Cultivation of recombinant Aspergillus niger strains on dairy whey as a carbohydrate source

Abstract Agricultural waste valorisation provides a sustainable solution to waste management, and combining waste utilisation with commodity production allows for responsible production processes. Recombinant Aspergillus niger D15 strains expressing fungal endoglucanases (Trichoderma reesei eg1 and eg2 and Aspergillus carneus aceg) were evaluated for their ability to utilise lactose as a carbon source to determine whether dairy waste could be used as a feedstock for enzyme production. The recombinant A. niger D15[eg1]PyrG, D15[eg2]PyrG, and D15[aceg]PyrG strains produced maximum endoglucanase activities of 34, 54, and 34 U/mL, respectively, on lactose and 23, 27, and 22 U/mL, respectively, on whey. The A. niger D15[eg2]PyrG strain was used to optimise the whey medium. Maximum endoglucanase activity of 46 U/mL was produced on 10% whey medium containing 0.6% NaNO3. The results obtained indicate that dairy whey can be utilised as a feedstock for recombinant enzyme production. However, variations in enzyme activities were observed and require further investigation.


Introduction
The rise in the world population requires an increase in the food supply without disruption of the existing food-producing practices.The primary obstacles that need to be addressed are twofold: First, to enhance food production by implementing strategies to minimise agricultural product losses, specifically reducing unnecessary wastage.Second, there is a need to reduce waste generation across various processes related to food production and consumption.Approximately 906 million tonnes of milk were produced globally in 2020, making it one of the most widely consumed beverages in the world (FAO, 2021 ).Milk is used to produce a variety of food products, such as butter, ice cream, cheese, etc. Whey is an abundant and inexpensive by-product of the cheese industry, with approximately 1 kg of manufactured cheese generating 9 L of whey (Fernández-Gutiérrez et al., 2017 ).Sweet whey has a pH of 6-7 and is produced when chymosin is used for enzymatic coagulation.Acid whey is a by-product of acidified milk coagulation and has a pH of less than 5 (Fernández-Gutiérrez et al., 2017 ).Whey contains 93-95% (w/w) water, 50% milk nutrients, and 60-70 g/L solids (Fernández-Gutiérrez et al., 2017 ).The dry matter fraction contains 66-77% (w/w) lactose, 8-15% (w/w) proteins, and 7-15% (w/w) minerals and vitamins.Whey is an environmental pollutant due to the high biochemical (30-50 g/L) and chemical (60-80 g/L) oxygen demand resulting from the high lactose (39-45 g/L) and protein (9-14 g/L) content (Fernández-Gutiérrez et al., 2017 ;Lech, 2020 ).The biotechnological challenge is (1) to decrease the waste produced in the agriculture industry without disruption of the existing food-producing practices and (2) to minimise the loss of agricultural products through unnecessary waste production.The whey proteins are highly desirable due to their health benefits and are recovered using micro-and ultra-filtration methods, leaving the sterile whey permeate for lactose recovery (Lech, 2020 ).These are expensive technologies resulting in valuable products, both of which are not feasible options for developing countries.Whey is supplied to local pig farms as an animal feed additive; however, the whey supply outweighs the demand.Proper waste treatment of huge volumes is costly, and untreated whey is discarded in sewer systems and rivers, which affects ecosystems and leads to the loss of biodiversity (Fernández-Gutiérrez et al., 2017 ).
A large market exists for cellulases, ranging from biofuel production to the food and feed industries.As with all industrial processes, the cost of the feedstock for enzyme production is a critically important determinant of the profit margin of the enzyme industry.Therefore, using a waste product as feedstock for a biorefinery can lower the production cost.Since whey is  readily available at a low cost in developing countries, it is fitting to investigate the possible use of whey as a feedstock, with the additional advantage of having a positive environmental impact.The use of Aspergillus spp.for the large-scale production of industrially relevant enzymes is well documented (Cairns et al., 2018 ;Li et al., 2020 ).Specific features that favour Aspergillus spp.for industrial use include its GRAS (g enerally r egarded a s s afe) status, high secretion capacity, proper folding of proteins without hyperglycosylation and rapid growth on inexpensive media.Aspergillus niger for example, is used for the large-scale production of enzymes such as pectinases, β-galactosidases, cellulases, etc., of which GlaA glucoamylase is produced at titres of 30 g/L (Cairns et al., 2021 ).The genus also produces a wide range of organic acids, with some strains producing up to 200 g/L citric acid (Cairns et al., 2021 ). A. niger can utilise lactose as a carbon source due to the expression and secretion of five β-galactosidases (EC 3.2.1.23)that hydrolyse the lactose to glucose and galactose (Niu et al., 2017 ).The ability to metabolise lactose makes the genus Aspergillus the ideal organism for cultivation on whey with simultaneous production of recombinant enzymes for the food or animal feed industry.
The study was designed in alignment with the goals for sustainable development.Instead of disposing whey into the environment, which could pose significant environmental risks and lead to nutrient loss, the research aimed to explore an economically viable approach for managing whey waste.Additionally, the study sought to create a valuable resource for the agriculture or food industry.To achieve these goals, the research focused on examining the constitutive expression of the endoglucanase-encoding genes from Trichoderma reesei ( eg1 and eg2 ) and Aspergillus carneus ( aceg1 ) in A. niger on lactose using a chemically defined and whey medium.

Strains
The relevant genotypes of the filamentous fungal strains and vectors used in this study are summarized in Table 1 .The A. niger D15#26 strain is a protease-deficient strain that does not acidify the growth media (Gordon et al., 2000 ;Wiebe et al., 2001 ), resulting in high protein titres with diminished degradation by natively produced acid proteases.The fungal strains were constructed using spheroplasts generated by lysing enzymes (Punt & van den Hondel, 1992 ).The construction of the A. niger D15[GT], D15[eg1] and D15[eg2] and D15[aceg1] strains were previously described in Rose & van Zyl (2002, 2008) .The endoglucanase encoding genes are expressed under the control of the constitutive A. niger glyceraldehyde 3-phosphate ( gpd ) promoter and Aspergillus awamori glucoamylase ( glaA ) terminator.The pUC-PyrG vector was introduced and integrated into the genomes of A. niger PyrG,and D15[aceg]PyrG strains (Table 1 ).The A. niger D15[GT]PyrG strain served as a reference strain.The A. niger D15[eg1] and D15[eg2] and D15[aceg1] strains were selected based on the pH optima of the recombinant endoglucanases (Table 2 ).

Cultivation Medium and Conditions
All reagents and media components were supplied by Merck (Darmstadt, Germany) unless stated otherwise.The Escherichia coli DH5 α strain (Takara Bio Inc., Kusatsu, Shiga, Japan) was used for plasmid propagation with transformants maintained and selected on Luria Bertani agar (5 g/L yeast extract, 10 g/L tryptone, 10 g/L NaCl, and 20 g/L agar) containing 100 μg/mL ampicillin.The E. coli DH5 α [pUC-PyrG] strain was cultured at 37°C in Terrific Broth (24 g/L yeast extract, 12 g/L tryptone, 4 mL/L glycerol, and 0.1 M potassium phosphate buffer) containing 100 mg/L ampicillin.

Qualitative Plate Assays
Transformants were screened on Ostazin brilliant red hydroxyethyl cellulose (OBR-HEC) agar plates (6.7 g/L yeast nitrogen base without amino acids [BD-Diagnostic Systems, Sparks, MD], 100 g/L glucose, 10 g/L OBR-HEC and 20 g/L agar).Spores were transferred to the OBR-HEC, agar plates, and the development of the clearing zone was monitored for 24 hr.

Quantitative Liquid Assays
Volumetric endoglucanase activity was determined using the reducing sugar assay (Miller, 1959 ) and 1% CMC (in 0.05 M citrate phosphate buffer, pH 5.0) as substrate.Recombinant fungal strains were cultivated aerobically in 125 mL Erlenmeyer flasks containing 20 mL 2 ×MM at 30°C for 7 days at 200 rpm.Supernatant samples were collected at 24 hr intervals.All assays were conducted in triplicate, and the absorbance was measured at 540 nm using the xMark Microplate Spectrophotometer (Biorad, Hercules, CA).

SDS-PAGE
Recombinant fungal strains were cultivated in 20 mL 2 ×MM medium containing 10% glucose in 125 mL Erlenmeyer flasks for 48 hr at 30°C and 200 rpm.Crude supernatant protein samples (15 mL) were separated using an 8% polyacrylamide separation gel (Laemmli, 1970 ;Sambrook et al., 1989 ) at 100 V for 90 min.Proteins were visualised using the silver staining method (O'Connell and Stults, 1997 ).The prestained PageRuler TM (Thermo Fischer Scientific) was used for size estimation of the recombinant endoglucanases.

Results and Discussion
Aspergillus niger is a desirable host for large-scale production of proteins or enzymes due to the ability to secrete large titres (grams per litre) of proteins or enzymes (Cairns et al., 2018 ;Li et al., 2020 ).The production of a diverse range of proteases (especially acid proteases) remains a hurdle in enzyme production since medium acidification naturally occurs during cultivation.
Spores  (2002 ).The Eg1 and Eg2 are produced as heterogeneous protein species as a result of differential glycosylation.The Eg1 and Eg2 display a molecular mass of 60-120 and 45-65 kDa, respectively (Fig. 1 b).The sizes are consistent with previous studies (Rose and van Zyl, 2002 ).The AcEg is secreted as a single protein species of approximately 26 kDa, which is consistent with the predicted unglycosylated mass.
The recombinant A. niger D15[GT]PyrG, D15[egI]PyrG, D15[eg2]PyrG, and D15[aceg]PyrG strains were inoculated to a spore density of 1 × 10 6 spores/mL in 2 ×MM (glucose or lactose at equal molar carbon concentrations).Supernatant samples were collected at 24-hr intervals and used to quantify the extracellular endoglucanase activity over 7 days (Fig. 2 ).The activity was determined at a pH of 4.5 because the pH optima of the recombinant Eg1, Eg2, and AcEg enzymes range from pH 4 to 5 (Rose and van Zyl, 2008 ).Maximum endoglucanase activity was detected with 4 days of cultivation on glucose, which coincided with the depletion of glucose in the medium (Fig. 2 a and c).The A. niger D15[egI]PyrG, D15[eg2]PyrG, and D15[aceg]PyrG strains produced a maximum of 50, 70, and 23 U/mL, respectively, whereas the A. niger D15[GT]PyrG reference strain produced 6.5 U/mL after 3 days.The activity remained constant for 7 days which is indicative of the stability of the hydrolases under cultivation conditions and the lack of extracellular protease activity.
The A. niger D15[eg2]PyrG strain displayed 54 U/mL on lactose (Fig. 2 b), which is notably less than with cultivation on glucose (Fig. 2 a).The A. niger D15[egI]PyrG and D15[aceg]PyrG strains produced around 34 U/mL of endoglucanase activity after 4 days of cultivation (Fig. 2 b), whereas the A. niger D15[GT]PyrG control strain consistently produced less than 10 U/mL throughout.The residual reducing sugars in the cultivation medium were monitored using the reducing sugar assay (Fig. 2 d).The reduced sugar concentration decreased over the first 4 days and was not completely utilised.In this study, a high lactose concentration was used to induce extracellular galactosidase production, but the hydrolysis of the lactose was inadequate to result in glucose accumulation.The gpd promoter requires high concentrations of glucose (10% glucose) for maximum expression (van Zyl et al., 2009 ).The reduced endoglucanase activity on lactose for the A.    PyrG displayed a maximum activity of 14 U/mL.The overall low level of activity is directly related to the lack of glucose accumulation for induction of the gpd promoter, as explained previously.The soy whey, Avonlac ® 282, contained a total sugar concentration of 5% (w/w), resulting in an initial concentration of 0.5% sugar in the cultivation medium.The low carbohydrate concentration is not suited for biomass production, resulting in low titres of secreted enzymes.An increase in the whey concentration results in an increase in viscosity in the medium, making it less ideal for oxygen transfer and fungal cultivation in shake flasks and was therefore not pursued.Eg1 is known to hydrolyse a range of organic polysaccharides, including cellulose and hemicelluloses, as well as chemical substrates (Bailey et al., 1993 ).This wide substrate range of Eg1 might result in improved hydrolysis of the soy content of the whey, releasing more carbon and nitrogen for metabolic processes, resulting in more Eg1 enzymes being produced.
The A. niger D15[eg1]PyrG, D15[eg2]PyrG, D15[aceg]PyrG, and D15[GT]PyrG strains produced maximum endoglucanase activities of 23, 27, 22, and 2 U/mL, respectively with cultivation on 10% bovine whey (Fig. 3 b).The A. niger D15[eg2]PyrG strain performed better on the bovine whey, which is in contrast to the observation in Fig. 3 a.During the strain construction, the gene cassettes are integrated at random throughout the genome, and the effect of the integration is unpredictable.This emphasizes the importance of screening multiple transformants under different cultivation conditions.The general decrease in endoglucanase activity points to the instability of the enzymes in the bovine whey medium after 3 days of cultivation.The effectiveness of the erythromycin started to diminish after 3 days, resulting in bacterial growth and the possible production of bacterial proteases.
Bovine whey (Roulou, Stellenbosch, SA) was autoclaved, which resulted in the elimination of bacterial contamination and the coagulation of the remainder of the proteins.The proteins were removed by decanting the liquid whey to simulate lower-grade whey.AspA was added (0.6% NaNO 3 ) as an additional nitrogen source after autoclaving.An increase in the permeate or whey concentration increased the extracellular endoglucanase activity displayed by the A. niger D15[eg2]PyrG strain (Fig. 4 a, b).Maximum activities of 12, 23, and 35 U/mL were obtained on 4, 6, and 10% lower-grade whey medium, respectively after 3-4 days of cultivation.Similarly, 14, 22, and 46 U/mL were obtained in the 4, 6, and 10% whey media, respectively.The proteins in the whey medium can act as a nitrogen source in addition to the exogenous nitrogen added, resulting in higher levels of activity in the whey medium.The parental strain used in this study is a protease-deficient strain, and therefore, has a diminished ability to utilise the extracellular proteins present in the whey.The addition of an external nitrogen source may not be required when a different A. niger strain is used as a host.An increase in the whey concentration results in an increase in viscosity in the medium, making it less ideal for oxygen transfer and fungal cultivation in shake flasks and was therefore not pursued.The A. niger D15[eg2] strain had previously been cultivated on different forms of potato waste, displaying activities ranging from 20 to 42 U/mL (Rose et al., 2018 ).The soy whey Avonlac ® 282 provides similar results to that of the potato waste but has the advantage that it is easier to transport and can be stored at room temperature.

Conclusion
Waste products can be used as a substrate for the production of value-added products (valorisation of the waste), which provides economic and environmental benefits for society.With Aspergillus being the main industrial enzyme producer and whey being the main waste product produced by the dairy industry, it seems fitting to bring the two together.The whey used in this study supports the growth of the A. niger strains without the requirement of additional nutrients.However, it is still advisable to investigate the optimisation of the whey as a cultivation medium or blend the whey with another waste stream to increase the carbohydrate content for increased expression when using the gpd promoter.To the best of our knowledge, this is the first report of the evaluation of the gpd promoter on lactose as the carbohydrate source.The use of constitutive promoters results in pure enzyme production due to the catabolite repression of the native hydrolases.However, the high levels of glucose required for the induction of the gpd promoter are problematic when other carbohydrate sources are used.Therefore, other promoters should be evaluated for use on agricultural waste that contains little to no glucose.
from the A. niger D15[GT]PyrG, D15[eg1]PyrG, D15[eg2]PyrG, and D15[aceg]PyrG strains were transferred to OBR-HEC agar plates containing 10% (w/v) glucose.The A. niger D15[eg1]PyrG, D15[eg2]PyrG, and D15[aceg]PyrG strains started to develop a clearing zone within 5 hr of germination, which is indicative of extracellular endoglucanase activity (Fig. 1 a).The use of the constitutive gpd promoter resulted in the expression of the genes in the presence of glucose.The A. niger D15[GT]PyrG only started to produce a zone after approximately 24 hr.The depletion of glucose in the medium surrounding the A. niger D15[GT]PyrG strain results in the relief of catabolite repression of the native endoglucanases, leading to delayed zone formation.The A. niger D15[GT]PyrG, D15[egI]PyrG, D15[eg2]PyrG, and D15[aceg]PyrG strains were cultivated in 2 ×MM containing 10% glucose for 48 hr.Supernatant samples were obtained and used directly for SDS-PAGE without further purification.The Eg1, Eg2, and AcEg recombinant enzymes are the prominent protein species in the supernatant samples obtained from the A. niger D15[egI]PyrG, D15[eg2]PyrG, and D15[aceg]PyrG cultures, respectively (Fig. 1 b), which supports the finding of Rose and van Zyl

Fig. 1 .
Fig. 1.Endoglucanase expression in the recombinant A. niger strains.The A. niger D15[GT]PyrG, D15[egI]PyrG, D15[eg2]PyrG, and D15[aceg]PyrG strains were cultivated on OBR-HEC agar plates for 24 hr at 30°C.(a) The clearing zone around the strains is indicative of endoglucanase activity.(b) SDS-PAGE analysis of the extracellular proteins produced by recombinant strains cultivated aerobically for 48 hr in 2 × MM containing 10% glucose.The molecular mass marker with the sizes (kDa) is depicted in the far-left lane.A. niger D15[GT]PyrG is the reference strain and serves as the negative control.

Fig. 2 .
Fig. 2. Endoglucanase activity of the recombinant A. niger strains on defined media.The extracellular endoglucanase activity (a, b) displayed by -•-A.niger D15[GT]PyrG, --D15[eg1]PyrG; --D15[eg2]PyrG and --D15[aceg]PyrG, as well as the residual carbohydrate source (c, d), was monitored for 7 days in 2 × MM medium containing glucose (a, c) and lactose (b, d), respectively.The values in this figure were obtained using three biological repeats with the error bars indicating the deviation between the biological repeats.A. niger D15[GT]PyrG is the reference strain and serves as the negative control.

Table 1 .
The Relevant Genotype and Sources of the Strains and Vector

Table 2 .
The Relevant Information of the Endoglucanases

Gene Enzyme Accession nr Predicted mass (kDa) unglycosylated pH optimum 1
2 aceg has a 100% DNA sequence homology to the A. niger egA .