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Lovastatin production by an oleaginous fungus, Aspergillus terreus KPR12 using sago processing wastewater (SWW)

Lovastatin production by an oleaginous fungus, Aspergillus terreus KPR12 using sago processing... Background fermentation (SSF), and submerged fermentation (SmF) Hypercholesterolemia is a well-studied metabolic disor- [14, 15]. For large-scale commercial production, SmF is der associated with cardiovascular morbidity and mor- used in batch and fed-batch modes [15]. A rich nutrient tality in human adults [1]. Statins are widely used as broth could be used for the production of lovastatin in cholesterol-lowering drugs that hinder the activity of the the SmF process. Although several agro-wastes are used critical catalyst, 3-hydroxy-3-methylglutaryl coenzyme A as substrates in the SSF process owing to their low cost, (HMG-CoA) reductase (mevalonate: NADP1 enzyme EC eco-safety, long-term availability, and easy downstream 1.1.1.34), which is involved in the endogenous biosynthe- processing [16], no research has been conducted on the sis of LDL cholesterol [2, 3]. Among statins, lovastatin is use of industrial wastewater. the first drug approved by the US Food and Drug Admin - India is one of the world’s largest producers of cassava, istration (FDA) in 1987 for the treatment of hypercho- which results in a wastewater discharge of about 40,000 lesterolemia [4]. Lovastatin has been reported to possess to 50,000 L and 15 to 30 tons of sludge per unit per day to anticancer properties, immunomodulatory function, produce flour and starch [17, 18]. Sago processing indus- and anti-inflammatory activity. In addition, it is known tries produce two types of wastewaters. The first type is to play a significant role in preventing neurological dis - released by the washing and peeling of cassava tubers orders and bone problems [5–7]. Lovastatin is a fungal and has low chemical oxygen demand (COD). The sec - secondary metabolite produced through the polyketide ond type is released during the extraction of starch; it has pathway. Several fungal genera such as Aspergillus, Peni- a high pollution load due to a high COD and biological cillium, Monascus, Paecilomyces, Trichoderma, Scopu- or biochemical oxygen demand (BOD); contains starch lariopsis, Doratomyces, Phoma, Pythium, Gymnoascus, up to 7% [19] and low concentrations of cytotoxic com- Hypomyces, and Pleurotus are known as lovastatin pro- pounds or growth inhibitors [20]. The reported  starch −1 ducers [8–12]. Of which, Monascus ruber and Aspergillus content of SWW was 4.82 g  L [21]. terreus are the foremost and targeted industrial produc- Applications of SWW include biogas [22, 23], hydrogen ers of lovastatin [4, 13]. [24, 25], microbial lipid and biodiesel production using Lovastatin is produced using different fermentation oleaginous yeast and fungi [21, 26–29]. Several oleagi- strategies, including surface fermentation, solid-state nous fungi and yeasts were isolated previously from this Sr inivasan et al. Microbial Cell Factories (2022) 21:22 Page 3 of 14 wastewater for biodiesel production with simultane- with sterile solution (0.05% Tween 80 and 0.9% NaCl), ous removal of pollutants [27–31]. Furthermore, certain washed twice with 0.1  M sterile phosphate buffer (pH 7 –1 hyper oleaginous fungi such as A. terreus KPR12 and A. 6), and adjusted to contain 10 spores mL . An aliquot caespitosus ASEF14 accumulate more than 20% of their of a spore suspension of each culture (1 mL) was inocu- dry weight lipid [26]. lated into 50  mL of potato dextrose broth (pH 6.5) in a We produced lovastatin with a high therapeutic value 250 mL Erlenmeyer flask and incubated at 30 °C under a using these known lovastatin-producing fungal strains static condition for 72 h until the exponential growth was and low-cost or zero-cost waste stream sago process- reached. ing wastewater (SWW) and simultaneously performed its decontamination. The produced lovastatin in SWW Fermentation conditions and lovastatin production was characterized and quantified using ultraviolet (UV) The two fungal strains grown under SmF conditions spectrometry, Fourier transform infrared (FTIR) spec- in SM and SWW were tested for lovastatin produc- troscopy, and high-performance liquid chromatography tion. About 100  mL of sterile SM and SWW were taken (HPLC). The lovastatin biogenesis of A. terreus KPR12 in in a 250  mL Erlenmeyer flask, and 10% of prepared liq - SWW was explained through a simple kinetic model. uid seed inoculum of A. caespitosus ASEF14 and A. ter- To the best of our knowledge, this is the first report on reus KPR12 was inoculated separately to the production lovastatin production using SWW. This study indicates media in the flasks. Before inoculation, the pH of both the prospect of exploiting cheaper, large, and underu- liquid substrates was adjusted to 6.5 using 0.1  N HCl tilized industrial effluent as a potential resource for the or 0.1  N NaOH. The initial starch content of SWW was production of lovastatin in addition to the sequestration −1 −1 4.82 g  L and adjusted to 10 g  L . The flasks were incu - of hazardous pollutants present in SWW. bated at 30  °C for 6  days under non-shaking conditions. The composition of the SM media (per L) was as follows: Materials and methods 10 g starch, 0.5 g ammonium sulfate, 7 g potassium dihy- Fungal strains and culture conditions drogen phosphate, 2.5  g disodium hydrogen phosphate, A. caespitosus ASEF14 and A. terreus KPR12 were iso- 1.5 g magnesium sulfate, 0.15 g ferric chloride, 0.15 g cal- lated, identified, characterized, and screened for ole - cium chloride, 0.02 g zinc sulfate, and 0.06 g manganese aginicity, amylase secretion, and cyanide degradation in sulfate. SWW [27, 28, 30]. In addition to biolipid production, these two fungal strains were screened for the production Biomass estimation of co-metabolite, lovastatin in synthetic medium (SM), as After fermentation, fungal mats in SM and SWW were well as SWW [26]. The GenBank accession numbers of separated by filtration through pre-weighed Whatman these strains are MF599090 and MF599091. The cultures grade 1 filter paper. The biomass obtained by filtration were maintained on potato dextrose agar (PDA) slants at was washed twice with distilled water and subjected to 4 °C. drying at 50 °C until it reached a constant weight. The dry weight of biomass was calculated by gravimetric analysis Physicochemical characterization of SWW [12]. The collection and characterization of SWW used in the present work have been reported in our previous work [28]. The initial starch concentration of SWW Extraction of intracellular lovastatin −1 was adjusted to 10  g  L , and other physicochemi- To measure the intracellular concentrations of statin, the cal parameters included pH 4.6, electrical conductiv- dry mycelium (0.5  g) was ruptured by ultrasonication −1 –1 ity (EC) 6.3  dS  m , salinity 4.86  g  L , total solids (TS) for 5 min (PCI Analytics; Mumbai, India). The sonicated –1 −1 4.57 g  L , total dissolved solids (TDS) 4.16  g  L , samples were adjusted to pH 3.0 using 2  NH PO and 3 4 −1 −1 nitrate 3.10 mg  L , ammonia 5.48  mg  L , phos- extracted with 10 mL of ethyl acetate in a shaker incuba- −1 phate 611.67 mg  L , biological oxygen demand (BOD) tor at 180 rpm at 30 °C for 2 h. The organic and aqueous −1 −1 5.04 g  L , chemical oxygen demand (COD) 70.67 g  L , phases of the filtrates were separated by cold centrifuga - −1 and cyanide 4.46 mg  L . tion (4  °C) at 6000  rpm for 10  min. The organic phases were collected, lactonized with 1% trifluoroacetic acid, Preparation of seed inoculant and concentrated under reduced pressure. The dried resi - The fungal strains of A. terreus KPR12 and A. caespito - due was dissolved in 1  mL acetonitrile, filtered through sus ASEF14 were grown on PDA incubated at 30  °C for a 0.45 µm filter, collected in clean brown glass vials, and 5 days, and stored under refrigeration at 4 °C. The conidi - stored at 4  °C for ultraviolet (UV) spectrophotometry, ospores from the above strains were harvested separately Fourier transform infrared (FTIR) spectroscopy, and Srinivasan et al. Microbial Cell Factories (2022) 21:22 Page 4 of 14 Kinetics of lovastatin production in SWW by A. terreus high-performance liquid chromatography (HPLC) analy- KPR12 sis [32]. A 250  mL Erlenmeyer flask containing approximately 100  mL of SWW was sterilized, inoculated with 10% of Extraction of extracellular lovastatin A. terreus KPR12 inoculum, and incubated at 30 °C. The To measure the extracellular concentrations of lovas- culture broth was harvested from day 1 until day 9 to tatin, the fermentation broths of SM and SWW were monitor the growth of strains and production of lovas- acidified to pH 3.0 by the addition of 10% 1  N HCl. The tatin. The cell dry weight was determined by gravimetric acidified broths were extracted with an equal volume of analysis. The amount of lovastatin was determined using ethyl acetate under shaking conditions (180 rpm) at 30 °C HPLC as mentioned in the analytical methods section. for 2 h. The organic and aqueous phases of filtrates were Residual starch in SWW was analyzed using the phe- separated by cold centrifugation (4  °C) at 6000  rpm for nol sulfuric acid method [35]. The following kinetic and 10  min. The organic phases were collected, lactonized, stoichiometric parameters used to describe the growth of concentrated, and analyzed as intracellular lovastatin strains and production of lovastatin by A. terreus KPR12 [32]. was determined. The substrate consumption rate (r) is expressed in days. Analytical methods r = (S −S )/�t, (1) i o UV spectrophotometric method The filtered fungal extracts were analyzed qualitatively where S is the initial concentration and S is the final i o for the presence of lovastatin using UV–visible spectro- concentration of substrate (s). photometer (SpectraMax i3x, Sunnyvale, California, US) The lovastatin yield coefficient (Y) was determined rel - [33]. The radiation source was a deuterium lamp emitting ative to the production of biomass (X) or the consump- a continuous UV spectrum between 210 and 360  nm. tion of total substrate (S) in the reaction. The lovastatin spectrum was recorded in the absorbance Y = (P −P )/(X −X ) LOV/X max i max i (2) mode at 247 nm and 258 nm, respectively. Pure lovasta- tin (Sigma Aldrich, St. Louis, Missouri, US) was used as a Y = (P −P )/(S −S ) standard for comparison. LOV/S max i i o (3) P is the maximum concentration of lovastatin, and max Fourier transform‑infrared spectroscopy P is the initial concentration of lovastatin in the above FTIR measurements of the samples were performed equation. µ is the maximum specific growth rate max using attenuated total reflectance (ATR) equipped with obtained from a plot of the specific biomass concentra - a deuterated triglycine sulfate (DTGS) detector (JASCO tion versus time. FT/IR-6300, Japan). The crude sample (10  µL) was directly placed on the surface of the diamond crystal. Samples were scanned using absorbance spectra at wave- Bioassay −1 −1 numbers 400 to 4000  cm at a resolution of 1  cm for The yeast growth inhibition bioassay was performed each interferogram. using the agar well diffusion method [36]. Candida tropicalis ASY2 (Acc no. MH011502) was used as a test organism. Cells of the C. tropicalis ASY2 were suspended High‑performance liquid chromatography in phosphate-buffered saline and spread onto the yeast The sample extracts were quantitatively analyzed for the extract peptone dextrose (YEPD) medium. Wells were presence of lovastatin using HPLC device, Shimadzu made using a sterile cork borer of 6  mm diameter. Fur- Nexera X2 (Shimadzu, Prominence HPLC, Kyoto, Japan) ther, 100 µL of intra- and extracellular extract of the fun- with a UV detector and a C18 column. Acetonitrile and gus KPR12 was loaded into separate wells. Ethyl acetate water (acidified with 1.1% phosphoric acid) in the ratio and the standard solution of lovastatin (10  mg dissolved of 70:30 v/v were used as mobile phase. The eluent flow in 100 mL of ethyl acetate) (Sigma Aldrich) were used as rate and the column temperature were maintained at negative and positive controls, respectively. The stand - –1 1 mL  min and 40  °C, respectively. The detection was ard was prepared according to the method of Friedrich performed at 238 nm wavelengths, with an injection vol- et  al. [37] with a slight modification, in which the lovas - ume of 20 µL. Lovastatin standard was prepared accord- tatin was suspended in ethyl acetate followed by sonica- ing to the manufacturer’s instructions [34]. Lovastatin tion and filtration. All plates were incubated at 30 °C for was identified in the sample by comparing the retention 16 to 24  h. A clear inhibition zone around the indicator times with the standards. Sr inivasan et al. Microbial Cell Factories (2022) 21:22 Page 5 of 14 organisms was observed, and the diameter of the inhibi- trifluoroacetic acid. This process can transform the acid tion zone is proportional to the concentration of lovasta- form of lovastatin into the lactone form. tin in samples. Generally, lovastatin exists in both open-ring β-hydroxy acid (active) and closed-ring β-lactone forms (inactive) (Fig.  1). The physicochemical and pharmaceutical prop - Characterization of decontaminated SWW erties of these two forms are different and interchange - The nutrient and toxicant removal efficiency of A. terreus able [6, 41]. In the broth culture media, the filamentous KPR12 in the SWW was studied along with lovastatin fungi secrete lovastatin mostly in its hydroxy acid form. kinetics. After fermentation, the spent SWW was filtered, However, the lactone form of industrial lovastatin makes and the physicochemical parameters were determined it a viable option for subsequent quantification analy - according to the standard method of water and wastewa- ses. Therefore, the reduction in pH and lactonization ter analysis [38]. The cyanide content in SWW was esti - converts the acid form to lactone for the quantification mated using the modified picric acid method [39]. of lovastatin [13, 41]. In the present investigation, the adopted techniques ensured the accurate quantification Statistical analysis of lovastatin in fermentation broth samples. Data were subjected to statistical analysis using the Microsoft Excel for Windows 2007 add-ins with XLSTAT Analysis of lovastatin in fungal crude extracts version 2010.5.05 [40], and all experiments were per- The lactonized lovastatin extracts from the samples formed in triplicate. Statistically significant differences were qualitatively analyzed using the UV–visible spec- between the means of groups and their interactions trophotometer and compared to the lovastatin absorp- were determined using one-way and two-way analysis tion spectrum (Fig.  2A). The lovastatin compound of variance (ANOVA) and Duncan’s multiple range test had a UV-absorbing peak at 247 nm (Fig. 2A). Such an (DMRT) at the 5% significance level. absorption band corresponds to the π–π transition due to the conjugated double bonds. As seen in Fig.  2A, Geolocation information intra- and extracellular fractions of A. terreus KPR12 The Tamil Nadu Agricultural University’s global position - from SM and SWW had the same UV absorption spec- ing system (GPS) coordinates are latitude: 11° 07′ 3.36ʺ N tra as the lovastatin standard (λ = 247, 258  nm). max and longitude: 76° 59′ 39.91ʺ E. The UV absorption spectra of intra- and extracellular fractions of A. caespitosus ASEF14 (Fig.  2B) revealed Results and discussion that the intracellular fraction exhibited an absorp- In the present study, we produced cholesterol-reduc- tion spectrum similar to that of the lovastatin stand- ing lovastatin using SWW under SmF using oleaginous ard. In contrast, the extracellular fraction of SM and fungal strains A. caespitosus ASEF14 and A. terreus SWW revealed a distinct pattern, such as stationary KPR12. Initially, the fermentation was performed for phase lines indicated the presence of non-lovastatin 6  days. After the extraction of lovastatin from the broth compounds. It has been reported three different maxi- of SM and SWW (extracellular) and fungal mycelium mum absorptions at 232, 238, and 247 nm of pure lov- (intracellular), it was acidified and lactonized with 1% astatin, suggesting its better identification from other Fig. 1 Closed-ring lactone (inactive) and open-ring hydroxy form (active) of lovastatin produced by filamentous fungi Srinivasan et al. Microbial Cell Factories (2022) 21:22 Page 6 of 14 λ = 247nm max λ = 258nm max Fig. 2 UV spectrophotometric analysis of intra- and extracellular fractions of A. terreus KPR12 (A) and A. caespitosus ASEF14 (B) grown in SM and SWW under SmF FTIR spectral analysis of lovastatin The FTIR spectra of fungal extracts were analyzed by interferometry using the pure lovastatin standard (Fig.  3). All spectra were recorded in the range of 400 −1 −1 to 4000  cm . A narrow band at 3400 to 3500  cm indicated the presence of non-hydrogen  bonded O–H stretches. Vibration often occurs to the left of this peak, suggesting the alcoholic/phenolic hydroxyl groups. The olefinic C–H stretching vibration band observed −1 at 2941.88  cm is a particular characteristic of chi- tin, a crucial component of the cell wall, and ergosterol −1 [43]. The peaks between 2900 and 3000  cm are ali- phatic and vinylic C–H stretching. Similarly, a band at −1 1447.31  cm represented two carbonyl ester groups for bending vibrations in methyl and methylene groups. The symmetric bending of the C–O–C ester and alkane C–H −1 −1 bonds at 1020.16  cm and between 680 and 610  cm , respectively, corresponds to specific functional peaks of lovastatin (Fig.  3). The C–H stretching absorptions −1 were observed below 3000  cm . Certain band struc- −1 tures observed between 3150 and 3000  cm represents Fig. 3 Functional groups corresponding to lovastatin identified unsaturation (C=C–H) and aromatic rings. The other in intracellular and extracellular fractions of A. terreus KPR12 and A. most important bands were aromatic ring vibrations at caespitosus ASEF14 in SM and SWW by FTIR spectral analysis and −1 around 1500 to 1600  cm , which usually appeared as a compared with the pure lovastatin standard pair of band structures in the lovastatin [44]. These FTIR spectra confirmed the presence of lovastatin in the fungal extracts and fractions (Fig. 3). compounds, which is due to the presence of dienes [6, 42]. The spectrophotometric analysis of lovastatin is easy, quick, eco-friendly, and less laborious than other Quantification of lovastatin analytical techniques. Based on these observations, Lovastatin produced by two different fungal strains the UV absorption spectrum of A. terreus KPR12 con- grown in SM and SWW was quantified using HPLC. The firmed the synthesis of lovastatin. retention time (5.124) of the first peak for both fungal extracts was similar to the standard lovastatin, and the Sr inivasan et al. Microbial Cell Factories (2022) 21:22 Page 7 of 14 mAU 238nm,4nm 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 min Retention time (min) mAU 238nm,4nm Lovastatin compounds eluted in samples at same retention time of pure lovastatin standard 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 min Retention time (min) mAU 238nm,4nm 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 min Retention time (min) Fig. 4 HPLC chromatograms of pure lovastatin standard (A), fermented extract of A. terreus KPR12 (B), and A. caespitosus ASEF14 (C) Absorbance (mAU) Absorbance (mAU) Absorbance (mAU) 0.301 0.685 0.768 1.261 1.439 1.439 1.452 1.739 1.846 1.833 1.863 2.220 2.242 2.250 2.388 2.598 2.608 2.784 2.933 2.974 3.026 3.292 3.957 4.147 4.139 4.203 4.345 5.037 5.091 5.124 6.005 6.304 6.412 6.883 6.959 7.834 7.848 Srinivasan et al. Microbial Cell Factories (2022) 21:22 Page 8 of 14 appearance of other peaks in the samples might be due lovastatin as an extracellular fraction. In this study, a 2.04 to the presence of impurities or unidentified compounds fold increase and 1.2 fold decrease was observed in extra- produced during the fermentation  process (Fig.  4A–C). cellular and intracellular concentrations of lovastatin by The concentrations of lovastatin in the intra- and extra - KPR12 in the SM compared to SWW, respectively. This cellular fractions of KPR12 grown in the SM were 117.93 lower secretion could be attributed to the mass transfer −1 and 883.28 mg  L , respectively; however, in SWW, resistance limits in SWW [46]. In addition, SWW con- −1 lovastatin yield were 142.23 and 429.98  mg  L , respec- tains hydrogen cyanide (HCN), which is generated dur- tively (Fig.  5A). Moreover, the lovastatin concentrations ing milling processes such as peeling, slicing, squeezing, in intra- and extracellular fractions of ASEF14 grown in and crushing cassava tubers. At high concentrations, −1 SM were 7.64 and 2.94 mg  L , and in SWW, these were cyanide becomes toxic to living organisms. Apart from −1 13.57 and 0.62 mg  L , respectively (Fig. 5B). The results its toxic nature, cyanide is well-known for its metabolic demonstrated that the fungal strain KPR12 was superior inhibitory effects [47, 48]. This may also affect the extra - to ASEF14 in terms of intra- and extracellular fractions, cellular secretion by fungi. Ultimately, the extraction of irrespective of SM and SWW. Therefore, a further experi - lovastatin from the intracellular portion of fungal cul- mental study focused only on the high lovastatin-yielding tures complicates the downstream processing due to fungus KPR12. the presence of structural analogs and intermediates The media conditions and compositions exert varying [49]. The production of lovastatin is highly influenced by effects on the production of fungal secondary metabolites slowly metabolized carbon sources (lactose, glycerol, and [45]. The synthesis of such secondary metabolites occurs fructose) compared to glucose [8, 50]. The pathway lead - at the end of the logarithmic (log) growth phase, in which ing to lovastatin synthesis using carbon is slower than the the essential nutrients are in low supply. The secretion of one that uses carbon for biomass production (glucose) accumulated metabolites into the surrounding medium because lovastatin is a product of secondary metabolism. is necessary. Fungal strains grown on a synthetic starch- u Th s, starch, a slowly metabolized carbon source present based substrate medium can secrete a high amount of in SWW and SM, could affect the production of biomass and lovastatin. Although the lovastatin content was lower in SWW than in SM; it was selected for kinetic analysis owing to its low-cost nature, high availability, economic factors, and environmental impact. All data were analyzed using a two-way ANOVA, and the results indicated significant differences (p < 0.05) in the localization pattern, fungal strains, growth media, and their interactions (Table 1). Kinetics of lovastatin production by A. terreus KPR12 in SWW The fermentation cycle was conducted for 9  days. The lovastatin content, dry cell weight, residual starch, and other physicochemical changes were measured periodi- cally in SWW (Fig.  6). The results revealed that lovasta - tin was not detected in the first 2  days of fermentation. Lovastatin, a product of secondary metabolism, is pro- duced at the end of the log or during  stationary growth phase of fungi. It cannot secrete or synthesize at the early growth stage of fungi [15]. The secretion of lovastatin in SWW started on the third day of fermentation using −1 −1 5.13 g  L starch and produced biomass of 1.82  g  L . The maximum extracellular concentration of lovastatin −1 −1 was 451 mg  L with a dry weight of 2.86 g  L on the 6th day of fermentation. The lovastatin synthesis pathway consumes carbon Fig. 5 Lovastatin content in intracellular and extracellular fractions more slowly than the biomass growth process. The syn - of A. terreus KPR12 (A) and A. caespitosus ASEF14 (B) grown in SM and thesis of building blocks for biomass synthesis is hin- SWW dered by nitrogen limitation, and the extra carbon is Sr inivasan et al. Microbial Cell Factories (2022) 21:22 Page 9 of 14 Table 1 Statistical parameters of two-factor ANOVA of lovastatin production as affected by cultivation medium, strains, and localization Effect SS DF MS F Prob F Sign –9 Growth medium 45,240.80 1 45,240.80 607.68 7.8354 × 10 ** –13 Strains 599,563.59 1 599,563.59 8053.44 2.65301 × 10 ** –12 Fraction 268,032.72 1 268,032.72 3600.26 6.61319 × 10 ** –9 Growth medium × strains 46,782.23 1 46,782.23 628.39 6.86322 × 10 ** –9 Growth medium × fraction 59,010.85 1 59,010.85 792.64 2.73655 × 10 ** –12 Strains × fraction 286,637.61 1 286,637.61 3850.17 5.05888 × 10 ** –9 Growth medium × strains × fraction 55,069.78 1 55,069.78 739.71 3.59881 × 10 ** Residual 595.58 8 74.45 Total 1,360,933.16 15 90,728.88 CV (%) 4.32 Growth medium—SM and SWW; Strains—A. terreus KPR12 and A. caespitosus ASEF14; Fraction—Intracellular and extracellular fraction; SS—sum of the squares; DF— degrees of freedom; MS—Mean sum of the squares; F—F test; Prob F—F probability; Sign.—significant at *p < 0.05; **p < 0.01 Table 2 Kinetic parameters of lovastatin production by A. terreus KPR12 grown in SWW Kinetic parameters Values −1 Lovastatin to biomass yield coefficient (Y ) 0.153 g g LOV/X −1 Lovastatin to starch yield coefficient (Y ) 0.043 g g LOV/S −1 −1 Maximum specific formation rate of lovastatin (Q ) 0.0011 g g h max −1 Biomass to starch yield coefficient (Y ) 0.278 g g X/S −1 Final biomass weight (X ) 3.20 g L Final −1 −1 higher productivity (0.093  mg  g  h ) was achieved at −1 lower growth rates (0.052  h ). Starvation due to a lack of essential nutrients (no residual starch content in SWW) Fig. 6 Growth kinetics and lovastatin production by A. terreus KPR12 in this study appeared to block fungal growth and lovas- in SWW tatin production. The kinetic parameters of A. terreus KPR12 grown in SWW are shown in Table  2. The adjusted initial starch channeled into lovastatin production. Polyketide syn- −1 content used for this kinetic study was 10  g  L . The thase (PKS: non-aketide synthase [LNKS] + diketide final biomass (X ) (on a dry weight basis) obtained synthase [LDKS]), a multifunctional enzyme complex, FINAL −1 by the fungi in SWW was 3.20 g  L . The lovastatin yield is involved in the biosynthesis of lovastatin [51]. This coefficients on biomass (Y ) and on the substrate enzyme followed the hyperbolic relationship when the LOV/X −1 (Y ) were found to be 0.153 and 0.043 g  g , respec- substrate concentration was low. In the current investi- LOV/S tively. Bizukojc and Ledakowicz [54] documented lovas- gation, a steep increase in the rate of reaction (lovastatin −1 tatin yield coefficients of 0.0065 and 0.0050  g  g by A. synthesis) with the availability of substrate was observed, terreus using lactose and glycerol in the culture, respec- i.e., starch (Fig.  6). When starch is unavailable, the tively. Lovastatin to biomass yield coefficient was 0.0052, enzyme catalytic site becomes vacant [52]. Thus, the rate and the initial lactose and glycerol contents were 10 and at which lovastatin synthesis drops dramatically (Fig. 6). −1 5 g  L , respectively. The results showed that a higher The results showed that the lovastatin yield would yield of lovastatin was obtained using pure sugar. be enhanced if essential nutrients were present in the In the current study, the biomass to starch yield medium. Such findings were consistent with Hajjaj et al. coefficient and the maximum specific lovastatin for - [53] and observed that the relatively low levels of lov- −1 −1 −1 mation rates (Q ) in SWW were 0.278  g  g and astatin produced (0.034  mg  g  h ) in cultures grow- max −1 −1 −1 0.0011 g  g  h , respectively. Pawlak and Bizukojć [55] ing at a high specific growth rate (0.070  h ), whereas Srinivasan et al. Microbial Cell Factories (2022) 21:22 Page 10 of 14 reported that biomass to lactose and biomass to glycerol terreus KSVL-SUCP-75. When compared to this value −1 yield coefficients by A. terreus were 0.55 g biomass/g lac -(360 mg  L ), the yield obtained from our research was tose and 0.55 g biomass/g glycerol in fed-batch fermenta- 1.25 fold higher. In other studies, strain improvement tion with an initial lactose and glycerol concentrations of techniques were adopted [60, 61] or supplements were −1 10 and 5 g  L , respectively. The maximum specific lovas - added to the culture medium [31, 62] to increase the −1 −1 tatin formation rate (Q ) was 0.00178 g  g  h . Based lovastatin titer. The results of the present study demon - max on the kinetic results obtained in the present study, strated that A. terreus KPR12 can be a potential lovas- SWW can be used as the growth substrate for the effec - tatin-producing strain, which effectively utilizes a waste tive production of various biomolecules. Moreover, the stream to produce therapeutic metabolites. synthesis of secondary molecules depends on strains and culture conditions. The lovastatin yield of A. terreus KPR12 under sub - Yeast growth inhibition bioassay merged fermentation in SWW was compared with Aspergillus species have proven to be a prolific source of other studies using diverse carbon sources (Table 3). In secondary metabolites with interesting biological activi- a study, Jaivel and Marimuthu [56] demonstrated that ties, including antibacterial activity [63, 64]. Lovastatin glucose was used as a sole carbon source to evaluate the is known for its antifungal activity; it inhibits the growth ability of 10 fungal strains from various natural sources of several fungal genera, including Saccharomyces cer- for the production of lovastatin and identified A. ter - evisiae, Candida spp., Aspergillus spp., and Cryptococ- reus (JPM3) as a better producer of lovastatin with a cus spp., by inhibiting HMG-CoA reductase that depletes −1 yield of 138.4  mg  L . In our study, the fungal strain ergosterol, the fungal counterpart of cholesterol [65–67]. A. terreus KPR12 produced nearly 3.3 fold higher yield Both ergosterol and cholesterol are important for cell than the previous  report [56]. Jia et  al. [57] used solu- viability and membrane fluidity, and they follow a simi - ble starch as a source of carbon and reported a 0.8 fold lar mechanism. The ethyl acetate extract of Aspergillus increase in the yield compared to the present study. contains several antimicrobial compounds such as hel- Pecyna and Bizukojc [58] analyzed specific lovastatin volic acid, monomethylsulochrin, ergosterol, terreic acid, yield during SmF using the lactose-to-glycerol ratio butyrolactone, tensyuic acids, emodin, kojic acid, fumiga- −1 and found a lovastatin yield of 161.8 mg  L . However, clavine, pseurotin, oleic acid, and n-hexadecanoic acid, in the current study indicates a 2.7 fold higher lovastatin addition to lovastatin [68–71]. output than the above studies. Sridevi and Charya [59] In the present study, a yeast growth inhibition bioas- isolated various strains of A. terreus from soil samples say was performed to verify the antifungal potential of and screened for the production of lovastatin using the intra- and extracellular fractions of A. terreus KPR12 agar plug assay method, and the maximum produc- against Candida tropicalis ASY2. The growth of C. trop - −1 tion of lovastatin (360  mg  L ) was obtained using A. icalis ASY2 was inhibited in both control and fungal Table 3 Lovastatin production by A. terreus KPR12 compared with other reports −1 A. terreus strain Carbon source Specific supplements/factorsYield (mg  L ) References ATCC 20542 Lactose, glycerol – 161.8 Bizukojc and Pecyna [80] JPM3 Glucose – 138.4 Jaivel and Marimuthu [56] Z15-7 Glycerol Mutant 916.7 Li et al. [61] LA414 Soluble starch Polyketide antibiotic 952.7 Jia et al. [62] NRRL 255 Glucose malt extract milk Reactor 920 Gupta et al. [81] powder GD13 Lactose Cyclic mutagenesis 1242 Kaur et al. [60] LA414 Soluble starch – 523.9 Jia et al. [57] MUCL 38669 Lactose, glucose Linoleic acid supplements 212.5 Sorrentino et al. [31] KPR12 Starch-based SWW – 450.79 (kinetic study) Present study Monascus strain MTCC 369 Glucose – 737 Ahmad et al. [82] MTCC 369 Glucose – 351 Sayyad et al. [83] Sr inivasan et al. Microbial Cell Factories (2022) 21:22 Page 11 of 14 effects of lovastatin and unidentified antimicrobial compounds present in the fungal extracts. Simultaneous decontamination of SWW In addition to lovastatin production, SWW can be treated and reused in the same industry for tuber wash- a b ing or irrigation, or recreation purposes. The compara - tive evaluation of raw and spent SWW with national standards is presented in Table  4. We detected a slight increase in pH from 6.5 to 7.1 of the treated SWW, sug- gesting that alkalinization may be due to the secretion of ammonia and its related compounds by the fungus during its growth in SWW [72] (Fig.  8). The very high −1 −1 EC (6.2 dS  m ) of raw SWW was reduced (4.1 dS  m ) after the fermentation due to the soluble salts metabo- lized by the growth of fungi. The salinity, TS, and TDS contents were also reduced in SWW (Table 4). The high organic matter content in SWW (COD and BOD) could be effectively fermented by oleaginous fungi by oxy - gen consumption, and the level reduced to 30.27 and Each well (6 mm diameter) loaded with 100 µl of −1 1.03 g  L , respectively. Our earlier report by Candida a- Lovastatin standard (Positive control) tropicalis ASY2 also supported the present investiga- b- Intracellular fraction tion [28]. Almost all nitrogen content in the SWW was c-Extracellularfraction d- Ethyl acetate(Negative control) used as a nitrogen source for fungal growth. A small −1 amount of phosphate (72.1 mg  L ) was available in the Fig. 7 Lovastatin extract of A. terreus KPR12 inhibiting the growth of treated SWW. The bound cyanide in the tapioca roots yeast observed by the zone of inhibition around a colony was hydrolyzed by linamerase during the starch extrac- tion process and left free cyanide in the waste stream. The microbe can grow and use cyanide-containing extracts, and clearing zones were observed (Fig. 7). The substrates through anaerobic metabolism, respiratory diameters of inhibition zones for both intra- and extra- chain metabolism, and their ability to detoxify cyanide cellular fractions in SWW were 12 and 14 mm, respec- by splitting the CN radical into carbon and nitrogen tively. The clear zone may be due to the combined [73, 74]. In the present study, the cyanide content was Table 4 Parametric comparison of raw and spent SWW with national standards Properties Raw SWW parameters Treated SWW parameters Different parameters of National effluent standards estimated in our study estimated in our study SWW reported in other for sago and starch industry studies pH 4.67 ± 0.03 8.1 ± 0.02 4.5–5.5 6.5–8.5 −1 EC (dS m ) 6.3 ± 0.04 4.11 ± 0.0 1.7–3.3 – −1 Salinity (g L ) 4.86 ± 0.09 2.15 ± 0.03 – – −1 Total solids (g L ) 4.57 ± 0.01 1.86 ± 0.01 0.8–12.45 0.1 −1 Total dissolved solids (g L ) 4.16 ± 0.02 1.32 ± 0.04 1.5–3.7 – −1 Starch (g L ) 10.00 ± 0.07 0.002 ± 0.0 4–7 – −1 BOD (g L ) 5.04 ± 0.08 1.03 ± 0.12 6.2–23.1 0.03 −1 COD (g L ) 70.22 ± 1.1 30.27 ± 1.2 11.08–19.08 0.25 −1 NO (mg L ) 3.10 ± 0.02 ND – 10 −1 NH (mg L ) 5.48 ± 0.05 ND – 50 −1 PO (mg L ) 611.67 ± 0.01 72.1 ± 0.04 – 5 −1 Cyanide (mg L ) 4.46 ± 0.02 1.54 ± 0.32 3.5–5.3 0.2 Adopted from Sujatha and Kumar [74]; Bhaskar and Prasada Rao [84], and Priya et al. [85] Srinivasan et al. Microbial Cell Factories (2022) 21:22 Page 12 of 14 Authors’ contributions SU: Conceptualized the idea and received research grants from DBT. SU, NS, and KT: Designed the experiments. NS and KT: Conducted the experiments. SU and NS: Analyzed the data and discussed the results. NS and KT: Wrote the manuscript. All authors helped in reviewing the manuscript. All authors read and approved the final manuscript. Funding We would like to thank DBT-GoI for the financial support for the project enti- tled “Biodiesel production: Sago processing industrial wastewater as feedstock for the microbial production of oil and derived co-products” granted to SU for financial support (File no. BT/PR8280/PBD/26/382/2013 dated 20.03.2015). Availability of data and materials All data generated or analyzed during this study are included in this published article. Fig. 8 Decontamination of SWW during lovastatin production by Declarations A. terreus KPR12. EC, electrical conductivity; TS, total solids; BOD, biochemical/biological oxygen demand; COD, chemical oxygen Ethics approval and consent to participate −1 −1 Not applicable. demand. * dS m ; units for all the other parameters are g L Consent for publication All authors have participated in the research study and manuscript −1 preparation. reduced to 1.54  mg  L . The results were supported by preliminary works of Kandasamy [75], who isolated Competing interests The authors hereby declare that they have no conflict of interest. bacterial isolates that could tolerate up to 5  mM cya- nide. However, further secondary treatment such as Author details anaerobic digestion [76, 77] and extended aeration [78, Biocatalysts Laboratory, Department of Agricultural Microbiology, Tamil Nadu Agricultural University, Coimbatore, Tamil Nadu 641 003, India. Depar t- 79] will further reduce the pollutant content of SWW ment of Renewable Energy Engineering, Agricultural Engineering College and would pave the way for the reuse of spent SWW for and Research Institute, Tamil Nadu Agricultural University, Coimbatore, Tamil various applications. Nadu 641 003, India. Present Address: Department of Agriculture Engineer- ing, Mahendra Engineering College, Namakkal, Tamil Nadu 637 503, India. Conclusion Received: 21 September 2021 Accepted: 25 January 2022 A. terreus KPR12 produced an optimal titer of −1 450.79 mg  L lovastatin in SWW without additional nutritional input or strain improvement techniques. These findings pave the way for the cost-effective and effi - References cient production of lovastatin by microbial fermentation, 1. Jeong S-M, Choi S, Kim K, Kim S-M, Lee G, Son JS, Yun J-M, Park SM. 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Lovastatin production by an oleaginous fungus, Aspergillus terreus KPR12 using sago processing wastewater (SWW)

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Abstract

Background fermentation (SSF), and submerged fermentation (SmF) Hypercholesterolemia is a well-studied metabolic disor- [14, 15]. For large-scale commercial production, SmF is der associated with cardiovascular morbidity and mor- used in batch and fed-batch modes [15]. A rich nutrient tality in human adults [1]. Statins are widely used as broth could be used for the production of lovastatin in cholesterol-lowering drugs that hinder the activity of the the SmF process. Although several agro-wastes are used critical catalyst, 3-hydroxy-3-methylglutaryl coenzyme A as substrates in the SSF process owing to their low cost, (HMG-CoA) reductase (mevalonate: NADP1 enzyme EC eco-safety, long-term availability, and easy downstream 1.1.1.34), which is involved in the endogenous biosynthe- processing [16], no research has been conducted on the sis of LDL cholesterol [2, 3]. Among statins, lovastatin is use of industrial wastewater. the first drug approved by the US Food and Drug Admin - India is one of the world’s largest producers of cassava, istration (FDA) in 1987 for the treatment of hypercho- which results in a wastewater discharge of about 40,000 lesterolemia [4]. Lovastatin has been reported to possess to 50,000 L and 15 to 30 tons of sludge per unit per day to anticancer properties, immunomodulatory function, produce flour and starch [17, 18]. Sago processing indus- and anti-inflammatory activity. In addition, it is known tries produce two types of wastewaters. The first type is to play a significant role in preventing neurological dis - released by the washing and peeling of cassava tubers orders and bone problems [5–7]. Lovastatin is a fungal and has low chemical oxygen demand (COD). The sec - secondary metabolite produced through the polyketide ond type is released during the extraction of starch; it has pathway. Several fungal genera such as Aspergillus, Peni- a high pollution load due to a high COD and biological cillium, Monascus, Paecilomyces, Trichoderma, Scopu- or biochemical oxygen demand (BOD); contains starch lariopsis, Doratomyces, Phoma, Pythium, Gymnoascus, up to 7% [19] and low concentrations of cytotoxic com- Hypomyces, and Pleurotus are known as lovastatin pro- pounds or growth inhibitors [20]. The reported  starch −1 ducers [8–12]. Of which, Monascus ruber and Aspergillus content of SWW was 4.82 g  L [21]. terreus are the foremost and targeted industrial produc- Applications of SWW include biogas [22, 23], hydrogen ers of lovastatin [4, 13]. [24, 25], microbial lipid and biodiesel production using Lovastatin is produced using different fermentation oleaginous yeast and fungi [21, 26–29]. Several oleagi- strategies, including surface fermentation, solid-state nous fungi and yeasts were isolated previously from this Sr inivasan et al. Microbial Cell Factories (2022) 21:22 Page 3 of 14 wastewater for biodiesel production with simultane- with sterile solution (0.05% Tween 80 and 0.9% NaCl), ous removal of pollutants [27–31]. Furthermore, certain washed twice with 0.1  M sterile phosphate buffer (pH 7 –1 hyper oleaginous fungi such as A. terreus KPR12 and A. 6), and adjusted to contain 10 spores mL . An aliquot caespitosus ASEF14 accumulate more than 20% of their of a spore suspension of each culture (1 mL) was inocu- dry weight lipid [26]. lated into 50  mL of potato dextrose broth (pH 6.5) in a We produced lovastatin with a high therapeutic value 250 mL Erlenmeyer flask and incubated at 30 °C under a using these known lovastatin-producing fungal strains static condition for 72 h until the exponential growth was and low-cost or zero-cost waste stream sago process- reached. ing wastewater (SWW) and simultaneously performed its decontamination. The produced lovastatin in SWW Fermentation conditions and lovastatin production was characterized and quantified using ultraviolet (UV) The two fungal strains grown under SmF conditions spectrometry, Fourier transform infrared (FTIR) spec- in SM and SWW were tested for lovastatin produc- troscopy, and high-performance liquid chromatography tion. About 100  mL of sterile SM and SWW were taken (HPLC). The lovastatin biogenesis of A. terreus KPR12 in in a 250  mL Erlenmeyer flask, and 10% of prepared liq - SWW was explained through a simple kinetic model. uid seed inoculum of A. caespitosus ASEF14 and A. ter- To the best of our knowledge, this is the first report on reus KPR12 was inoculated separately to the production lovastatin production using SWW. This study indicates media in the flasks. Before inoculation, the pH of both the prospect of exploiting cheaper, large, and underu- liquid substrates was adjusted to 6.5 using 0.1  N HCl tilized industrial effluent as a potential resource for the or 0.1  N NaOH. The initial starch content of SWW was production of lovastatin in addition to the sequestration −1 −1 4.82 g  L and adjusted to 10 g  L . The flasks were incu - of hazardous pollutants present in SWW. bated at 30  °C for 6  days under non-shaking conditions. The composition of the SM media (per L) was as follows: Materials and methods 10 g starch, 0.5 g ammonium sulfate, 7 g potassium dihy- Fungal strains and culture conditions drogen phosphate, 2.5  g disodium hydrogen phosphate, A. caespitosus ASEF14 and A. terreus KPR12 were iso- 1.5 g magnesium sulfate, 0.15 g ferric chloride, 0.15 g cal- lated, identified, characterized, and screened for ole - cium chloride, 0.02 g zinc sulfate, and 0.06 g manganese aginicity, amylase secretion, and cyanide degradation in sulfate. SWW [27, 28, 30]. In addition to biolipid production, these two fungal strains were screened for the production Biomass estimation of co-metabolite, lovastatin in synthetic medium (SM), as After fermentation, fungal mats in SM and SWW were well as SWW [26]. The GenBank accession numbers of separated by filtration through pre-weighed Whatman these strains are MF599090 and MF599091. The cultures grade 1 filter paper. The biomass obtained by filtration were maintained on potato dextrose agar (PDA) slants at was washed twice with distilled water and subjected to 4 °C. drying at 50 °C until it reached a constant weight. The dry weight of biomass was calculated by gravimetric analysis Physicochemical characterization of SWW [12]. The collection and characterization of SWW used in the present work have been reported in our previous work [28]. The initial starch concentration of SWW Extraction of intracellular lovastatin −1 was adjusted to 10  g  L , and other physicochemi- To measure the intracellular concentrations of statin, the cal parameters included pH 4.6, electrical conductiv- dry mycelium (0.5  g) was ruptured by ultrasonication −1 –1 ity (EC) 6.3  dS  m , salinity 4.86  g  L , total solids (TS) for 5 min (PCI Analytics; Mumbai, India). The sonicated –1 −1 4.57 g  L , total dissolved solids (TDS) 4.16  g  L , samples were adjusted to pH 3.0 using 2  NH PO and 3 4 −1 −1 nitrate 3.10 mg  L , ammonia 5.48  mg  L , phos- extracted with 10 mL of ethyl acetate in a shaker incuba- −1 phate 611.67 mg  L , biological oxygen demand (BOD) tor at 180 rpm at 30 °C for 2 h. The organic and aqueous −1 −1 5.04 g  L , chemical oxygen demand (COD) 70.67 g  L , phases of the filtrates were separated by cold centrifuga - −1 and cyanide 4.46 mg  L . tion (4  °C) at 6000  rpm for 10  min. The organic phases were collected, lactonized with 1% trifluoroacetic acid, Preparation of seed inoculant and concentrated under reduced pressure. The dried resi - The fungal strains of A. terreus KPR12 and A. caespito - due was dissolved in 1  mL acetonitrile, filtered through sus ASEF14 were grown on PDA incubated at 30  °C for a 0.45 µm filter, collected in clean brown glass vials, and 5 days, and stored under refrigeration at 4 °C. The conidi - stored at 4  °C for ultraviolet (UV) spectrophotometry, ospores from the above strains were harvested separately Fourier transform infrared (FTIR) spectroscopy, and Srinivasan et al. Microbial Cell Factories (2022) 21:22 Page 4 of 14 Kinetics of lovastatin production in SWW by A. terreus high-performance liquid chromatography (HPLC) analy- KPR12 sis [32]. A 250  mL Erlenmeyer flask containing approximately 100  mL of SWW was sterilized, inoculated with 10% of Extraction of extracellular lovastatin A. terreus KPR12 inoculum, and incubated at 30 °C. The To measure the extracellular concentrations of lovas- culture broth was harvested from day 1 until day 9 to tatin, the fermentation broths of SM and SWW were monitor the growth of strains and production of lovas- acidified to pH 3.0 by the addition of 10% 1  N HCl. The tatin. The cell dry weight was determined by gravimetric acidified broths were extracted with an equal volume of analysis. The amount of lovastatin was determined using ethyl acetate under shaking conditions (180 rpm) at 30 °C HPLC as mentioned in the analytical methods section. for 2 h. The organic and aqueous phases of filtrates were Residual starch in SWW was analyzed using the phe- separated by cold centrifugation (4  °C) at 6000  rpm for nol sulfuric acid method [35]. The following kinetic and 10  min. The organic phases were collected, lactonized, stoichiometric parameters used to describe the growth of concentrated, and analyzed as intracellular lovastatin strains and production of lovastatin by A. terreus KPR12 [32]. was determined. The substrate consumption rate (r) is expressed in days. Analytical methods r = (S −S )/�t, (1) i o UV spectrophotometric method The filtered fungal extracts were analyzed qualitatively where S is the initial concentration and S is the final i o for the presence of lovastatin using UV–visible spectro- concentration of substrate (s). photometer (SpectraMax i3x, Sunnyvale, California, US) The lovastatin yield coefficient (Y) was determined rel - [33]. The radiation source was a deuterium lamp emitting ative to the production of biomass (X) or the consump- a continuous UV spectrum between 210 and 360  nm. tion of total substrate (S) in the reaction. The lovastatin spectrum was recorded in the absorbance Y = (P −P )/(X −X ) LOV/X max i max i (2) mode at 247 nm and 258 nm, respectively. Pure lovasta- tin (Sigma Aldrich, St. Louis, Missouri, US) was used as a Y = (P −P )/(S −S ) standard for comparison. LOV/S max i i o (3) P is the maximum concentration of lovastatin, and max Fourier transform‑infrared spectroscopy P is the initial concentration of lovastatin in the above FTIR measurements of the samples were performed equation. µ is the maximum specific growth rate max using attenuated total reflectance (ATR) equipped with obtained from a plot of the specific biomass concentra - a deuterated triglycine sulfate (DTGS) detector (JASCO tion versus time. FT/IR-6300, Japan). The crude sample (10  µL) was directly placed on the surface of the diamond crystal. Samples were scanned using absorbance spectra at wave- Bioassay −1 −1 numbers 400 to 4000  cm at a resolution of 1  cm for The yeast growth inhibition bioassay was performed each interferogram. using the agar well diffusion method [36]. Candida tropicalis ASY2 (Acc no. MH011502) was used as a test organism. Cells of the C. tropicalis ASY2 were suspended High‑performance liquid chromatography in phosphate-buffered saline and spread onto the yeast The sample extracts were quantitatively analyzed for the extract peptone dextrose (YEPD) medium. Wells were presence of lovastatin using HPLC device, Shimadzu made using a sterile cork borer of 6  mm diameter. Fur- Nexera X2 (Shimadzu, Prominence HPLC, Kyoto, Japan) ther, 100 µL of intra- and extracellular extract of the fun- with a UV detector and a C18 column. Acetonitrile and gus KPR12 was loaded into separate wells. Ethyl acetate water (acidified with 1.1% phosphoric acid) in the ratio and the standard solution of lovastatin (10  mg dissolved of 70:30 v/v were used as mobile phase. The eluent flow in 100 mL of ethyl acetate) (Sigma Aldrich) were used as rate and the column temperature were maintained at negative and positive controls, respectively. The stand - –1 1 mL  min and 40  °C, respectively. The detection was ard was prepared according to the method of Friedrich performed at 238 nm wavelengths, with an injection vol- et  al. [37] with a slight modification, in which the lovas - ume of 20 µL. Lovastatin standard was prepared accord- tatin was suspended in ethyl acetate followed by sonica- ing to the manufacturer’s instructions [34]. Lovastatin tion and filtration. All plates were incubated at 30 °C for was identified in the sample by comparing the retention 16 to 24  h. A clear inhibition zone around the indicator times with the standards. Sr inivasan et al. Microbial Cell Factories (2022) 21:22 Page 5 of 14 organisms was observed, and the diameter of the inhibi- trifluoroacetic acid. This process can transform the acid tion zone is proportional to the concentration of lovasta- form of lovastatin into the lactone form. tin in samples. Generally, lovastatin exists in both open-ring β-hydroxy acid (active) and closed-ring β-lactone forms (inactive) (Fig.  1). The physicochemical and pharmaceutical prop - Characterization of decontaminated SWW erties of these two forms are different and interchange - The nutrient and toxicant removal efficiency of A. terreus able [6, 41]. In the broth culture media, the filamentous KPR12 in the SWW was studied along with lovastatin fungi secrete lovastatin mostly in its hydroxy acid form. kinetics. After fermentation, the spent SWW was filtered, However, the lactone form of industrial lovastatin makes and the physicochemical parameters were determined it a viable option for subsequent quantification analy - according to the standard method of water and wastewa- ses. Therefore, the reduction in pH and lactonization ter analysis [38]. The cyanide content in SWW was esti - converts the acid form to lactone for the quantification mated using the modified picric acid method [39]. of lovastatin [13, 41]. In the present investigation, the adopted techniques ensured the accurate quantification Statistical analysis of lovastatin in fermentation broth samples. Data were subjected to statistical analysis using the Microsoft Excel for Windows 2007 add-ins with XLSTAT Analysis of lovastatin in fungal crude extracts version 2010.5.05 [40], and all experiments were per- The lactonized lovastatin extracts from the samples formed in triplicate. Statistically significant differences were qualitatively analyzed using the UV–visible spec- between the means of groups and their interactions trophotometer and compared to the lovastatin absorp- were determined using one-way and two-way analysis tion spectrum (Fig.  2A). The lovastatin compound of variance (ANOVA) and Duncan’s multiple range test had a UV-absorbing peak at 247 nm (Fig. 2A). Such an (DMRT) at the 5% significance level. absorption band corresponds to the π–π transition due to the conjugated double bonds. As seen in Fig.  2A, Geolocation information intra- and extracellular fractions of A. terreus KPR12 The Tamil Nadu Agricultural University’s global position - from SM and SWW had the same UV absorption spec- ing system (GPS) coordinates are latitude: 11° 07′ 3.36ʺ N tra as the lovastatin standard (λ = 247, 258  nm). max and longitude: 76° 59′ 39.91ʺ E. The UV absorption spectra of intra- and extracellular fractions of A. caespitosus ASEF14 (Fig.  2B) revealed Results and discussion that the intracellular fraction exhibited an absorp- In the present study, we produced cholesterol-reduc- tion spectrum similar to that of the lovastatin stand- ing lovastatin using SWW under SmF using oleaginous ard. In contrast, the extracellular fraction of SM and fungal strains A. caespitosus ASEF14 and A. terreus SWW revealed a distinct pattern, such as stationary KPR12. Initially, the fermentation was performed for phase lines indicated the presence of non-lovastatin 6  days. After the extraction of lovastatin from the broth compounds. It has been reported three different maxi- of SM and SWW (extracellular) and fungal mycelium mum absorptions at 232, 238, and 247 nm of pure lov- (intracellular), it was acidified and lactonized with 1% astatin, suggesting its better identification from other Fig. 1 Closed-ring lactone (inactive) and open-ring hydroxy form (active) of lovastatin produced by filamentous fungi Srinivasan et al. Microbial Cell Factories (2022) 21:22 Page 6 of 14 λ = 247nm max λ = 258nm max Fig. 2 UV spectrophotometric analysis of intra- and extracellular fractions of A. terreus KPR12 (A) and A. caespitosus ASEF14 (B) grown in SM and SWW under SmF FTIR spectral analysis of lovastatin The FTIR spectra of fungal extracts were analyzed by interferometry using the pure lovastatin standard (Fig.  3). All spectra were recorded in the range of 400 −1 −1 to 4000  cm . A narrow band at 3400 to 3500  cm indicated the presence of non-hydrogen  bonded O–H stretches. Vibration often occurs to the left of this peak, suggesting the alcoholic/phenolic hydroxyl groups. The olefinic C–H stretching vibration band observed −1 at 2941.88  cm is a particular characteristic of chi- tin, a crucial component of the cell wall, and ergosterol −1 [43]. The peaks between 2900 and 3000  cm are ali- phatic and vinylic C–H stretching. Similarly, a band at −1 1447.31  cm represented two carbonyl ester groups for bending vibrations in methyl and methylene groups. The symmetric bending of the C–O–C ester and alkane C–H −1 −1 bonds at 1020.16  cm and between 680 and 610  cm , respectively, corresponds to specific functional peaks of lovastatin (Fig.  3). The C–H stretching absorptions −1 were observed below 3000  cm . Certain band struc- −1 tures observed between 3150 and 3000  cm represents Fig. 3 Functional groups corresponding to lovastatin identified unsaturation (C=C–H) and aromatic rings. The other in intracellular and extracellular fractions of A. terreus KPR12 and A. most important bands were aromatic ring vibrations at caespitosus ASEF14 in SM and SWW by FTIR spectral analysis and −1 around 1500 to 1600  cm , which usually appeared as a compared with the pure lovastatin standard pair of band structures in the lovastatin [44]. These FTIR spectra confirmed the presence of lovastatin in the fungal extracts and fractions (Fig. 3). compounds, which is due to the presence of dienes [6, 42]. The spectrophotometric analysis of lovastatin is easy, quick, eco-friendly, and less laborious than other Quantification of lovastatin analytical techniques. Based on these observations, Lovastatin produced by two different fungal strains the UV absorption spectrum of A. terreus KPR12 con- grown in SM and SWW was quantified using HPLC. The firmed the synthesis of lovastatin. retention time (5.124) of the first peak for both fungal extracts was similar to the standard lovastatin, and the Sr inivasan et al. Microbial Cell Factories (2022) 21:22 Page 7 of 14 mAU 238nm,4nm 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 min Retention time (min) mAU 238nm,4nm Lovastatin compounds eluted in samples at same retention time of pure lovastatin standard 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 min Retention time (min) mAU 238nm,4nm 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 min Retention time (min) Fig. 4 HPLC chromatograms of pure lovastatin standard (A), fermented extract of A. terreus KPR12 (B), and A. caespitosus ASEF14 (C) Absorbance (mAU) Absorbance (mAU) Absorbance (mAU) 0.301 0.685 0.768 1.261 1.439 1.439 1.452 1.739 1.846 1.833 1.863 2.220 2.242 2.250 2.388 2.598 2.608 2.784 2.933 2.974 3.026 3.292 3.957 4.147 4.139 4.203 4.345 5.037 5.091 5.124 6.005 6.304 6.412 6.883 6.959 7.834 7.848 Srinivasan et al. Microbial Cell Factories (2022) 21:22 Page 8 of 14 appearance of other peaks in the samples might be due lovastatin as an extracellular fraction. In this study, a 2.04 to the presence of impurities or unidentified compounds fold increase and 1.2 fold decrease was observed in extra- produced during the fermentation  process (Fig.  4A–C). cellular and intracellular concentrations of lovastatin by The concentrations of lovastatin in the intra- and extra - KPR12 in the SM compared to SWW, respectively. This cellular fractions of KPR12 grown in the SM were 117.93 lower secretion could be attributed to the mass transfer −1 and 883.28 mg  L , respectively; however, in SWW, resistance limits in SWW [46]. In addition, SWW con- −1 lovastatin yield were 142.23 and 429.98  mg  L , respec- tains hydrogen cyanide (HCN), which is generated dur- tively (Fig.  5A). Moreover, the lovastatin concentrations ing milling processes such as peeling, slicing, squeezing, in intra- and extracellular fractions of ASEF14 grown in and crushing cassava tubers. At high concentrations, −1 SM were 7.64 and 2.94 mg  L , and in SWW, these were cyanide becomes toxic to living organisms. Apart from −1 13.57 and 0.62 mg  L , respectively (Fig. 5B). The results its toxic nature, cyanide is well-known for its metabolic demonstrated that the fungal strain KPR12 was superior inhibitory effects [47, 48]. This may also affect the extra - to ASEF14 in terms of intra- and extracellular fractions, cellular secretion by fungi. Ultimately, the extraction of irrespective of SM and SWW. Therefore, a further experi - lovastatin from the intracellular portion of fungal cul- mental study focused only on the high lovastatin-yielding tures complicates the downstream processing due to fungus KPR12. the presence of structural analogs and intermediates The media conditions and compositions exert varying [49]. The production of lovastatin is highly influenced by effects on the production of fungal secondary metabolites slowly metabolized carbon sources (lactose, glycerol, and [45]. The synthesis of such secondary metabolites occurs fructose) compared to glucose [8, 50]. The pathway lead - at the end of the logarithmic (log) growth phase, in which ing to lovastatin synthesis using carbon is slower than the the essential nutrients are in low supply. The secretion of one that uses carbon for biomass production (glucose) accumulated metabolites into the surrounding medium because lovastatin is a product of secondary metabolism. is necessary. Fungal strains grown on a synthetic starch- u Th s, starch, a slowly metabolized carbon source present based substrate medium can secrete a high amount of in SWW and SM, could affect the production of biomass and lovastatin. Although the lovastatin content was lower in SWW than in SM; it was selected for kinetic analysis owing to its low-cost nature, high availability, economic factors, and environmental impact. All data were analyzed using a two-way ANOVA, and the results indicated significant differences (p < 0.05) in the localization pattern, fungal strains, growth media, and their interactions (Table 1). Kinetics of lovastatin production by A. terreus KPR12 in SWW The fermentation cycle was conducted for 9  days. The lovastatin content, dry cell weight, residual starch, and other physicochemical changes were measured periodi- cally in SWW (Fig.  6). The results revealed that lovasta - tin was not detected in the first 2  days of fermentation. Lovastatin, a product of secondary metabolism, is pro- duced at the end of the log or during  stationary growth phase of fungi. It cannot secrete or synthesize at the early growth stage of fungi [15]. The secretion of lovastatin in SWW started on the third day of fermentation using −1 −1 5.13 g  L starch and produced biomass of 1.82  g  L . The maximum extracellular concentration of lovastatin −1 −1 was 451 mg  L with a dry weight of 2.86 g  L on the 6th day of fermentation. The lovastatin synthesis pathway consumes carbon Fig. 5 Lovastatin content in intracellular and extracellular fractions more slowly than the biomass growth process. The syn - of A. terreus KPR12 (A) and A. caespitosus ASEF14 (B) grown in SM and thesis of building blocks for biomass synthesis is hin- SWW dered by nitrogen limitation, and the extra carbon is Sr inivasan et al. Microbial Cell Factories (2022) 21:22 Page 9 of 14 Table 1 Statistical parameters of two-factor ANOVA of lovastatin production as affected by cultivation medium, strains, and localization Effect SS DF MS F Prob F Sign –9 Growth medium 45,240.80 1 45,240.80 607.68 7.8354 × 10 ** –13 Strains 599,563.59 1 599,563.59 8053.44 2.65301 × 10 ** –12 Fraction 268,032.72 1 268,032.72 3600.26 6.61319 × 10 ** –9 Growth medium × strains 46,782.23 1 46,782.23 628.39 6.86322 × 10 ** –9 Growth medium × fraction 59,010.85 1 59,010.85 792.64 2.73655 × 10 ** –12 Strains × fraction 286,637.61 1 286,637.61 3850.17 5.05888 × 10 ** –9 Growth medium × strains × fraction 55,069.78 1 55,069.78 739.71 3.59881 × 10 ** Residual 595.58 8 74.45 Total 1,360,933.16 15 90,728.88 CV (%) 4.32 Growth medium—SM and SWW; Strains—A. terreus KPR12 and A. caespitosus ASEF14; Fraction—Intracellular and extracellular fraction; SS—sum of the squares; DF— degrees of freedom; MS—Mean sum of the squares; F—F test; Prob F—F probability; Sign.—significant at *p < 0.05; **p < 0.01 Table 2 Kinetic parameters of lovastatin production by A. terreus KPR12 grown in SWW Kinetic parameters Values −1 Lovastatin to biomass yield coefficient (Y ) 0.153 g g LOV/X −1 Lovastatin to starch yield coefficient (Y ) 0.043 g g LOV/S −1 −1 Maximum specific formation rate of lovastatin (Q ) 0.0011 g g h max −1 Biomass to starch yield coefficient (Y ) 0.278 g g X/S −1 Final biomass weight (X ) 3.20 g L Final −1 −1 higher productivity (0.093  mg  g  h ) was achieved at −1 lower growth rates (0.052  h ). Starvation due to a lack of essential nutrients (no residual starch content in SWW) Fig. 6 Growth kinetics and lovastatin production by A. terreus KPR12 in this study appeared to block fungal growth and lovas- in SWW tatin production. The kinetic parameters of A. terreus KPR12 grown in SWW are shown in Table  2. The adjusted initial starch channeled into lovastatin production. Polyketide syn- −1 content used for this kinetic study was 10  g  L . The thase (PKS: non-aketide synthase [LNKS] + diketide final biomass (X ) (on a dry weight basis) obtained synthase [LDKS]), a multifunctional enzyme complex, FINAL −1 by the fungi in SWW was 3.20 g  L . The lovastatin yield is involved in the biosynthesis of lovastatin [51]. This coefficients on biomass (Y ) and on the substrate enzyme followed the hyperbolic relationship when the LOV/X −1 (Y ) were found to be 0.153 and 0.043 g  g , respec- substrate concentration was low. In the current investi- LOV/S tively. Bizukojc and Ledakowicz [54] documented lovas- gation, a steep increase in the rate of reaction (lovastatin −1 tatin yield coefficients of 0.0065 and 0.0050  g  g by A. synthesis) with the availability of substrate was observed, terreus using lactose and glycerol in the culture, respec- i.e., starch (Fig.  6). When starch is unavailable, the tively. Lovastatin to biomass yield coefficient was 0.0052, enzyme catalytic site becomes vacant [52]. Thus, the rate and the initial lactose and glycerol contents were 10 and at which lovastatin synthesis drops dramatically (Fig. 6). −1 5 g  L , respectively. The results showed that a higher The results showed that the lovastatin yield would yield of lovastatin was obtained using pure sugar. be enhanced if essential nutrients were present in the In the current study, the biomass to starch yield medium. Such findings were consistent with Hajjaj et al. coefficient and the maximum specific lovastatin for - [53] and observed that the relatively low levels of lov- −1 −1 −1 mation rates (Q ) in SWW were 0.278  g  g and astatin produced (0.034  mg  g  h ) in cultures grow- max −1 −1 −1 0.0011 g  g  h , respectively. Pawlak and Bizukojć [55] ing at a high specific growth rate (0.070  h ), whereas Srinivasan et al. Microbial Cell Factories (2022) 21:22 Page 10 of 14 reported that biomass to lactose and biomass to glycerol terreus KSVL-SUCP-75. When compared to this value −1 yield coefficients by A. terreus were 0.55 g biomass/g lac -(360 mg  L ), the yield obtained from our research was tose and 0.55 g biomass/g glycerol in fed-batch fermenta- 1.25 fold higher. In other studies, strain improvement tion with an initial lactose and glycerol concentrations of techniques were adopted [60, 61] or supplements were −1 10 and 5 g  L , respectively. The maximum specific lovas - added to the culture medium [31, 62] to increase the −1 −1 tatin formation rate (Q ) was 0.00178 g  g  h . Based lovastatin titer. The results of the present study demon - max on the kinetic results obtained in the present study, strated that A. terreus KPR12 can be a potential lovas- SWW can be used as the growth substrate for the effec - tatin-producing strain, which effectively utilizes a waste tive production of various biomolecules. Moreover, the stream to produce therapeutic metabolites. synthesis of secondary molecules depends on strains and culture conditions. The lovastatin yield of A. terreus KPR12 under sub - Yeast growth inhibition bioassay merged fermentation in SWW was compared with Aspergillus species have proven to be a prolific source of other studies using diverse carbon sources (Table 3). In secondary metabolites with interesting biological activi- a study, Jaivel and Marimuthu [56] demonstrated that ties, including antibacterial activity [63, 64]. Lovastatin glucose was used as a sole carbon source to evaluate the is known for its antifungal activity; it inhibits the growth ability of 10 fungal strains from various natural sources of several fungal genera, including Saccharomyces cer- for the production of lovastatin and identified A. ter - evisiae, Candida spp., Aspergillus spp., and Cryptococ- reus (JPM3) as a better producer of lovastatin with a cus spp., by inhibiting HMG-CoA reductase that depletes −1 yield of 138.4  mg  L . In our study, the fungal strain ergosterol, the fungal counterpart of cholesterol [65–67]. A. terreus KPR12 produced nearly 3.3 fold higher yield Both ergosterol and cholesterol are important for cell than the previous  report [56]. Jia et  al. [57] used solu- viability and membrane fluidity, and they follow a simi - ble starch as a source of carbon and reported a 0.8 fold lar mechanism. The ethyl acetate extract of Aspergillus increase in the yield compared to the present study. contains several antimicrobial compounds such as hel- Pecyna and Bizukojc [58] analyzed specific lovastatin volic acid, monomethylsulochrin, ergosterol, terreic acid, yield during SmF using the lactose-to-glycerol ratio butyrolactone, tensyuic acids, emodin, kojic acid, fumiga- −1 and found a lovastatin yield of 161.8 mg  L . However, clavine, pseurotin, oleic acid, and n-hexadecanoic acid, in the current study indicates a 2.7 fold higher lovastatin addition to lovastatin [68–71]. output than the above studies. Sridevi and Charya [59] In the present study, a yeast growth inhibition bioas- isolated various strains of A. terreus from soil samples say was performed to verify the antifungal potential of and screened for the production of lovastatin using the intra- and extracellular fractions of A. terreus KPR12 agar plug assay method, and the maximum produc- against Candida tropicalis ASY2. The growth of C. trop - −1 tion of lovastatin (360  mg  L ) was obtained using A. icalis ASY2 was inhibited in both control and fungal Table 3 Lovastatin production by A. terreus KPR12 compared with other reports −1 A. terreus strain Carbon source Specific supplements/factorsYield (mg  L ) References ATCC 20542 Lactose, glycerol – 161.8 Bizukojc and Pecyna [80] JPM3 Glucose – 138.4 Jaivel and Marimuthu [56] Z15-7 Glycerol Mutant 916.7 Li et al. [61] LA414 Soluble starch Polyketide antibiotic 952.7 Jia et al. [62] NRRL 255 Glucose malt extract milk Reactor 920 Gupta et al. [81] powder GD13 Lactose Cyclic mutagenesis 1242 Kaur et al. [60] LA414 Soluble starch – 523.9 Jia et al. [57] MUCL 38669 Lactose, glucose Linoleic acid supplements 212.5 Sorrentino et al. [31] KPR12 Starch-based SWW – 450.79 (kinetic study) Present study Monascus strain MTCC 369 Glucose – 737 Ahmad et al. [82] MTCC 369 Glucose – 351 Sayyad et al. [83] Sr inivasan et al. Microbial Cell Factories (2022) 21:22 Page 11 of 14 effects of lovastatin and unidentified antimicrobial compounds present in the fungal extracts. Simultaneous decontamination of SWW In addition to lovastatin production, SWW can be treated and reused in the same industry for tuber wash- a b ing or irrigation, or recreation purposes. The compara - tive evaluation of raw and spent SWW with national standards is presented in Table  4. We detected a slight increase in pH from 6.5 to 7.1 of the treated SWW, sug- gesting that alkalinization may be due to the secretion of ammonia and its related compounds by the fungus during its growth in SWW [72] (Fig.  8). The very high −1 −1 EC (6.2 dS  m ) of raw SWW was reduced (4.1 dS  m ) after the fermentation due to the soluble salts metabo- lized by the growth of fungi. The salinity, TS, and TDS contents were also reduced in SWW (Table 4). The high organic matter content in SWW (COD and BOD) could be effectively fermented by oleaginous fungi by oxy - gen consumption, and the level reduced to 30.27 and Each well (6 mm diameter) loaded with 100 µl of −1 1.03 g  L , respectively. Our earlier report by Candida a- Lovastatin standard (Positive control) tropicalis ASY2 also supported the present investiga- b- Intracellular fraction tion [28]. Almost all nitrogen content in the SWW was c-Extracellularfraction d- Ethyl acetate(Negative control) used as a nitrogen source for fungal growth. A small −1 amount of phosphate (72.1 mg  L ) was available in the Fig. 7 Lovastatin extract of A. terreus KPR12 inhibiting the growth of treated SWW. The bound cyanide in the tapioca roots yeast observed by the zone of inhibition around a colony was hydrolyzed by linamerase during the starch extrac- tion process and left free cyanide in the waste stream. The microbe can grow and use cyanide-containing extracts, and clearing zones were observed (Fig. 7). The substrates through anaerobic metabolism, respiratory diameters of inhibition zones for both intra- and extra- chain metabolism, and their ability to detoxify cyanide cellular fractions in SWW were 12 and 14 mm, respec- by splitting the CN radical into carbon and nitrogen tively. The clear zone may be due to the combined [73, 74]. In the present study, the cyanide content was Table 4 Parametric comparison of raw and spent SWW with national standards Properties Raw SWW parameters Treated SWW parameters Different parameters of National effluent standards estimated in our study estimated in our study SWW reported in other for sago and starch industry studies pH 4.67 ± 0.03 8.1 ± 0.02 4.5–5.5 6.5–8.5 −1 EC (dS m ) 6.3 ± 0.04 4.11 ± 0.0 1.7–3.3 – −1 Salinity (g L ) 4.86 ± 0.09 2.15 ± 0.03 – – −1 Total solids (g L ) 4.57 ± 0.01 1.86 ± 0.01 0.8–12.45 0.1 −1 Total dissolved solids (g L ) 4.16 ± 0.02 1.32 ± 0.04 1.5–3.7 – −1 Starch (g L ) 10.00 ± 0.07 0.002 ± 0.0 4–7 – −1 BOD (g L ) 5.04 ± 0.08 1.03 ± 0.12 6.2–23.1 0.03 −1 COD (g L ) 70.22 ± 1.1 30.27 ± 1.2 11.08–19.08 0.25 −1 NO (mg L ) 3.10 ± 0.02 ND – 10 −1 NH (mg L ) 5.48 ± 0.05 ND – 50 −1 PO (mg L ) 611.67 ± 0.01 72.1 ± 0.04 – 5 −1 Cyanide (mg L ) 4.46 ± 0.02 1.54 ± 0.32 3.5–5.3 0.2 Adopted from Sujatha and Kumar [74]; Bhaskar and Prasada Rao [84], and Priya et al. [85] Srinivasan et al. Microbial Cell Factories (2022) 21:22 Page 12 of 14 Authors’ contributions SU: Conceptualized the idea and received research grants from DBT. SU, NS, and KT: Designed the experiments. NS and KT: Conducted the experiments. SU and NS: Analyzed the data and discussed the results. NS and KT: Wrote the manuscript. All authors helped in reviewing the manuscript. All authors read and approved the final manuscript. Funding We would like to thank DBT-GoI for the financial support for the project enti- tled “Biodiesel production: Sago processing industrial wastewater as feedstock for the microbial production of oil and derived co-products” granted to SU for financial support (File no. BT/PR8280/PBD/26/382/2013 dated 20.03.2015). Availability of data and materials All data generated or analyzed during this study are included in this published article. Fig. 8 Decontamination of SWW during lovastatin production by Declarations A. terreus KPR12. EC, electrical conductivity; TS, total solids; BOD, biochemical/biological oxygen demand; COD, chemical oxygen Ethics approval and consent to participate −1 −1 Not applicable. demand. * dS m ; units for all the other parameters are g L Consent for publication All authors have participated in the research study and manuscript −1 preparation. reduced to 1.54  mg  L . The results were supported by preliminary works of Kandasamy [75], who isolated Competing interests The authors hereby declare that they have no conflict of interest. bacterial isolates that could tolerate up to 5  mM cya- nide. However, further secondary treatment such as Author details anaerobic digestion [76, 77] and extended aeration [78, Biocatalysts Laboratory, Department of Agricultural Microbiology, Tamil Nadu Agricultural University, Coimbatore, Tamil Nadu 641 003, India. Depar t- 79] will further reduce the pollutant content of SWW ment of Renewable Energy Engineering, Agricultural Engineering College and would pave the way for the reuse of spent SWW for and Research Institute, Tamil Nadu Agricultural University, Coimbatore, Tamil various applications. Nadu 641 003, India. Present Address: Department of Agriculture Engineer- ing, Mahendra Engineering College, Namakkal, Tamil Nadu 637 503, India. Conclusion Received: 21 September 2021 Accepted: 25 January 2022 A. terreus KPR12 produced an optimal titer of −1 450.79 mg  L lovastatin in SWW without additional nutritional input or strain improvement techniques. These findings pave the way for the cost-effective and effi - References cient production of lovastatin by microbial fermentation, 1. Jeong S-M, Choi S, Kim K, Kim S-M, Lee G, Son JS, Yun J-M, Park SM. 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Colin X, Farinet JL, Rojas O, Alazard D. Anaerobic treatment of cassava Re Read ady y to to submit y submit your our re researc search h ? Choose BMC and benefit fr ? Choose BMC and benefit from om: : starch extraction wastewater using a horizontal flow filter with bamboo as support. Bioresour Technol. 2007;98:1602–7. fast, convenient online submission 77. Priya M, Meenambal T, Balasubramanian N, Perumal B. Comparative study thorough peer review by experienced researchers in your field of treatment of sago wastewater using HUASB reactor in the presence and absence of effective microorganisms. Procedia Earth Planet Sci. rapid publication on acceptance 2015;11:483–90. support for research data, including large and complex data types 78. Kandasamy S, Dananjeyan B, Krishnamurthy K. Potential of continu- • gold Open Access which fosters wider collaboration and increased citations ous and intermittent aeration for sago wastewater treatment. Ecoscan. 2013;7:129–32. maximum visibility for your research: over 100M website views per year 79. Wahi AR, Hamdan M, King W, Kopli B. The potential of extended aeration system for sago effluent treatment. Am J Appl Sci. 2010;7:616–9. At BMC, research is always in progress. 80. Bizukojc M, Pecyna M. Lovastatin and (+)-geodin formation by Aspergil- Learn more biomedcentral.com/submissions lus terreus ATCC 20542 in a batch culture with the simultaneous use of lactose and glycerol as carbon sources. Eng Life Sci. 2011;11:272–82.

Journal

Microbial Cell Factories – Springer Journals

Published: Feb 14, 2022

Keywords: Aspergillus; Lovastatin; Sago wastewater; Bioassay

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