input variables Morphology (−) pel Glucose l -Arabinose Sucrose N:C ratio pH Oleic acid Tributirine CaCl FeCl Olive oil Specific lipase 2 3 lets (+) dispersed activity (U/g ) dry weight Level (g/L) (−) 0.5 CaCl 0 0 5.0 0.4 5.0 0 0 0 0 10.0 (+) 1.0 FeCl 10.0 10.0 10.0 0.6 7.0 1.0 1.0 0.05 0.1 20.0 Trial number 1 + − + + − + − − − + + 33.92 ± 1.53 2 − − + + + − + + − + − 17.32 ± 3.72 3 − − − + + + − + + − + 17.88 ± 3.22 4 + + − + − − − + + + − 11.84 ± 1.94 5 − + − − − + + + − + + 15.33 ± 0.04 6 + − − − + + + − + + − 7.61 ± 0.28 7 − − − − − − − − − − − 3.12 ± 0.07 8 − + + + − + + − + − − 17.19 ± 3.68 9 + + − + + − + − − − + 24.47 ± 6.66 10 − + + − + − − − + + − 11.41 ± 1.51 11 + + + − + + − + − − − 20.84 ± 5.88 12 + − + − − − + + + − + 4.75 ± 1.81 Effect 3.531 2.747 4.198 9.923 2.232 6.643 − 2.053 − 1.625 − 7.390 1.530 3.978 R = 92.73% p-value 0.022 0.062 0.009 < 0.001 0.121 < 0.001 0.151 0.248 < 0.001 0.275 0.003 R = 86.07% adj Letters in bold show the experimental procedure, variables and statistical analysis corresponding to the pre-growth stage Concentration of medium components Salvatierra et al. AMB Expr (2021) 11:42 Page 6 of 11 Table 3 Central composite design: input variables and their the lipase activity being negligible the presence of either levels oleic acid or tributyrin. Besides, the initial pH 7 of cul- ture medium was more favorable to the enzyme produc- Input variables Levels tion than the initial pH 5. Regarding the influence of ions, − α − 1 0 + 1 + α the supplementation of 0.05 g/L CaCl displayed a highly negative and significant effect on the enzyme produc - tion (E = − 7.390). The linear effect corresponding to the N:C ratio 0.200 0.281 0.400 0.519 0.600 input variable F eCl was not significant (p = 0.275). FeCl (g/L) 0.050 0.080 0.125 0.170 0.200 Two‑stage lipase production 2: central composite design Considering that our next objective was to explore the Olive oil (g/L) 10.000 14.000 20.000 26.000 30.000 relationship between the enzyme production and the input variables N:C ratio (X ), FeCl (X ) and olive oil 1 3 2 (X ), it was decided to apply the central composite design Results for 20 trials expressing the enzyme activity as U/L. As Previously, it was reported an olive oil-induced extracel- it was mentioned before, with this kind of experimental lular lipase activity by the native A. niger MYA 135 using design is possible to estimate linear, quadratic and inter- a mineral culture medium (Colin et  al. 2010). In this action effects. To conduct this experiment, the biomass work, the employ of statistically designed experiments was developed in the presence of 1  g/L FeCl ; and then, was proposed to obtain knowledge about the relation- it was collected, washed, weighted and added to differ - ship among some input factors and the production of this ent lipase production media according to the experimen- extracellular enzyme. The results of the three two-stage tal design (Table  4). Data on enzyme activity showed a fermentations are described below. wide variation covering the range from 1.57 ± 0.41 to 324.57 ± 19.86 U/L. Considering the p-value of the corre- Two‑stage lipase production 1: Plackett–Burman sponding coefficients (0.007 or smaller), the lipase activ - experimental design ity production depended on two linear effects (X and Firstly, linear effects of fungal morphology and several X ), one two-way interaction (X X ) and two quadratic 3 2 3 physiological parameters on a lipase activity production 2 2 effects (X and X ) (Table 5). The second-order polyno - 1 2 were explored using a fractional experimental design. As mial equation is shown below: both fungal morphology and physiology are affected by Y = 195.0−61.9X + 48.8X + 39.8X X the same environmental conditions, the biomass and the (U/L) 1 3 2 3 lipase production was uncouple using the replacement 2 2 −64.3X + 30.7X 1 3 technique. Considering that our main objective in this assay was to evaluate the impact of biomass morphol- As it can be seen, both linear and quadratic terms cor- ogy on the lipase activity production, it was decided to responding to the N:C ratio were negative. While, those determine the enzyme activity expressed as U/g . terms associated to olive oil as well as its interaction with dry weight Table  2 shows the Plackett- Burman design for 12 trials FeCl were positive. In addition, the R value indicated and the corresponding response variable. The R value that 80.10% of data variation was explained by the input indicated that 92.73% of data variation was explained by variables. The adj R was 73.24%. The lack-of-fit test is the input variables. The adj R was 86.07%. Thus, data testing the lack of fit for the quadratic model; the p-value on specific enzyme activity exhibited a wide variation for this test is large (p = 0.578) implying that the quad- covering the range from 3.12 ± 0.07 to 33.92 ± 1.53 U/ ratic model was adequate (Table 5). g . The fungal morphology displayed a significant dry weight effect on the final response being the dispersed myce - Two‑stage lipase production 3: a prove of concept lia more favorable than the pelleted form of growth for Taking in mind the results obtained, a third two-stage to increase the specific lipase activity. Concerning the lipase production was conducted as a prove of concept. impact of the carbon source on the biomass pre-growth, The biomass was pre-grown at an initial pH 7, in the both the sucrose and the l-arabinose presented posi - presence of 1  g/L FeCl and with a starting N:C ratio of tive and significant effects on the enzyme production, 0.2 as described in the material and methods section. while the effect of glucose was not significant under our After 24  h of cultivation, the entire culture was trans- assay conditions. In relation to the effect of physiologi - ferred to another reactor without any washed procedure cal parameters on the lipase production, as expected, getting an initial olive oil concentration of 20  g/L. The the olive oil had a positive and a significant effect on highest value of lipase activity (U/L) (Fig. 1a) and specific S alvatierra et al. AMB Expr (2021) 11:42 Page 7 of 11 Table 4 Central composite design: experimental procedure and variables Two-stage fermentation Pre-growth stage (24 h) in the presense of 1.0 g/L FeCl Biomass morphology: dispersed mycelium Lipase production stage (72 h), initial biomass concentration: 15 g wet /L weight Trial number Input variables Response variable N:C ratio FeCl Olive oil Lipase activity (U/L) CP 0 0 0 153.73 ± 2.17 CP 0 0 0 82,94 ± 0.29 CP 0 0 0 219.61 ± 6.50 b CP 0 0 0 191.01 ± 5.78 CP 0 0 0 238.76 ± 14.80 CP 0 0 0 239.78 ± 5.42 1 + 1 + 1 − 1 3.33 ± 0.21 2 − 1 − 1 + 1 294.90 ± 25.94 3 0 0 − α 89.59 ± 1.73 4 − 1 + 1 + 1 324.57 ± 19.86 5 + 1 − 1 − 1 20.13 ± 0.87 6 + 1 − 1 + 1 6.56 ± 4.27 Fig. 1 Time course of an extracellular lipase activity from Aspergillus 7 0 + α 0 315.63 ± 32.50 niger MYA 135 during the lipase production stage expressed as 8 0 0 + α 251.28 ± 10.47 volumetric activity (U/L) (a) or specific activity (U/mg ) (b) protein 9 − 1 + 1 − 1 135.60 ± 10.11 10 + 1 + 1 + 1 170.84 ± 7.95 11 0 − α 0 276.05 ± 48.76 96  h of cultivation. In addition, the culture supernatant 12 + α 0 0 1.57 ± 0.41 was analyzed by native PAGE. Two lipolytic bands were 13 − α 0 0 30.16 ± 11.66 detected using α-naphtyl acetate as substrate being the 14 − 1 − 1 − 1 242.98 ± 30.52 intensity signal of the top one also increased at the time of 96 h (Fig. 2). On the other hand, SEM micrographs of lipase activity (U/mg ) (Fig.  1b) was obtained after protein the harvested biomass displayed a dispersed mycelium (lax clumps mixed with free mycelium) showing scarcely Table 5 Central composite design: terms and the corresponding branched and not fragmentated hyphae; in addition, the coefficients presence of conidiophores was not observed (Fig. 3). Term Coefficient p-value Constant 195.0 < 0.001 Discussion Linear < 0.001 The impact of biomass morphology and culture condi - X (N:C ratio) − 61.9 < 0.001 tions on fungal fermentation was analyzed in several X (FeCl ) 12.7 0.251 investigations as it was briefly mentioned in “Introduc - 2 3 X (Olive oil) 48.8 < 0.001 tion” section. Interaction < 0.001 Here, through the application of designed experiments, X X 28.1 0.057 we evaluated the influence of some of those input factors 1 2 X X − 10.9 0.450 on a lipase activity production separately by using the A. 1 3 X X 39.8 0.009 niger MYA 135 as a model and the two-stage fermenta- 2 3 Quadratic 0.015 tion technique as the culture modality. X − 64.3 < 0.001 X 30.7 0.007 Biomass morphology and lipase production X − 9.7 0.368 3 In coincidence with the results reviewed by Krull et  al. Lack-of-fit 0.578 (2010), the macroscopic characteristic of fungal mor- R (%) 80.10 phology displaying by A. niger MYA 135 was already R (%) 73.24 adj apparent after about 18 h of incubation (data not shown). Salvatierra et al. AMB Expr (2021) 11:42 Page 8 of 11 biomass morphology on the specific lipase production expressed as U/g was, firstly, evaluated. Dispersed dry weight or pelleted mycelia were harvested, washed by vacuum filtration, weighted as indicated in the material and methods section, and then added to different produc - tion media. The Plackett–Burman experimental design identified the significant effect of the biomass morphol - ogy being the dispersed mycelium preferred to increase the specific lipase production. In fact, the same culture medium at an initial pH 7 without FeCl supplementa- tion favours the pelleted form of growth; and, under that environmental condition, the maximum specific activity using p-NPP as substrate was 1.6 U/mg after 96 h of protein cultivation (Pera et  al. 2006). Here, as it can be seen in the prove of concept assay, that value was increased ten times in the presence of dispersed mycelium (Fig.  1b). In concordance with our observation, a freely dispersed mycelium from A. niger SKAn1015 induced by the sup- plementation of silicate microparticles was linked to an Fig. 2 Native PAGE showing the time course of an extracellular lipase activity from Aspergillus niger MYA 135 during the lipase production increase of a fructofuranosidase activity displaying the stage. The lipolytic bands were detected using α-naphtyl acetate as microparticles itself an important physiological role (Dri- substrate ouch et al. 2010). On the contrary, the performance of a synthetic mycelium-bound lipase activity from Rhizopus chinensis was favoured by fully entangled mycelial fila - ments (Teng et al. 2009). However, as it will be discussed later, the lipase production by filamentous fungi should be analyzed from a holistic point of view. Fungal physiology and lipase production Firstly, the impact of several physiological factors on the lipase production activity expressed as U/g dry weight (Two-stage lipase production 1) was evaluated. Thus, besides the fungal biomass morphology, the kind of car- bon source using during the pre-growth stage also had a relevant role on the specific lipase production. In fact, according to the Plackett–Burman experimental design, the presence of sucrose during the first-stage of fermen - tation exhibited the most significant and positive effect Fig. 3 Image of dispersed mycelium obtained with a scanning on the response variable. On the other hand, the olive electron microscope showing the harvested biomass from Aspergillus oil concentration also displayed a positive effect on the niger MYA 135 after 96 h of cultivation response variable being the effect associated with the supplementation of either oleic acid or tributyrin not significant. So, the hydrophilic substrate could have a For that reason, it was decided to conduct a biomass pre- relevant role during the fungal growth stage, while the growth stage of 24  h. So, culture media at initial pH 5 hydrophobic one acts mainly as an inducer of the lipo- were supplemented with morphological inducers in the lytic activity synthesis. These results were compatible pre-growth fermentation stage. As previously reported, with those that reported the use of a combination of dispersed mycelium is induced in the presence of 1  g/L carbohydrate source and lipid inducer as a strategy to FeCl ; while, the pelleted form of growth is favoured by increase the biocatalyst production. However, the proper the addition of 0.5 g/L C aCl (Colin et al. 2013). In addi- mixture of those culture components varies according to tion, under our culture conditions, before that time of the microorganism used. To give examples, the optimal cultivation, no significant extracellular lipase activity substrates for a lipase activity production from Pichia was found (data not shown). Thus, the influence of the lynferdii Y-7723 were soybean oil and sucrose (Kim S alvatierra et al. AMB Expr (2021) 11:42 Page 9 of 11 et  al. 2010); while, olive oil and xylose were preferred by replacement technique it was demonstrated that a low Rhizopus oryzae ZAC3 (Ayinla et  al. 2017). Concerning N:C ratio was preferable to increase the lipase produc- A. niger MYA 135, the olive oil was the best hydropho- tion by A. niger MYA 135. This effect was also observed bic substrate using in the presence of sucrose (Colin et al. in the prove of concept assay where the initial N:C ratio 2010). Additionally, as it was also shown in this work, the was 0.2 (or 5 C:N ratio). As expected, the next important initial pH value of the production medium is an impor- factor was the olive oil concentration. Not only the linear tant physiological factor involved in the lipase produc- term corresponding to this input variable was positive tion being this parameter a fungi-dependent variable. In (p < 0.001) but also a positive interaction between olive this connection, it was reported the production of fun- oil and FeCl (p = 0.009) was found. Thus, as the linear gal lipases under acidic (Turati et  al. 2019), near neutral term corresponding to olive oil was positive, the effect on (Colla et al. 2016) or alkaline (Rajan and Nair 2011) cul- the response will tend to be more positive increasing the ture conditions. Finally, the impact of metal ions on the value of FeCl concentration. Thus, the FeCl acted not 3 3 lipolytic enzyme synthesis has been evaluated in several only as a morphological effector but also as an additive studies during the selection of significant input variables that contributed to increase the lipase production. How- (Rajendran and Thangavelu 2009; Salihu et  al. 2011). In ever, according to ours results, the N:C ratio always has this work, the supplementation of 0.1  g/L F eCl did not to be taken into account when evaluating the impact of has a significant effect on the specific enzyme activity; other input variables on the lipase activity. other aspects concerning to this salt are discussed in the Finally, the dispersed mycelium produced at least two next paragraph. On the contrary, the presence of 0.05 g/L lipases as it was observed in native PAGE being the inten- CaCl exhibited a highly negative impact on the response sity signal of the top one increased after 72 h of incuba- (E = − 7.390). Similarly, Geoffry and Achur (2018a), tion. In addition, the lipase production pattern of A. niger reported that the variable CaCl influences negatively on MYA 135 was similar to those reported for Aspergil- the production of a lipase activity from Fusarium solani. lus carbonarius (Ire and Ike 2014) and Rhizopus oryzae Oppositely, the supplementation of this salt is favorable ZAC3 (Ayinla et  al. 2017) achieving all of them an opti- for the synthesis of a lipase activity using Rhizopus arrhi- mum incubation time of 96 h. zus MTCC 2233 (Rajendran and Thangavelu 2009). And, In summary, analysing the three independent assays the effect of CaCl is no significant to produce a lipase conducted during this work, it can be highlighted the activity from Yarrowia lipolytica MTCC 35 (Kishan et al. usefulness of the replacement technique to study how 2013). Thus, reported data clearly indicate the wide varia - environmental conditions affected a lipase activity pro - tion in effects of CaCl on the lipase production. duction evaluating the influence of biomass morphol - Secondly, the central composite design was adopted to ogy and other physiological effectors separately. The study the relationship between the lipase production and dispersed morphology was confirmed as a proper qual - the input variables N:C ratio, F eCl and olive oil. In this ity characteristic for producing a lipase activity from A. case, the response variable was the volumetric enzyme niger MYA 135. In addition, the bifunctional role of FeCl activity expressed as U/L. This parameter was measured on this fungus was presented. Finally, according to ours after 96 h of cultivation (Two-stage lipase production 2). results, the N:C ratio always has to be taken into account Concerning the effect of nitrogen source on the lipase when evaluating the impact of other input variables on production, there is a general agreement in the literature this process using this filamentous fungus. that this enzyme synthesis is favoured at high nitrogen concentration, and therefore, lower C:N ratios, being Authors’ contributions this substrate either organic or inorganic (Das et al. 2016; MDB and LMP conceived and designed research. HNS, and ELR conducted Geoffry and Achur 2018b). In this connection, Coradi experiments. HNS and LMP analyzed data. MDB and LMP wrote the manu- script. All authors read and approved the manuscript. et  al. (2013) reported that Trichoderma harzianum dis- played the highest lipase activity in a culture medium Funding containing 0.5% yeast extract and 1% olive oil (2.5 C:N This work was supported by FONCyT (PICT 2015-2596) and UNT (PIUNT D 606). ratio). Here, among the model terms, the N:C ratio of Availability of data and materials the production culture medium had the most significant Authors can confirm that all relevant data are included in the article. influence on the lipase activity production. A negative linear coefficient suggests that as the N:C ratio increases, Declarations the independent variable tends to decrease. In addition, Ethics approval and consent to participate as the corresponding quadratic term was also negative, This article does not contain any studies with human participants or animals the net effect of increasing the N:C ratio was an accelera - performed by any of the authors. tion of the response variable decrease. u Th s, by using the Salvatierra et al. AMB Expr (2021) 11:42 Page 10 of 11 Consent for publication Griebeler N, Polloni AE, Remonatto D, Arber F, Vardanega R, Cechet JL, Di Not applicable. Luccio M, de Oliveira D, Treichel H, Cansian RL, Rigo E, Ninow JL (2011) Isolation and screening of lipase-producing fungi with hydrolytic Competing interests activity. Food Bioprocess Technol 4:578–586. https:// doi. org/ 10. 1007/ The authors declare that they have no conflict of interest.s11947- 008- 0176-5 Ire FS, Ike VC (2014) Screening and optimization of process parameters for the Author details production of lipase in submerged fermentation by Aspergillus carbon- Morphogenesis and Fermentation Lab, PROIMI-CONICET, T4001 MVB San arius (Bainer) IMI 366159. Annu Res Rev Biol 4:2587–2602. https:// doi. org/ Miguel de Tucumán, Argentina. Facultad de Bioquímica, Química y Farmacia, 10. 9734/ ARRB/ 2014/ 9879 Cátedra de Microbiología Superior, Universidad Nacional de Tucumán, Karahalil E, Coban HB, Turhan I (2019) A current approach to the control of T4000INI San Miguel de Tucumán, Argentina. filamentous fungal growth in media: microparticle enhanced cultiva- tion technique. Crit Rev Biotechnol 39:192–201. https:// doi. org/ 10. 1080/ Received: 14 December 2020 Accepted: 7 March 202107388 551. 2018. 15318 21 Kim HR, Kim IH, Hou CT, Kwon KI, SHIN BS, (2010) Production of a novel cold- active lipase from Pichia lynferdii Y-7723. J Agric Food Chem 58:1322– 1326. https:// doi. org/ 10. 1021/ jf903 430t Kishan G, Gopalakannan P, Muthukumaran C, Thirumalai Muthukumaresan K, Dharmendira Kumar M, Tamilarasan K (2013) Statistical optimization References of critical medium components for lipase production from Yarrowia Ayinla ZA, Ademakinwa AN, Agboola FK (2017) Studies on the optimization of lipolytica (MTCC 35). J Genet Eng Biotechnol 11:111–116. https:// doi. org/ lipase production by Rhizopus sp. ZAC3 isolated from the contaminated 10. 1016/j. jgeb. 2013. 06. 001 soil of a palm oil processing shed. J App Biol Biotechnol 5:030–037. Krull R, Cordes C, Horn H, Kampen I, Kwade A, Neu TR, Nörtemann B (2010) https:// doi. org/ 10. 7324/ JABB. 2017. 50205 Morphology of filamentous fungi: linking cellular biology to process engi- Bradford MM (1976) A rapid and sensitive method for the quantification of neering using Aspergillus niger. Adv Biochem Eng Biotechnol 121:1–21. microgram quantities of protein utilizing the principle of protein-dye https:// doi. org/ 10. 1007/ 10_ 2009_ 60 binding. Anal Biochem 72:248–254. https:// doi. org/ 10. 1016/ 0003- Lanka S, Pydipalli M, Latha JNL (2015) Optimization of process variables for 2697(76) 90527-3 extracellular lipase production from Emericella nidulans NFCCI 3643 iso- Cairns TC, Zheng X, Zheng P, Sun J, Meyer V (2019) Moulding the mould: lated from palm oil mill effluent (POME) dump sites using OFAT method. understanding and reprogramming filamentous fungal growth and mor - Res J Microbiol 10:38–53. https:// doi. org/ 10. 3923/ jm. 2015. 38. 53 phogenesis for next generation cell factories. Biotechnol Biofuels 12:77. Myers R, Montgomery D (2002) Response surface methodology. Process and https:// doi. org/ 10. 1186/ s13068- 019- 1400-4 product optimization using designed experiments. John Wiley & Sons, Colin VL, Baigori MD, Pera LM (2010) Eec ff t of environmental conditions on INC., Hoboken extracellular lipases production and fungal morphology from Aspergillus Papagianni M (2004) Fungal morphology and metabolite production in sub- niger MYA 135. J Basic Microbiol 50:52–58. https:// doi. org/ 10. 1002/ jobm. merged mycelial processes. Biotechnol Adv 22:189–259. https:// doi. org/ 20090 0162 10. 1016/j. biote chadv. 2003. 09. 005 Colin VL, Baigori MD, Pera LM (2013) Tailoring fungal morphology of Aspergillus Pera LM, Romero CM, Baigori MD, Castro GR (2006) Catalytic properties of niger MYA 135 by altering the hyphal morphology and the conidia adhe- lipase extracts from Aspergillus niger. Food Technol Biotechnol 44:247–252 sion capacity: biotechnological applications. AMB Expr 3:27. https:// doi. Plackett RL, Burman JP (1946) Trust the design of optimum multifactorial org/ 10. 1186/ 2191- 0855-3- 27 experiments. Biometrika 33:305–325. https:// doi. org/ 10. 1093/ biomet/ Colla LM, Primaz AL, Benedetti S, Loss RA, Lima M, Reinehr CO, Bertolin TE, 33.4. 305 Vieira Costa JA (2016) Surface response methodology for the optimiza- Pokorny D, Friedrich J, Cimerman A (1994) Eec ff t of nutritional factors on lipase tion of lipase production under submerged fermentation by filamentous biosynthesis by Aspergillus niger. Biotechnol Lett 16:363–366. https:// doi. fungi. Braz J Microbiol 47:461–467. https:// doi. org/ 10. 1016/j. bjm. 2016. org/ 10. 1007/ BF002 45052 01. 028 Quintanilla D, Hagemann T, Hansen K, Gernaey KV (2015) Fungal morphology Coradi GV, da Visitação VL, de Lima EA, Saito LY T, Palmieri DA, Takita MA, Neto in industrial enzyme production—modelling and monitoring. Adv Bio- PO, Gomes de Lima VM (2013) Comparing submerged and solid-state chem Eng Biotechnol 149:29–54. https:// doi. org/ 10. 1007/ 10_ 2015_ 309 fermentation of agro-industrial residues for the production and charac- Rajan A, Nair AJ (2011) A comparative study on alkaline lipase production by a terization of lipase by Trichoderma harzianum. Ann Microbiol 63:533–540. newly isolated Aspergillus fumigatus MTCC 9657 in submerged and solid- https:// doi. org/ 10. 1007/ s13213- 012- 0500-1 state fermentation using economically and industrially feasible substrate. Das A, Bhattacharya S, Shivakumar S, Shakya S, Sogane SS (2016) Coconut oil Turk J Biol 35:569–574. https:// doi. org/ 10. 3906/ biy- 0912-6 induced production of a surfactant-compatible lipase from Aspergillus Rajendran A, Thangavelu V (2009) Statistical experimental design for evalua- tamarii under submerged fermentation. J Basic Microbiol 57:114–120. tion of medium components for lipase production by Rhizopus arrhizus https:// doi. org/ 10. 1002/ jobm. 20160 0478 MTCC 2233. LWT-Food Sci Technol 42:985–992. https:// doi. org/ 10. 1016/j. Driouch H, Sommer B, Wittmann C (2010) Morphology engineering of lwt. 2008. 12. 009 Aspergillus niger for improved enzyme production. Biotechnol Bioeng Salihu A, Alam MDZ, AbdulKarim MI, Salleh HM (2011) Optimization of lipase 105:1058–1068. https:// doi. org/ 10. 1002/ bit. 22614 production by Candida cylindracea in palm oil mill effluent based Feng K, Huang Z, Peng B, Dai W, Li Y, Zhu X, Chen Y, Tong X, Lan Y, Cao Y (2020) medium using statistical experimental design. J Mol Catal B-Enzym Immobilization of Aspergillus niger lipase onto a novel macroporous 69:66–73. https:// doi. org/ 10. 1016/j. molca tb. 2010. 12. 012 acrylic resin: Stable and recyclable biocatalysis for deacidification of high- Sternberg D, Mandels GR (1979) Induction of cellulolytic enzymes in Tricho- acid soy sauce residue oil. Bioresour Technol 298:122553. https:// doi. org/ derma reesei by sophorose. J Bacteriol 139:761–769. https:// doi. org/ 10. 10. 1016/j. biort ech. 2019. 122553 1128/ JB. 139.3. 761- 769. 1979 Geoffry K, Achur RN (2018a) Optimization of novel halophilic lipase production Teng Y, Xu Y, Wang D (2009) Changes in morphology of Rhizopus chinensis in by Fusarium solani strain NFCCL 4084 using palm oil mill effluent. J Genet submerged fermentation and their effect on production of mycelium- Eng Biotechnol 16:327–334. https:// doi. org/ 10. 1016/j. jgeb. 2018. 04. 003 bound lipase. Bioprocess Biosyst Eng 32:397–405. https:// doi. org/ 10. Geoffry K, Achur RN (2018b) Screening and production of lipase from fungal 1007/ s00449- 008- 0259-8 organisms. Biocatal Agric Biotechnol 14:241–253. https:// doi. org/ 10. Turati DFM, Almeida AF, Terrone CC, Nascimento JMF, Terrasan CRF, Fernandez- 1016/j. bcab. 2018. 03. 009 Lorente G, Pessela BC, Guisan JM, Carmona EC (2019) Thermotolerant Gordillo MA, Obradors N, Montesinos JL, Valero F, Lafuente J, Solà C (1995) lipase from Penicillium sp. Section Gracilenta CBMAI 1583: effect of carbon Stability studies and effect of the initial oleic acid concentration on lipase sources on enzyme production, biochemical properties of crude and production by Candida rugose. Appl Microbiol Biotechnol 43:38–41. purified enzyme and substrate specificity. Biocatal Agric Biotechnol https:// doi. org/ 10. 1007/ BF001 70620 17:15–24. https:// doi. org/ 10. 1016/j. bcab. 2018. 10. 002 S alvatierra et al. AMB Expr (2021) 11:42 Page 11 of 11 Verma S, Meghwanshi GK, Kumar R (2021) Current perspectives for microbial Würleitner E, Pera L, Wacenovsky C, Cziferszky A, Zeilinger S, Kubicek CP, Mach lipases from extremophiles and metagenomics. Biochimie 182:23–36. RL (2003) Transcriptional regulation of xyn2 in Hypocrea jecorina. Eukariot https:// doi. org/ 10. 1016/j. biochi. 2020. 12. 027 Cell 2:150–158. https:// doi. org/ 10. 1128/ EC.2. 1. 150- 158. 2003 Winkler UK, Stuckmann M (1979) Glycogen, hyaluronate, and some other polysaccharides greatly enhance the formation of exolipase by Serratia Publisher’s Note marcescens. J Bacteriol 138:663–670. https:// doi. org/ 10. 1128/ JB. 138.3. Springer Nature remains neutral with regard to jurisdictional claims in pub- 663- 670. 1979 lished maps and institutional affiliations. Wucherpfennig T, Hestler T, Krull R (2011) Morphology engineering—osmolal- ity and its effect on Aspergillus niger morphology and productivity. Microb Cell Fact 10:58. https:// doi. org/ 10. 1186/ 1475- 2859- 10- 58 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png AMB Express Springer Journals

Orchestration an extracellular lipase production from Aspergillus niger MYA 135: biomass morphology and fungal physiology

Loading next page...
 
/lp/springer-journals/orchestration-an-extracellular-lipase-production-from-aspergillus-00PNu5PYk9

References (35)

Publisher
Springer Journals
Copyright
Copyright © The Author(s) 2021
eISSN
2191-0855
DOI
10.1186/s13568-021-01202-y
Publisher site
See Article on Publisher Site

Abstract

The impact of biomass morphology and culture conditions on fungal fermentation was widely reviewed in the literature. In this work, we presented three independent experiments in order to evaluate the influence of some of those input factors on a lipase production separately by using the Aspergillus niger MYA 135 and the two-stage fer- mentation technique. Regarding the culture modality, the biomass was pre-grown in a first reactor. Then, the washed mycelium was transferred to a second reactor to continue the study. Firstly, linear effects of fungal morphology and several physiological parameters on a lipase production were explored using the Plackett–Burman design. The dispersed fungal morphology was confirmed as a proper quality characteristic for producing an extracellular lipase activity. Concerning the impact of the carbon source on the biomass pre-growth, the sucrose (E = 9.923, p < 0.001) and the l -arabinose (E = 4.198, p = 0.009) presented positive and significant effects on the enzyme production. On the contrary, the supplementation of 0.05 g/L CaCl displayed a highly negative and significant effect on this process (E = − 7.390, p < 0.001). Secondly, the relationship between the enzyme production and the input variables N:C ratio, FeCl and olive oil was explored applying the central composite design. Among the model terms, the N:C ratio of the production medium had the most negative and significant influence on the enzyme synthesis. Thus, it was concluded that a low N:C ratio was preferable to increase its production. In addition, the bifunctional role of F eCl on this fungus was presented. Thirdly, a prove of concept assay was also discussed. Keywords: Lipase production, Aspergillus niger, Biomass morphology, Fungal physiology Introduction documents related to pharmaceutical industries and Lipases (EC 3.1.1.3) are versatile catalysts that have been medical diagnostic. The oleochemical and waste-related used in hydrolytic and synthetic reactions (Verma et  al. lipase applications exhibited an accelerated growing dur- 2021). As shown in Table  1, a search made by our team ing the last 10 years. In addition, the biosensor field was covering academic and invention patent documents dis- detected as an emerging category involving the utiliza- played an increasing interest of this enzyme in several tion of this enzyme. industrial applications, with a significant growth over Filamentous fungi as enzyme sources are widely used the last decade. Food and flavour industries were the because they are able to produce a large amount of pro- most relevant sectors for products using lipases. In the teins. Native or recombinant biocatalysts from Aspergil- second place, it can mention the number of total patent lus niger, A. oryzae and Trichoderma reesei have been frequently reported. As an example, a lipase from A. niger was immobilized onto a novel macroporous acrylic resin getting a low-cost, stable and recyclable biocatalyst for *Correspondence: lymb@arnet.com.ar Morphogenesis and Fermentation Lab, PROIMI-CONICET, T4001 deacidification of high-acid soy sauce residue oil (Feng MVB San Miguel de Tucumán, Argentina et  al. 2020). However, fermentations involving these Full list of author information is available at the end of the article © The Author(s) 2021. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. Salvatierra et al. AMB Expr (2021) 11:42 Page 2 of 11 Table 1 General interest of lipase-related applications during the last four decades Search methodology (field combinations) Database PubMed Patentscope Lipase Decade Total Decade Total AND (lipase application combined with OR) 1980–1989 1990–1999 2000–2009 2010–2019 1980–2020 1980–1989 1990–1999 2000–2009 2010–2019 1980–2020 AND Date of publication Lipase applications Number of documents Pharmaceutical, Medical diagnostic 2 8 50 170 240 2 76 211 302 602 Oleochemical, Bio/fuel 0 1 15 346 369 0 1 22 84 105 Agrochemical 0 0 0 4 5 0 4 1 3 8 Food, Flavour 233 289 616 1,299 2509 13 100 289 584 1035 Detergent 49 65 65 134 322 32 179 100 211 535 Biosensor 0 0 8 46 56 0 4 2 6 12 Waste, Valorization, Waste management, 2 20 127 354 529 2 18 58 153 233 Bioremediation Cosmetic 0 4 28 117 156 0 26 99 49 171 To represent the scope of this article, the keyword biofuel was more appropriated in PubMed database; while, the keyword fuel was more appropriated in Patentscope database S alvatierra et al. AMB Expr (2021) 11:42 Page 3 of 11 complexes microorganisms are affected by the interrela - Material and methods tion of several parameters, including biomass morphol- Microorganism and culture conditions ogy and culture conditions. In submerged fermentation, The native microorganism Aspergillus niger ATCC MYA filamentous fungi displayed a dispersed (freely hyphae 135, formerly known as A. niger 419 from our culture col- or lax clumps) or a pelletized (spherical agglomerates of lection (PROIMI-CONICET), was used throughout this hyphae) morphology (Quintanilla et  al. 2015). Besides, work. The strain was maintained by monthly transfer to quantitatively measure the effect of culture conditions onto glucose potato agar slants, incubated at 30  °C and on fungal growth, particle parameters such as projected stored at 4 °C. Submerged fermentation was also carried area, circularity, aspect ratio and surface roughness have out at 30  °C on an orbital shaker (INFORS) at 200  rpm. been described to generate a dimensionless morphology Flasks were inoculated with a conidial suspension to get a number. Thus, both fructofuranosidase and glucoamyl - final concentration of about 10 conidia m/L. ase production by A. niger has been negatively correlated with an increasing the morphological number; while, Biomass determination a hypothetical correlation between the morphological The biomass was estimated by drying washed mycelia at number and productivity is proposed for citric acid and 105 °C until constant weight (Colin et al. 2013). secondary metabolites (Cairns et  al. 2019). In the same way, the enzyme productions are influenced by physico- Protein determination and native PAGE chemical factors that should be optimized to reach a Protein concentration was determined according to maximum yield (Geoffry and Achur 2018a). Bradford (1976). Additionally, proteins were separated by In other to control fungal morphology, several strate- native-PAGE using 10% (by mass per volume) polyacryla- gies have been reported. The conventional procedure mide gel. The presence of a lipolytic band was detected is based on the adjustments of chemical, physical and using 1.3 mM of α-naphtyl acetate as substrate. Released biological parameters such as conidia concentration, naphthol was coupled with 1  mM Fast Blue to give a medium composition, temperature, pH, supplementation coloured product. Reactions were carried out at 37  °C of glass beads, agitation systems, fermenter geometry, in shaken plates containing 100  mM phosphate buffer etc. (Papagianni et  al. 2004). Furthermore, Wucherp- (pH = 7.0). fennig et  al. (2011) reported that the culture osmolality also affects fungal morphology and process productiv - Lipase determination ity. In another approach, the macroscopic fungal mor- The lipase activity was measured according to Winkler phology was tailoring by altering the hyphal morphology and Stuckman (1979). One unit of enzyme activity was and the conidia adhesion capacity (Colin et  al. 2013). defined as the amount of enzyme that released 1 μmol of More recently, Karahalil et  al. (2019) reviewed several p-nitro phenol per min. Lipase production was expressed aspects of an interesting technique named microparticle either as a volumetric activity (units per liter of culture enhanced cultivation. That method allows the control of supernatant, U/L) or a specific activity (units per gram of growth physically in submerged fermentation by block- biomass dry weight, U/g ; units per milligram of dry weight ing the aggregation of filamentous microorganisms using protein, U/mg ). protein microparticles such as talc, aluminium oxide, titanium silicom oxide, iron (II, III) and forsterite. Scanning electron microscopy u Th s, as the biomass morphology and the culture con - For observation with scanning electron microscope ditions can affect the process productivity, the influence (SEM), at the end of fermentation, the mycelium was of those variables should be study separately. To do that, collected by filtration, washed with 0.1  mM phosphate the two-stage fermentation strategy can be used being buffer (pH 7), fixed with 2.5% glutaraldehyde and post - the replacement technique one of this culture modal- fixed with 1% OsO . The mycelium was dehydrated in ity (Sternberg and Mandels 1979). Briefly, the biomass is acetone, dried in a critical point apparatus, coated with pre-grown in a first reactor. Then, the washed mycelium gold and observed by using a Zeiss Supra 55VP (Carl is transferred to a second reactor to continue the study. Zeiss, Oberkochen, Germany). For instance, this approach was reported to analyse the transcriptional regulation of xyn2 in Hypocrea jecorina Two‑stage lipase production: replacement technique (Würleitner et al. 2003). Assays were conducted in 50  mL conical flasks contain - In this work, the main objective was to study the effect ing 10  mL of culture medium. The use of this work - of biomass morphology and physiological factors on an ing volume was previously reported for an extracellular extracellular lipase production from Aspergillus niger lipase production by A. niger MYA 135 (Colin et al. 2010). MYA 135 by using a submerged two-stage fermentation. For evaluate both biomass morphology and physiological Salvatierra et al. AMB Expr (2021) 11:42 Page 4 of 11 effectors on the lipase production, the replacement 1994; Gordillo et  al. 1995; Colin et  al. 2010; Griebeler technique reported by Sternberg and Mandels (1979) et  al. 2011; Lanka et  al. 2015). The experimental error was used. Briefly, mycelia were pre-grown during 24  h, was determined by replication of the entire experimental at initial pH 5 and in the presence of 2  g/L NH NO . In matrix. 4 3 the case of Plackett–Burman experimental design, the pre-growth fungal stage was conducted using morpho- Two‑stage lipase production 2: central composite design logical inducers and carbon sources detailed in Table  2. The relationship between the three input variables N:C To induce pelleted or dispersed fungal morphology ratio, FeCl and olive oil, and the enzyme production the corresponding culture medium was supplemented 3 expressed as lipase units per liter of culture supernatant with 0.5  g/L C aCl or 1.0  g/L FeCl , respectively (Colin 2 3 (U/L) were evaluated after 96 h of cultivation by using the et  al. 2013). In the case of central composite design, the central composite design (Myers and Montgomery 2002). pre-growth fungal stage was conducted under a unique This is an experimental design for building a second condition using 1  g/L F eCl and 10  g/L sucrose as mor- order polynomial for the response variable. It involves phological inducer and carbon source, respectively. Bio- three types of trials: 2 k factorial trials, 2 k axial trials and masses were collected and washed by vacuum filtration; nc center point trials, where k is number of factors stud- then, they were weighted and added to different lipase ied in the assay. Values at center point provide informa- production culture media according to the experimental tion about the existence of curvature in the response; that design (Plackett–Burman experimental design or cen- is, they contribute to the estimation of quadratic terms. tral composite design). The initial biomass concentration Axial points are also used to estimate quadratic terms, was 8 and 15 g /L for Plackett–Burman and cen- wet weight while factorial points contribute to the estimation of lin- tral composite experimental designs, respectively. Flasks ear and interaction terms. Each factor was studied at five were further incubated for 72  h under the same condi- different levels (− α, − 1,0 , + 1, + α) (Table  3). Variable tions. Both biomass pre-growth media and lipase pro- settings that represent the axial point of the design were duction media contained the same inorganic supplement decided based on either our own experience or litera- (in g/L): KH PO 1.0, MgSO 7H O 0.2, CuSO 5H O 0.06. 2 4 4 2 4 2 ture reports (Pokorny et al. 1994; Colin et al. 2010; Salihu The nitrogen:carbon (N:C) ratio was studied in produc - et  al. 2011). The experimental error was determined by tion media by varying the concentration of NH NO 4 3 replication of the entire experimental matrix. keeping the concentration of sucrose at 5 g/L.The super - natant was obtained by vacuum filtration and used as enzyme source. Two‑stage lipase production 3: a prove of concept The assay was conducted in 500  mL conical flasks con - Two‑stage lipase production 1: Plackett–Burman taining 100  mL of culture medium. The fermentation experimental design medium comprised (in g/L): sucrose 10.0, KH PO 1.0, 2 4 The effect of 11 environmental factors on the enzyme NH NO 2.0, M gSO 7H O 2.0, C uSO 0.06 and F eCl 4 3 4 2 4 3 production expressed as lipase units per gram of bio- 1.0. The initial pH was adjusted to 7.0 with NaOH. After mass dry weight (U/g ) were evaluated after 96  h dry weight 24 h of incubation, the culture was transferred to another of cultivation by using the Plackett–Burman experimen- 500  mL flask containing 50  mL of 3% (by volume) and tal design (Plackett and Burman 1946) (Table  2). This is was further incubated for 168  h under the same condi- a two-level fractional factorial design for studying n-1 tions. The lipase production was monitored during fer - input variables (factors) using n runs, where n is a mul- mentation using as response variables either the enzyme tiple of 4 (n is the number of experiments). Each fac- activity expressed as lipase units per liter of culture tor is represented at two levels, high and low, which are supernatant (U/L) or the specific lipase activity expressed denoted by (+) and (−), respectively. The effect for a fac - as enzyme units per milligram of protein (U/mg ). protein tor is always described as the change in the response in going from the low level of that factor to the high level. A negative sign means that going from low level to high Statistical analysis level for a factor decreases the response. A positive sign All statistical analysis was performed using Minitab soft- means that going from the low level to the high level ware (Minitab Inc., State College, PA, USA). Data were increases the response. The effect (E) of each variable on expressed as means ± standard deviation. Differences the response was determined by subtracting the average were accepted as significant when p < 0.05. The fitness of response of the low level from that of the high level. Lev- models was checked by both the determination coeffi - els of each input variable were decided based on either cient (R ) and the adjusted determination coefficient (adj our own experience or literature reports (Pokorny et  al. R ). S alvatierra et al. AMB Expr (2021) 11:42 Page 5 of 11 Table 2 Plackett–Burman design: experimental procedure, variables and statistical analysis Two-stage Pre-growth stage (24 h) Lipase production stage (72 h) Response variable Fermentation Initial pH 5 Initial biomass concentration: 8 g /L wet weight Input variables Morphology (−) pel Glucose l -Arabinose Sucrose N:C ratio pH Oleic acid Tributirine CaCl FeCl Olive oil Specific lipase 2 3 lets (+) dispersed activity (U/g ) dry weight Level (g/L) (−) 0.5 CaCl 0 0 5.0 0.4 5.0 0 0 0 0 10.0 (+) 1.0 FeCl 10.0 10.0 10.0 0.6 7.0 1.0 1.0 0.05 0.1 20.0 Trial number 1 + − + + − + − − − + + 33.92 ± 1.53 2 − − + + + − + + − + − 17.32 ± 3.72 3 − − − + + + − + + − + 17.88 ± 3.22 4 + + − + − − − + + + − 11.84 ± 1.94 5 − + − − − + + + − + + 15.33 ± 0.04 6 + − − − + + + − + + − 7.61 ± 0.28 7 − − − − − − − − − − − 3.12 ± 0.07 8 − + + + − + + − + − − 17.19 ± 3.68 9 + + − + + − + − − − + 24.47 ± 6.66 10 − + + − + − − − + + − 11.41 ± 1.51 11 + + + − + + − + − − − 20.84 ± 5.88 12 + − + − − − + + + − + 4.75 ± 1.81 Effect 3.531 2.747 4.198 9.923 2.232 6.643 − 2.053 − 1.625 − 7.390 1.530 3.978 R = 92.73% p-value 0.022 0.062 0.009 < 0.001 0.121 < 0.001 0.151 0.248 < 0.001 0.275 0.003 R = 86.07% adj Letters in bold show the experimental procedure, variables and statistical analysis corresponding to the pre-growth stage Concentration of medium components Salvatierra et al. AMB Expr (2021) 11:42 Page 6 of 11 Table 3 Central composite design: input variables and their the lipase activity being negligible the presence of either levels oleic acid or tributyrin. Besides, the initial pH 7 of cul- ture medium was more favorable to the enzyme produc- Input variables Levels tion than the initial pH 5. Regarding the influence of ions, − α − 1 0 + 1 + α the supplementation of 0.05 g/L CaCl displayed a highly negative and significant effect on the enzyme produc - tion (E = − 7.390). The linear effect corresponding to the N:C ratio 0.200 0.281 0.400 0.519 0.600 input variable F eCl was not significant (p = 0.275). FeCl (g/L) 0.050 0.080 0.125 0.170 0.200 Two‑stage lipase production 2: central composite design Considering that our next objective was to explore the Olive oil (g/L) 10.000 14.000 20.000 26.000 30.000 relationship between the enzyme production and the input variables N:C ratio (X ), FeCl (X ) and olive oil 1 3 2 (X ), it was decided to apply the central composite design Results for 20 trials expressing the enzyme activity as U/L. As Previously, it was reported an olive oil-induced extracel- it was mentioned before, with this kind of experimental lular lipase activity by the native A. niger MYA 135 using design is possible to estimate linear, quadratic and inter- a mineral culture medium (Colin et  al. 2010). In this action effects. To conduct this experiment, the biomass work, the employ of statistically designed experiments was developed in the presence of 1  g/L FeCl ; and then, was proposed to obtain knowledge about the relation- it was collected, washed, weighted and added to differ - ship among some input factors and the production of this ent lipase production media according to the experimen- extracellular enzyme. The results of the three two-stage tal design (Table  4). Data on enzyme activity showed a fermentations are described below. wide variation covering the range from 1.57 ± 0.41 to 324.57 ± 19.86 U/L. Considering the p-value of the corre- Two‑stage lipase production 1: Plackett–Burman sponding coefficients (0.007 or smaller), the lipase activ - experimental design ity production depended on two linear effects (X and Firstly, linear effects of fungal morphology and several X ), one two-way interaction (X X ) and two quadratic 3 2 3 physiological parameters on a lipase activity production 2 2 effects (X and X ) (Table 5). The second-order polyno - 1 2 were explored using a fractional experimental design. As mial equation is shown below: both fungal morphology and physiology are affected by Y = 195.0−61.9X + 48.8X + 39.8X X the same environmental conditions, the biomass and the (U/L) 1 3 2 3 lipase production was uncouple using the replacement 2 2 −64.3X + 30.7X 1 3 technique. Considering that our main objective in this assay was to evaluate the impact of biomass morphol- As it can be seen, both linear and quadratic terms cor- ogy on the lipase activity production, it was decided to responding to the N:C ratio were negative. While, those determine the enzyme activity expressed as U/g . terms associated to olive oil as well as its interaction with dry weight Table  2 shows the Plackett- Burman design for 12 trials FeCl were positive. In addition, the R value indicated and the corresponding response variable. The R value that 80.10% of data variation was explained by the input indicated that 92.73% of data variation was explained by variables. The adj R was 73.24%. The lack-of-fit test is the input variables. The adj R was 86.07%. Thus, data testing the lack of fit for the quadratic model; the p-value on specific enzyme activity exhibited a wide variation for this test is large (p = 0.578) implying that the quad- covering the range from 3.12 ± 0.07 to 33.92 ± 1.53 U/ ratic model was adequate (Table 5). g . The fungal morphology displayed a significant dry weight effect on the final response being the dispersed myce - Two‑stage lipase production 3: a prove of concept lia more favorable than the pelleted form of growth for Taking in mind the results obtained, a third two-stage to increase the specific lipase activity. Concerning the lipase production was conducted as a prove of concept. impact of the carbon source on the biomass pre-growth, The biomass was pre-grown at an initial pH 7, in the both the sucrose and the l-arabinose presented posi - presence of 1  g/L FeCl and with a starting N:C ratio of tive and significant effects on the enzyme production, 0.2 as described in the material and methods section. while the effect of glucose was not significant under our After 24  h of cultivation, the entire culture was trans- assay conditions. In relation to the effect of physiologi - ferred to another reactor without any washed procedure cal parameters on the lipase production, as expected, getting an initial olive oil concentration of 20  g/L. The the olive oil had a positive and a significant effect on highest value of lipase activity (U/L) (Fig. 1a) and specific S alvatierra et al. AMB Expr (2021) 11:42 Page 7 of 11 Table 4 Central composite design: experimental procedure and variables Two-stage fermentation Pre-growth stage (24 h) in the presense of 1.0 g/L FeCl Biomass morphology: dispersed mycelium Lipase production stage (72 h), initial biomass concentration: 15 g wet /L weight Trial number Input variables Response variable N:C ratio FeCl Olive oil Lipase activity (U/L) CP 0 0 0 153.73 ± 2.17 CP 0 0 0 82,94 ± 0.29 CP 0 0 0 219.61 ± 6.50 b CP 0 0 0 191.01 ± 5.78 CP 0 0 0 238.76 ± 14.80 CP 0 0 0 239.78 ± 5.42 1 + 1 + 1 − 1 3.33 ± 0.21 2 − 1 − 1 + 1 294.90 ± 25.94 3 0 0 − α 89.59 ± 1.73 4 − 1 + 1 + 1 324.57 ± 19.86 5 + 1 − 1 − 1 20.13 ± 0.87 6 + 1 − 1 + 1 6.56 ± 4.27 Fig. 1 Time course of an extracellular lipase activity from Aspergillus 7 0 + α 0 315.63 ± 32.50 niger MYA 135 during the lipase production stage expressed as 8 0 0 + α 251.28 ± 10.47 volumetric activity (U/L) (a) or specific activity (U/mg ) (b) protein 9 − 1 + 1 − 1 135.60 ± 10.11 10 + 1 + 1 + 1 170.84 ± 7.95 11 0 − α 0 276.05 ± 48.76 96  h of cultivation. In addition, the culture supernatant 12 + α 0 0 1.57 ± 0.41 was analyzed by native PAGE. Two lipolytic bands were 13 − α 0 0 30.16 ± 11.66 detected using α-naphtyl acetate as substrate being the 14 − 1 − 1 − 1 242.98 ± 30.52 intensity signal of the top one also increased at the time of 96 h (Fig. 2). On the other hand, SEM micrographs of lipase activity (U/mg ) (Fig.  1b) was obtained after protein the harvested biomass displayed a dispersed mycelium (lax clumps mixed with free mycelium) showing scarcely Table 5 Central composite design: terms and the corresponding branched and not fragmentated hyphae; in addition, the coefficients presence of conidiophores was not observed (Fig. 3). Term Coefficient p-value Constant 195.0 < 0.001 Discussion Linear < 0.001 The impact of biomass morphology and culture condi - X (N:C ratio) − 61.9 < 0.001 tions on fungal fermentation was analyzed in several X (FeCl ) 12.7 0.251 investigations as it was briefly mentioned in “Introduc - 2 3 X (Olive oil) 48.8 < 0.001 tion” section. Interaction < 0.001 Here, through the application of designed experiments, X X 28.1 0.057 we evaluated the influence of some of those input factors 1 2 X X − 10.9 0.450 on a lipase activity production separately by using the A. 1 3 X X 39.8 0.009 niger MYA 135 as a model and the two-stage fermenta- 2 3 Quadratic 0.015 tion technique as the culture modality. X − 64.3 < 0.001 X 30.7 0.007 Biomass morphology and lipase production X − 9.7 0.368 3 In coincidence with the results reviewed by Krull et  al. Lack-of-fit 0.578 (2010), the macroscopic characteristic of fungal mor- R (%) 80.10 phology displaying by A. niger MYA 135 was already R (%) 73.24 adj apparent after about 18 h of incubation (data not shown). Salvatierra et al. AMB Expr (2021) 11:42 Page 8 of 11 biomass morphology on the specific lipase production expressed as U/g was, firstly, evaluated. Dispersed dry weight or pelleted mycelia were harvested, washed by vacuum filtration, weighted as indicated in the material and methods section, and then added to different produc - tion media. The Plackett–Burman experimental design identified the significant effect of the biomass morphol - ogy being the dispersed mycelium preferred to increase the specific lipase production. In fact, the same culture medium at an initial pH 7 without FeCl supplementa- tion favours the pelleted form of growth; and, under that environmental condition, the maximum specific activity using p-NPP as substrate was 1.6 U/mg after 96 h of protein cultivation (Pera et  al. 2006). Here, as it can be seen in the prove of concept assay, that value was increased ten times in the presence of dispersed mycelium (Fig.  1b). In concordance with our observation, a freely dispersed mycelium from A. niger SKAn1015 induced by the sup- plementation of silicate microparticles was linked to an Fig. 2 Native PAGE showing the time course of an extracellular lipase activity from Aspergillus niger MYA 135 during the lipase production increase of a fructofuranosidase activity displaying the stage. The lipolytic bands were detected using α-naphtyl acetate as microparticles itself an important physiological role (Dri- substrate ouch et al. 2010). On the contrary, the performance of a synthetic mycelium-bound lipase activity from Rhizopus chinensis was favoured by fully entangled mycelial fila - ments (Teng et al. 2009). However, as it will be discussed later, the lipase production by filamentous fungi should be analyzed from a holistic point of view. Fungal physiology and lipase production Firstly, the impact of several physiological factors on the lipase production activity expressed as U/g dry weight (Two-stage lipase production 1) was evaluated. Thus, besides the fungal biomass morphology, the kind of car- bon source using during the pre-growth stage also had a relevant role on the specific lipase production. In fact, according to the Plackett–Burman experimental design, the presence of sucrose during the first-stage of fermen - tation exhibited the most significant and positive effect Fig. 3 Image of dispersed mycelium obtained with a scanning on the response variable. On the other hand, the olive electron microscope showing the harvested biomass from Aspergillus oil concentration also displayed a positive effect on the niger MYA 135 after 96 h of cultivation response variable being the effect associated with the supplementation of either oleic acid or tributyrin not significant. So, the hydrophilic substrate could have a For that reason, it was decided to conduct a biomass pre- relevant role during the fungal growth stage, while the growth stage of 24  h. So, culture media at initial pH 5 hydrophobic one acts mainly as an inducer of the lipo- were supplemented with morphological inducers in the lytic activity synthesis. These results were compatible pre-growth fermentation stage. As previously reported, with those that reported the use of a combination of dispersed mycelium is induced in the presence of 1  g/L carbohydrate source and lipid inducer as a strategy to FeCl ; while, the pelleted form of growth is favoured by increase the biocatalyst production. However, the proper the addition of 0.5 g/L C aCl (Colin et al. 2013). In addi- mixture of those culture components varies according to tion, under our culture conditions, before that time of the microorganism used. To give examples, the optimal cultivation, no significant extracellular lipase activity substrates for a lipase activity production from Pichia was found (data not shown). Thus, the influence of the lynferdii Y-7723 were soybean oil and sucrose (Kim S alvatierra et al. AMB Expr (2021) 11:42 Page 9 of 11 et  al. 2010); while, olive oil and xylose were preferred by replacement technique it was demonstrated that a low Rhizopus oryzae ZAC3 (Ayinla et  al. 2017). Concerning N:C ratio was preferable to increase the lipase produc- A. niger MYA 135, the olive oil was the best hydropho- tion by A. niger MYA 135. This effect was also observed bic substrate using in the presence of sucrose (Colin et al. in the prove of concept assay where the initial N:C ratio 2010). Additionally, as it was also shown in this work, the was 0.2 (or 5 C:N ratio). As expected, the next important initial pH value of the production medium is an impor- factor was the olive oil concentration. Not only the linear tant physiological factor involved in the lipase produc- term corresponding to this input variable was positive tion being this parameter a fungi-dependent variable. In (p < 0.001) but also a positive interaction between olive this connection, it was reported the production of fun- oil and FeCl (p = 0.009) was found. Thus, as the linear gal lipases under acidic (Turati et  al. 2019), near neutral term corresponding to olive oil was positive, the effect on (Colla et al. 2016) or alkaline (Rajan and Nair 2011) cul- the response will tend to be more positive increasing the ture conditions. Finally, the impact of metal ions on the value of FeCl concentration. Thus, the FeCl acted not 3 3 lipolytic enzyme synthesis has been evaluated in several only as a morphological effector but also as an additive studies during the selection of significant input variables that contributed to increase the lipase production. How- (Rajendran and Thangavelu 2009; Salihu et  al. 2011). In ever, according to ours results, the N:C ratio always has this work, the supplementation of 0.1  g/L F eCl did not to be taken into account when evaluating the impact of has a significant effect on the specific enzyme activity; other input variables on the lipase activity. other aspects concerning to this salt are discussed in the Finally, the dispersed mycelium produced at least two next paragraph. On the contrary, the presence of 0.05 g/L lipases as it was observed in native PAGE being the inten- CaCl exhibited a highly negative impact on the response sity signal of the top one increased after 72 h of incuba- (E = − 7.390). Similarly, Geoffry and Achur (2018a), tion. In addition, the lipase production pattern of A. niger reported that the variable CaCl influences negatively on MYA 135 was similar to those reported for Aspergil- the production of a lipase activity from Fusarium solani. lus carbonarius (Ire and Ike 2014) and Rhizopus oryzae Oppositely, the supplementation of this salt is favorable ZAC3 (Ayinla et  al. 2017) achieving all of them an opti- for the synthesis of a lipase activity using Rhizopus arrhi- mum incubation time of 96 h. zus MTCC 2233 (Rajendran and Thangavelu 2009). And, In summary, analysing the three independent assays the effect of CaCl is no significant to produce a lipase conducted during this work, it can be highlighted the activity from Yarrowia lipolytica MTCC 35 (Kishan et al. usefulness of the replacement technique to study how 2013). Thus, reported data clearly indicate the wide varia - environmental conditions affected a lipase activity pro - tion in effects of CaCl on the lipase production. duction evaluating the influence of biomass morphol - Secondly, the central composite design was adopted to ogy and other physiological effectors separately. The study the relationship between the lipase production and dispersed morphology was confirmed as a proper qual - the input variables N:C ratio, F eCl and olive oil. In this ity characteristic for producing a lipase activity from A. case, the response variable was the volumetric enzyme niger MYA 135. In addition, the bifunctional role of FeCl activity expressed as U/L. This parameter was measured on this fungus was presented. Finally, according to ours after 96 h of cultivation (Two-stage lipase production 2). results, the N:C ratio always has to be taken into account Concerning the effect of nitrogen source on the lipase when evaluating the impact of other input variables on production, there is a general agreement in the literature this process using this filamentous fungus. that this enzyme synthesis is favoured at high nitrogen concentration, and therefore, lower C:N ratios, being Authors’ contributions this substrate either organic or inorganic (Das et al. 2016; MDB and LMP conceived and designed research. HNS, and ELR conducted Geoffry and Achur 2018b). In this connection, Coradi experiments. HNS and LMP analyzed data. MDB and LMP wrote the manu- script. All authors read and approved the manuscript. et  al. (2013) reported that Trichoderma harzianum dis- played the highest lipase activity in a culture medium Funding containing 0.5% yeast extract and 1% olive oil (2.5 C:N This work was supported by FONCyT (PICT 2015-2596) and UNT (PIUNT D 606). ratio). Here, among the model terms, the N:C ratio of Availability of data and materials the production culture medium had the most significant Authors can confirm that all relevant data are included in the article. influence on the lipase activity production. A negative linear coefficient suggests that as the N:C ratio increases, Declarations the independent variable tends to decrease. In addition, Ethics approval and consent to participate as the corresponding quadratic term was also negative, This article does not contain any studies with human participants or animals the net effect of increasing the N:C ratio was an accelera - performed by any of the authors. tion of the response variable decrease. u Th s, by using the Salvatierra et al. AMB Expr (2021) 11:42 Page 10 of 11 Consent for publication Griebeler N, Polloni AE, Remonatto D, Arber F, Vardanega R, Cechet JL, Di Not applicable. Luccio M, de Oliveira D, Treichel H, Cansian RL, Rigo E, Ninow JL (2011) Isolation and screening of lipase-producing fungi with hydrolytic Competing interests activity. Food Bioprocess Technol 4:578–586. https:// doi. org/ 10. 1007/ The authors declare that they have no conflict of interest.s11947- 008- 0176-5 Ire FS, Ike VC (2014) Screening and optimization of process parameters for the Author details production of lipase in submerged fermentation by Aspergillus carbon- Morphogenesis and Fermentation Lab, PROIMI-CONICET, T4001 MVB San arius (Bainer) IMI 366159. Annu Res Rev Biol 4:2587–2602. https:// doi. org/ Miguel de Tucumán, Argentina. Facultad de Bioquímica, Química y Farmacia, 10. 9734/ ARRB/ 2014/ 9879 Cátedra de Microbiología Superior, Universidad Nacional de Tucumán, Karahalil E, Coban HB, Turhan I (2019) A current approach to the control of T4000INI San Miguel de Tucumán, Argentina. filamentous fungal growth in media: microparticle enhanced cultiva- tion technique. Crit Rev Biotechnol 39:192–201. https:// doi. org/ 10. 1080/ Received: 14 December 2020 Accepted: 7 March 202107388 551. 2018. 15318 21 Kim HR, Kim IH, Hou CT, Kwon KI, SHIN BS, (2010) Production of a novel cold- active lipase from Pichia lynferdii Y-7723. J Agric Food Chem 58:1322– 1326. https:// doi. org/ 10. 1021/ jf903 430t Kishan G, Gopalakannan P, Muthukumaran C, Thirumalai Muthukumaresan K, Dharmendira Kumar M, Tamilarasan K (2013) Statistical optimization References of critical medium components for lipase production from Yarrowia Ayinla ZA, Ademakinwa AN, Agboola FK (2017) Studies on the optimization of lipolytica (MTCC 35). J Genet Eng Biotechnol 11:111–116. https:// doi. org/ lipase production by Rhizopus sp. ZAC3 isolated from the contaminated 10. 1016/j. jgeb. 2013. 06. 001 soil of a palm oil processing shed. J App Biol Biotechnol 5:030–037. Krull R, Cordes C, Horn H, Kampen I, Kwade A, Neu TR, Nörtemann B (2010) https:// doi. org/ 10. 7324/ JABB. 2017. 50205 Morphology of filamentous fungi: linking cellular biology to process engi- Bradford MM (1976) A rapid and sensitive method for the quantification of neering using Aspergillus niger. Adv Biochem Eng Biotechnol 121:1–21. microgram quantities of protein utilizing the principle of protein-dye https:// doi. org/ 10. 1007/ 10_ 2009_ 60 binding. Anal Biochem 72:248–254. https:// doi. org/ 10. 1016/ 0003- Lanka S, Pydipalli M, Latha JNL (2015) Optimization of process variables for 2697(76) 90527-3 extracellular lipase production from Emericella nidulans NFCCI 3643 iso- Cairns TC, Zheng X, Zheng P, Sun J, Meyer V (2019) Moulding the mould: lated from palm oil mill effluent (POME) dump sites using OFAT method. understanding and reprogramming filamentous fungal growth and mor - Res J Microbiol 10:38–53. https:// doi. org/ 10. 3923/ jm. 2015. 38. 53 phogenesis for next generation cell factories. Biotechnol Biofuels 12:77. Myers R, Montgomery D (2002) Response surface methodology. Process and https:// doi. org/ 10. 1186/ s13068- 019- 1400-4 product optimization using designed experiments. John Wiley & Sons, Colin VL, Baigori MD, Pera LM (2010) Eec ff t of environmental conditions on INC., Hoboken extracellular lipases production and fungal morphology from Aspergillus Papagianni M (2004) Fungal morphology and metabolite production in sub- niger MYA 135. J Basic Microbiol 50:52–58. https:// doi. org/ 10. 1002/ jobm. merged mycelial processes. Biotechnol Adv 22:189–259. https:// doi. org/ 20090 0162 10. 1016/j. biote chadv. 2003. 09. 005 Colin VL, Baigori MD, Pera LM (2013) Tailoring fungal morphology of Aspergillus Pera LM, Romero CM, Baigori MD, Castro GR (2006) Catalytic properties of niger MYA 135 by altering the hyphal morphology and the conidia adhe- lipase extracts from Aspergillus niger. Food Technol Biotechnol 44:247–252 sion capacity: biotechnological applications. AMB Expr 3:27. https:// doi. Plackett RL, Burman JP (1946) Trust the design of optimum multifactorial org/ 10. 1186/ 2191- 0855-3- 27 experiments. Biometrika 33:305–325. https:// doi. org/ 10. 1093/ biomet/ Colla LM, Primaz AL, Benedetti S, Loss RA, Lima M, Reinehr CO, Bertolin TE, 33.4. 305 Vieira Costa JA (2016) Surface response methodology for the optimiza- Pokorny D, Friedrich J, Cimerman A (1994) Eec ff t of nutritional factors on lipase tion of lipase production under submerged fermentation by filamentous biosynthesis by Aspergillus niger. Biotechnol Lett 16:363–366. https:// doi. fungi. Braz J Microbiol 47:461–467. https:// doi. org/ 10. 1016/j. bjm. 2016. org/ 10. 1007/ BF002 45052 01. 028 Quintanilla D, Hagemann T, Hansen K, Gernaey KV (2015) Fungal morphology Coradi GV, da Visitação VL, de Lima EA, Saito LY T, Palmieri DA, Takita MA, Neto in industrial enzyme production—modelling and monitoring. Adv Bio- PO, Gomes de Lima VM (2013) Comparing submerged and solid-state chem Eng Biotechnol 149:29–54. https:// doi. org/ 10. 1007/ 10_ 2015_ 309 fermentation of agro-industrial residues for the production and charac- Rajan A, Nair AJ (2011) A comparative study on alkaline lipase production by a terization of lipase by Trichoderma harzianum. Ann Microbiol 63:533–540. newly isolated Aspergillus fumigatus MTCC 9657 in submerged and solid- https:// doi. org/ 10. 1007/ s13213- 012- 0500-1 state fermentation using economically and industrially feasible substrate. Das A, Bhattacharya S, Shivakumar S, Shakya S, Sogane SS (2016) Coconut oil Turk J Biol 35:569–574. https:// doi. org/ 10. 3906/ biy- 0912-6 induced production of a surfactant-compatible lipase from Aspergillus Rajendran A, Thangavelu V (2009) Statistical experimental design for evalua- tamarii under submerged fermentation. J Basic Microbiol 57:114–120. tion of medium components for lipase production by Rhizopus arrhizus https:// doi. org/ 10. 1002/ jobm. 20160 0478 MTCC 2233. LWT-Food Sci Technol 42:985–992. https:// doi. org/ 10. 1016/j. Driouch H, Sommer B, Wittmann C (2010) Morphology engineering of lwt. 2008. 12. 009 Aspergillus niger for improved enzyme production. Biotechnol Bioeng Salihu A, Alam MDZ, AbdulKarim MI, Salleh HM (2011) Optimization of lipase 105:1058–1068. https:// doi. org/ 10. 1002/ bit. 22614 production by Candida cylindracea in palm oil mill effluent based Feng K, Huang Z, Peng B, Dai W, Li Y, Zhu X, Chen Y, Tong X, Lan Y, Cao Y (2020) medium using statistical experimental design. J Mol Catal B-Enzym Immobilization of Aspergillus niger lipase onto a novel macroporous 69:66–73. https:// doi. org/ 10. 1016/j. molca tb. 2010. 12. 012 acrylic resin: Stable and recyclable biocatalysis for deacidification of high- Sternberg D, Mandels GR (1979) Induction of cellulolytic enzymes in Tricho- acid soy sauce residue oil. Bioresour Technol 298:122553. https:// doi. org/ derma reesei by sophorose. J Bacteriol 139:761–769. https:// doi. org/ 10. 10. 1016/j. biort ech. 2019. 122553 1128/ JB. 139.3. 761- 769. 1979 Geoffry K, Achur RN (2018a) Optimization of novel halophilic lipase production Teng Y, Xu Y, Wang D (2009) Changes in morphology of Rhizopus chinensis in by Fusarium solani strain NFCCL 4084 using palm oil mill effluent. J Genet submerged fermentation and their effect on production of mycelium- Eng Biotechnol 16:327–334. https:// doi. org/ 10. 1016/j. jgeb. 2018. 04. 003 bound lipase. Bioprocess Biosyst Eng 32:397–405. https:// doi. org/ 10. Geoffry K, Achur RN (2018b) Screening and production of lipase from fungal 1007/ s00449- 008- 0259-8 organisms. Biocatal Agric Biotechnol 14:241–253. https:// doi. org/ 10. Turati DFM, Almeida AF, Terrone CC, Nascimento JMF, Terrasan CRF, Fernandez- 1016/j. bcab. 2018. 03. 009 Lorente G, Pessela BC, Guisan JM, Carmona EC (2019) Thermotolerant Gordillo MA, Obradors N, Montesinos JL, Valero F, Lafuente J, Solà C (1995) lipase from Penicillium sp. Section Gracilenta CBMAI 1583: effect of carbon Stability studies and effect of the initial oleic acid concentration on lipase sources on enzyme production, biochemical properties of crude and production by Candida rugose. Appl Microbiol Biotechnol 43:38–41. purified enzyme and substrate specificity. Biocatal Agric Biotechnol https:// doi. org/ 10. 1007/ BF001 70620 17:15–24. https:// doi. org/ 10. 1016/j. bcab. 2018. 10. 002 S alvatierra et al. AMB Expr (2021) 11:42 Page 11 of 11 Verma S, Meghwanshi GK, Kumar R (2021) Current perspectives for microbial Würleitner E, Pera L, Wacenovsky C, Cziferszky A, Zeilinger S, Kubicek CP, Mach lipases from extremophiles and metagenomics. Biochimie 182:23–36. RL (2003) Transcriptional regulation of xyn2 in Hypocrea jecorina. Eukariot https:// doi. org/ 10. 1016/j. biochi. 2020. 12. 027 Cell 2:150–158. https:// doi. org/ 10. 1128/ EC.2. 1. 150- 158. 2003 Winkler UK, Stuckmann M (1979) Glycogen, hyaluronate, and some other polysaccharides greatly enhance the formation of exolipase by Serratia Publisher’s Note marcescens. J Bacteriol 138:663–670. https:// doi. org/ 10. 1128/ JB. 138.3. Springer Nature remains neutral with regard to jurisdictional claims in pub- 663- 670. 1979 lished maps and institutional affiliations. Wucherpfennig T, Hestler T, Krull R (2011) Morphology engineering—osmolal- ity and its effect on Aspergillus niger morphology and productivity. Microb Cell Fact 10:58. https:// doi. org/ 10. 1186/ 1475- 2859- 10- 58

Journal

AMB ExpressSpringer Journals

Published: Mar 17, 2021

There are no references for this article.