Research ArticlePretreatment Strategies to Improve Crude GlycerolUtilisation and Metabolite Production by Aspergillus terreus
MuhamadHafiz Abd Rahim ,1 HananHasan ,1 Elicia Jitming Lim,2 Phebe K. Samrani,3
and Ali Abbas3
1Faculty of Food Science and Technology, University Putra Malaysia, 43400 Serdang, Selangor, Malaysia2School of Medical Sciences, e University of Sydney, Sydney 2006, Australia3School of Chemical and Biomolecular Engineering, e University of Sydney, Sydney 2006, Australia
Correspondence should be addressed to Muhamad Hafiz Abd Rahim; [email protected]
Received 5 November 2018; Revised 10 January 2019; Accepted 19 February 2019; Published 1 April 2019
Crude glycerol (CG) can be used as a substrate for microbial bioconversion. However, due to presence of many impurities, manymicroorganisms are unable to utilise this substrate efficiently. +e present study is trying to improve CG using as the feedstock ofAspergillus terreus for the production of lovastatin, (+)-geodin, and sulochrin. +e CG was pretreated chemically (solvents) andphysically (activated carbon (AC) and water softener (WS)) to separate most of the impurities from the CG. For solventpretreatments, petroleum ether (PE) produced the largest increase of lovastatin (92.8%) when compared to positive control andpure glycerol (PG) and up to 820% when compared to negative control (CG). In contrast, diethyl ether (DE) produced the largestincrease in (+)-geodin at 80.81% (versus CG) and 176.23% (versus PG). +e largest increase in toluene (Tol) was observed insulochrin production, at 67.22% (versus CG) and 183.85% (versus PG). For physical pretreatments, the pattern of metaboliteproduction in AC (lovastatin: 20.65mg/L, (+)-geodin: 7.42mg/L, sulochrin: 11.74mg/L) resembled PG (lovastatin: 21.8mg/L,(+)-geodin: 8.60mg/L, sulochrin: 8.18mg/L), while WS (lovastatin: 11.25mg/L, (+)-geodin: 15.38mg/L, sulochrin: 16.85mg/L)resembled CG (lovastatin: 7.1mg/L, (+)-geodin: 17.10mg/L, sulochrin: 14.78mg/L) at day 6 of fermentation.+ese results indicatethat solvent pretreatments on CG are excellent for metabolites production in A. terreus, depending on the solvents used. Incontrast, physical pretreatments are only feasible for (+)-geodin and sulochrin production. +erefore, different strategies can beemployed to manipulate the A. terreus bioconversion using improved CG by using a few simple pretreatment strategies.
1. Introduction
CG waste was previously considered a valuable by-productderived from the biodiesel industry. However, the rapidgrowth of the biodiesel industry resulted in an over-production of CG, leading to its significant devaluation. CGwaste has become a burden, especially for small biodieselproducers, as improper disposal is harmful to the envi-ronment, and purification processes are not cost-effective[1]. +e bioconversion of CG is particularly challenging asbiodiesel-derived CG waste contains a significant amount ofimpurities. CG contents vary widely with the biodieselmanufacturer, ranging from 38% to 96% glycerol; with theremaining normally comprised of water, methanol (MeOH),free fatty acids, and salts [2]. MeOH and fatty acids are
formed during the transesterification process, while saltoriginating from the catalyst is used in the biodiesel pro-duction process. High contents of impurities in CG mayinhibit the production of metabolites in most microor-ganisms [3–6] and hence can reduce the economic potentialof chemical processes using CG as feedstock.
+emost commonmethod of crude glycerol purificationincludes laborious process of distillation, which involvesspecific temperature, pressure, and pH control. Further-more, such a process is employed to produce industriallypure glycerol, which is not required in microbial bio-conversion as microorganisms may be able to toleratecertain amount of impurities. Simple techniques, such as theaddition of solvent, water softener, and activated charcoal[7], may reduce the amount of impurities and enhance the
HindawiInternational Journal of Chemical EngineeringVolume 2019, Article ID 2504540, 6 pageshttps://doi.org/10.1155/2019/2504540
microbial conversion significantly. Solvent extraction iscapable of forming two heterogonous layers of impuritiesand glycerol, while water softener removes hard chemicalsand activated carbon is efficient in capturing ash andMONGof crude glycerol. Some microorganisms displayed excellentuse of improved CG, including Schizochytrium limacinum[5], yeast Yarrowia lipolytica [8], and bacteria Clostridiumbutyricum [9, 10].
Aspergillus terreus ATCC 20542 is a filamentous fungusthat can be found in soil. It is a well-known strain that canproduce lovastatin, a cholesterol-lowering drug. +e modeof action of lovastatin involved the inhibition of 3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) reductase, a rate-limiting enzyme involved in cholesterol biosynthesis inthe liver. Its lesser known metabolites, namely, (+)-geodinand sulochrin, can also be pharmaceutically important.(+)-Geodin has been shown to inhibit the plasminogenactivator inhibitor (PAI-1), a molecule that is important infibrinolysis mechanism and for the stimulation of glucoseuptake in rat cells [11]. Likewise, sulochrin can inhibit theactivation of human immune cell (eosinophil), endothelialcells, and certain cytokine release (IL-8 and LTC4) [12, 13].
A. terreus is of particular interest to be used with CG,mainly due to their fungus characteristic which is known fortheir ability to withstand different contaminants present inCG. +e cultivation of A. terreus in CG significantly reducesits abilities to produce certain metabolites, although thegrowth of the fungus was uninterrupted [14]. It was foundthat the contaminants in CG, namely, methanol, sodiumchloride, and fatty acids can lead to different responses of A.terreus. For lovastatin, certain fatty acids are inhibitory to A.terreus, while methanol and sodium chloride are stimulatoryup to a certain concentration [14]. In contrast, (+)-geodinwas inhibited in the presence of methanol and palmitic acid[14].
+is current study reports the efficiency of pretreated CGon the growth and metabolite production of A. terreusATCC 20542. +ree strategies of pretreatments are applied,by using nonpolar solvents, activated carbon, and watersoftener pillow. +e growth, in the form of dry cell weight,and the production of metabolites were compared againstthe CG (negative control) and PG (positive control). +isstudy is a continuation from the previous investigation byAbd Rahim et al. [14].+is is the first study involving the useof improved CG for the production of lovastatin, (+)-geodin,and sulochrin from A. terreus.
2. Materials and Methods
2.1. Description of Crude Glycerol Used in the Study. CGsamples were donated by Biodiesel Producers Limited(Melbourne, VIC). +e CG is a waste by-product derivedfrom a biodiesel process that uses tallow and uses cooking oilas feed. +e CG exhibits dark yellow colour and has a pH of7. It was stored in a sealed aluminium container at roomtemperature. Depending on the type of experiment con-ducted, the CG was either filtered to remove the undissolvedcontents, autoclaved to removeMeOH, or vigorously shakento evenly distribute all the contents.
+e pretreatment using solvent washing was performedto dissolve hydrocarbons present in CG. Different nonpolarsolvents (hexane, heptane, octane, and petroleum ether)were used to wash the CG at room temperature using 1 :1volume ratio, followed by shaking at 200 rpm. After threehours of mixing, it was centrifuged at 3000g for 2minutes toseparate the mixture into two distinct phases, namely, theupper phase of solvent and the lower aqueous phase ofglycerol containing residual impurities. +e lower phase wassubjected to washing again with fresh solvent, and theprocess was repeated to collect the final treated CG.
+e physical pretreatments involved the use of activatedcarbon and water softener pillow purchased from MARSFishcare North America (PA, USA). +e CG used in thispretreatment was diluted with the same volume of deionisedwater to reduce its viscosity. Around 20% (wt/v) of activatedcarbon or water softener pillow was added directly to thediluted samples, following the incubation at 200 rpm atroom temperature for 3 hours. +e samples were latercentrifuged at 3000 g for 15minutes, and the deposit (ac-tivated carbon or resins of water softener pillow) was re-moved to obtain the CG.
2.2. Culture Conditions. +e fungal strain used in this studyis Aspergillus terreus ATCC 20542. +e cultivation of A.terreus into the shake flask was done as previously described[15]. In short, the freeze-dried fungus was reactivated usingsterile deionised water and maintained on potato dextroseagar at 30°C for 7 days. +e number of spores used was 107spores/mL (counted using the haemocytometer) and in-oculated into the 125mL Erlenmeyer flasks and shaken at185± 5 rpm and a temperature of 30± 1°C. +e basic basalsalt medium was used for all experiments (0.4 g/L KH2PO4,0.2 g/L MgSO4·7H2O, 0.4 g/L NaCl, and 0.001 g/LZnSO4·7H2O). Carbon sources (pretreated CG, CG, andPG) were adjusted to 30 g/L, and yeast extract was used as thesole nitrogen source at 4.0 g/L. +e preculture was preparedin a similar basal salt medium, but with glycerol carbonsource (10 g/L) and yeast extract nitrogen source at 8 g/Lbetween 24 and 30 hours. +e fungus was used for culti-vation when the diameter of the fungal pellet reached1.5± 0.5mm, measured using a digital calliper. +e day ofthe preculture is considered as Day 1.
2.3. Analytical Methods. +e quantification of lovastatin,(+)-geodin, and sulochrin was carried out by high-performance liquid chromatography (HPLC), Agilent1200, using a C-18 column and UV detector at a wavelengthof 238 nm, with reference wavelength of 360 nm, as pre-viously described [15]. +e preparation of lovastatin stan-dard involved a treatment of methanol and sodiumhydroxide solution [14]. +e (+)-geodin and sulochrinstandards were purchased from Sapphire Bioscience (Syd-ney, Australia). Sulochrin, (+)-geodin, and lovastatinappeared at a retention time of around 4, 7, and 10minutes,respectively, in the HPLC chromatogram.+e quantificationof glycerol in the samples was performed using the glycerolcalorimetric detection reagent purchased from Sigma-
2 International Journal of Chemical Engineering
Aldrich (Sydney, Australia). +is reagent measures glycerolby using enzymatic reaction. As the detection is very sen-sitive, the acceptable range of glycerol concentration isaround 20mg/L. +e plate was read using a microplatereader (Biorad model 680) at 450 nm.+e biomass yield wasdetermined gravimetrically. Fungus biomass was recoveredby filtration using No. 2 Whatman filter paper and washedtwice with distilled water, followed by drying at 80°C for24 hours or until a constant weight is achieved.
2.4. Statistical Analysis. All experiments were conducted atleast using triplicates. Data obtained for metabolite pro-duction involving lovastatin, (+)-geodin, and sulochrin wereanalysed using one-way ANOVA with the Tukey post hoctest. +e results are considered significant when p< 0.05. Allstatistical analyses were performed using Graphpad Prism,version 6.01. For the plotting of the graph, 95% confidenceinterval was used for the error bar.
3. Results and Discussion
3.1. Comparison between theGrowth, Substrate Consumption,and Metabolite Production Using Crude, Pure, and NonpolarSolvent Pretreated Glycerol. Solvent washing is a simplemethod of separating a compound based on the differentsolubilities of two immiscible liquids. +is method is effi-cient in reducing certain fatty acids in the solution [16]. Inour investigation, we opted to use petroleum ether (PE),diethyl ether (DE), and toluene (Tol) as our solvent of in-terest. Rehman et al. were among the first team to use thistechnique on CG, achieving a good microbial bioconversion[17].
+e growth of A. terreus in PG exhibited better growththan in CG. +e use of pretreated CG produced goodbiomass, which was comparable or exceeded that observedwith PG (Table 1). DE showed the highest biomass pro-duction (11.28 g/L), followed by PE (10.60 g/L), PG (10.07 g/L), Tol (9.80 g/L), and CG (9.75 g/L). +e high production ofbiomass was likely from the residual fatty acids that are stillpresent in pretreated CG, given that the solvent washingcannot entirely remove them from the solution [16]. +eincrease in biomass in the presence of fatty acids in CG hasalso been shown previously [14]. Although in theory, CGshould contain the highest amount of fatty acids, the bio-mass produced was the lowest due to the presence of certaintype growth inhibitory fatty acids, as shown with the soap[9]. Nevertheless, the biomass production is not as im-portant in this type of cultivation, as the main goal is not toproduce biomass, but rather, to produce metabolites. Inmany previous studies, the increase or decrease in biomass isnot a direct indication of the metabolite production [18].
+ere was little change in the glycerol content betweenthe CG before and after the chemical solvent pretreatments.+e pretreatments of CG with these solvents improved theglycerol consumption significantly (up to 50% improve-ment) when compared to nontreated CG media (Figure 1).+is may be possible as a result of the solvent reducing mostof the free and methyl ester fatty acids in the CG. However,
the rate of consumption of pretreated CG still did not reachthe level of consumption when PG was used, as the treat-ments were still unable to remove other type of contami-nants such as MeOH and salts [16]. +e lower glycerolconsumption in CG is most likely due to the presence of fattyacids, which may lead to the substrate competition (as fattyacids may probably be the preferable carbon source) or otherinhibitory effect of fatty acids on carbon uptake [14].
+e production of lovastatin, (+)-geodin, and sulochrinusing pretreated CG is depicted in Figure 2. PE, in particular,showed a significant increase of 92.8% in lovastatin pro-duction at day 6 compared to the PG (positive control). Incontrast, the final titre of lovastatin at day 6 for both DE andTol was insignificant to the positive control. Interestingly, aset of different responses were observed in (+)-geodin andsulochrin production. Instead of PE (5.58mg/L), DE(8.95mg/L)-induced (+)-geodin is the strongest, whilesulohrin is more responsive to Tol pretreatment (24.78mg/L). +ese results are still considered excellent nonetheless,because substantial improvement in metabolite production,as well as glycerol consumption, was achieved in all pre-treated samples when compared to the CG (negative control)(Figure 2).
+e improvement of lovastatin production can be at-tributed to the ability of the solvent to remove some of thecontaminants, while leaving a number of stimulatory sub-stances. Previous investigation showed that certain solventscan remove several key fatty acids in CG, such as linoleic acidmethyl ester, palmitic acids, and oleic acids [16]. Althoughsome the beneficial fatty acids, such as oleic acid, are alsobeing removed, the removal of inhibiting saturated fattyacids such palmitic acids was shown to have a larger effect onthe lovastatin production by this fungus [14, 16]. It ispossible that the induction of lovastatin production in PE iscaused by the incomplete removal of unsaturated fatty acidsin the treatment. +e incomplete removal may also be thereason why we observed a mixed response of (+)-geodin andsulochrin as well (Figure 2). It might be that each of thesemetabolites is induced more strongly by different impurities,which resulted in a unique response in different solventpretreatments [14].
3.2. e Efficiency of Nonliquid Pretreatment Technique ofCrude Glycerol on Substrate Consumption, Growth, andLovastatin Production. In this section, activated carbon(AC) and water softener pillow (WS) were used to improvethe CG. AC is a form of carbonmaterial that has been treatedwith oxygen by thermal decomposition to create millions ofpores that increase the surface area available for adsorption.Solvent washing reduces the impurities by transferring theimpurities from one liquid to another. However, AC trapsand filters the impurities inside the CG. In contrast, WSreduces the “hardness” of the solution by reducing thepresence of certain minerals such as calcium and magne-sium. While there is no evidence that associate the effect ofhard water on the growth or production of metabolites byfungus, recent evidence showed that divalent metal cationscan influence lovastatin biosynthesis [19]. Moreover, the
International Journal of Chemical Engineering 3
presence of certain metal ions together in certain concen-trations may be inhibitory to the lovastatin production.
As in solvent pretreatment, these physical treatmentsproduced very little change in terms of glycerol content. Oursubsequent analysis showed an improvement in terms ofsubstrate consumption when compared to CG when AC andWS (42% and 22% improvement, respectively, at day 2) wasused (Figure 3). However, no improvement in biomassgrowth was detected under both treatments. �e higherconsumption improvement when AC was used instead ofWS indicated that the presence of nondissolved solids inside
the CG played a major role in the uptake of substrate(glycerol). Nevertheless, the removal of those cations stillimproves the substrate consumption by a signi�cant margin.
Figure 4 shows the production of lovastatin, (+)-geodin,and sulochrin using pretreated CGs (AC andWS), nontreatedCG, and PG during the 6-days cultivation. �e production oflovastatin (21.80mg/L), (+)-geodin (7.43mg/L), and sulo-chrin (11.74mg/L) following AC treatment was very similar toPG (20.65mg/L, 8.60mg/L, and 8.18mg/L, respectively). Incontrast, WS metabolite productions (lovastatin� 11.25mg/L, (+)-geodin� 12.34mg/L, and sulochrin� 12.85mg/L)
0
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Figure 2: �e (+)-geodin and sulochrin production under di�erent substrates. (+)-geodin is the highest under the DE pretreatment, whilesulochrin is the highest when Tol is used as pretreatment.
01020304050607080
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Figure 1: �e glycerol consumption and lovastatin production of A. terreus under di�erent substrates. �e glycerol consumption is theslowest in CG and the fastest in PG. Lovastatin is consistently the highest under PE pretreatment, while CG produced the lowest lovastatin.
Table 1: �e biomass production of A. terreus under di�erent pretreatments of nonpolar solvents. �e standard errors represent 95%con�dence.
Treatments DE PE Tol CG control PG controlBiomass (g/L) 11.28± 0.30 10.60± 0.27 9.80± 0.22 9.75± 0.12 10.07± 0.10
4 International Journal of Chemical Engineering
mirrored CG (lovastatin � 7.10mg/L, (+)-geodin�17.10mg/L, and sulochrin � 14.78mg/L). �is suggests thatthe AC treatment produced almost “pure” substrate,comparable to PG, while WS treatment is less e�cient in
improving the quality of the cultivation given that itsproduction pattern resembles that of CG. Nevertheless, WSstill produced signi�cantly higher lovastatin than that ofCG, although it is not as good as the production in PG. As
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Figure 3: �e glycerol consumption and biomass growth by A. terreus under the treatment of AC and WS. (a) Glycerol consumption. (b)Biomass production taken on day 6. In (a), the glycerol consumption in AC improved considerably, similar to positive control. In (b), thebiomass production was not a�ected by the pretreatments.
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Figure 4: �e production of lovastatin, (+)-geodin, and sulochrin by A. terreus under the treatment of AC andWS. Lovastatin, (+)-geodin,and sulochrin production in AC mirrored positive control (PG), while the metabolite production in WS mirrored negative control (CG).
International Journal of Chemical Engineering 5
expected, WS treatment and CG control produced higher(+)-geodin and sulochrin due to the higher presence ofimpurities. +is observation supports that (+)-geodin andsulochrin are more responsive towards impurities and maybe indicative of their role in stress-related mechanism in A.terreus [14].
4. Conclusion
+e use of pretreated CG can increase the production ofmetabolites, even higher than PG (positive control). Al-though CG may not be a better carbon source than PG forlovastatin production, it is indeed a promising economic andenvironmental alternative to make full use of easily acces-sible bio-wastes.
Data Availability
+e data used to support the findings of this study areavailable from the corresponding author upon request.
Disclosure
+is publication contains an excerpt from a previouslypublished work (PhD thesis) of the first author [20].
Conflicts of Interest
+e authors declare that they have no conflicts of interest.
Acknowledgments
+e authors would like to thank Universiti Putra Malaysiaand the Ministry of Higher Education, Malaysia (MOHE),for their scholarship support and assistance with this project.
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