Bioresource Technology 177 (2015) 28–33
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Harvesting freshwater Chlorella vulgaris with flocculant derived from spent brewer’s yeast Gita Prochazkova a, Petr Kastanek b, Tomas Branyik a,⇑ a b
Department of Biotechnology, Institute of Chemical Technology Prague, Technicka 5, 166 28 Prague, Czech Republic EcoFuel Laboratories Ltd., Ocelarska 392/9, 190 00 Prague, Czech Republic
h i g h l i g h t s Successful flocculation with novel agent derived from spent brewer’s yeast. Over 90% harvesting efficiency achieved at dosage P0.4 mg flocculant/g biomass. Dosage increase required when phosphates or algogenic organic matter are present.
a r t i c l e
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Article history: Received 2 September 2014 Received in revised form 10 November 2014 Accepted 12 November 2014 Available online 18 November 2014 Keywords: Microalgae harvesting Chlorella Flocculation Spent brewer’s yeast Surface modification
a b s t r a c t One of the key bottlenecks of the economically viable production of low added value microalgal products (food supplements, feed, biofuels) is the harvesting of cells from diluted culture medium. The main goals of this work were to prepare a novel flocculation agent based on spent brewer’s yeast, a brewery by-product, and to test its harvesting efficiency on freshwater Chlorella vulgaris in different environments. The yeast was first autolyzed/hydrolyzed and subsequently chemically modified with 2-chloro-N, N-diethylethylamine hydrochloride (DEAE). Second, optimal dosage of modified spent yeast (MSY) flocculant for harvesting C. vulgaris was determined in culture media of various compositions. It was found that the absence of phosphorus ions decreased (0.4 mg MSY/g biomass), while the presence of algogenic organic matter (AOM) increased (51 mg MSY/g biomass) the required dosage of flocculant as compared to complete mineral medium with phosphorus and without AOM (12 mg MSY/g biomass). Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Microalgae represent a vast group of microorganisms which can produce a range of various biotechnologically attractive products (e.g. polysaccharides, polyunsaturated fatty acids, phytohormones, vitamins). Due to a continuously rising demand worldwide on cheap and sustainable energy resources, many studies also focus on microalgal biomass rich in lipids or starch as an alternative source for biofuel production (Harun et al., 2010; Marsalkova et al., 2010; Milledge, 2011). For this purpose large-scale production systems are a necessity, but the current microalgal production cannot prove cost-efficient as it is hindered by various bottlenecks, one of them being the harvesting of microalgal biomass. The reasons for difficult harvesting are the low sedimentation velocity of microalgae given by their small cell sizes (typically 5–10 lm), colloidal character with repulsive negative surface ⇑ Corresponding author. Tel.: +420 220 444 126; fax: +420 224 311 082. E-mail address: [email protected] (T. Branyik). http://dx.doi.org/10.1016/j.biortech.2014.11.056 0960-8524/Ó 2014 Elsevier Ltd. All rights reserved.
charges and low biomass concentrations (Shelef et al., 1984). Before applying an energy consuming process that requires costly equipment (e.g. filtration), it is beneficial to pre-concentrate the microalgal culture using a less expensive technique such as flocculation (Granados et al., 2012; Vandamme et al., 2012a). Via flocculation concentration factors of 20–100 can be reached, thus a dilute suspension of e.g. 0.5 g/L dry matter is concentrated to a slurry of 10–50 g/L. An algal paste of 25% dry matter content can be obtained by further dewatering using a mechanical method such as centrifugation (Vandamme et al., 2013). Various (in)organic flocculation agents can be applied for this purpose e.g. synthetic and natural polymers/polyelectrolytes, polyvalent metal ions such as Al3+ or Fe3+ (Shelef et al., 1984) or magnetic particles (Cerff et al., 2012; Prochazkova et al., 2013a,b). Nevertheless, their use is frequently accompanied by high price of the agent, microalgal growth inhibition by alum (Lee et al., 2013) or risk of biomass contamination by metals. Some of these drawbacks could be eliminated by applying biodegradable and non-toxic agents based on positively charged starch (Vandamme
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et al., 2010) or cationic cassia gum (Banerjee et al., 2014). Other opportunities are presented by autoflocculation under alkaline conditions or bio-flocculation, i.e. the introduction of a flocculating microbial species (e.g. bacteria, fungi, diatoms or microalgae) to a non-flocculating one (Sukenik and Shelef, 1984; Zhang and Hu, 2012). But these phenomena cannot be applied upon all biotechnological processes as the appropriate algal species may be sensitive to high pH values or addition of other microbial species is undesirable. Further disadvantages of commonly applied flocculants have been reviewed (Pragya et al., 2013; Schlesinger et al., 2012). Spent brewer’s yeast represent a by-product which is currently used mainly as feed for livestock, or to a lesser extent the cells are also used to produce yeast extract or dried, compressed and sold as a food supplement (Huige, 2006; Chae et al., 2001). However, spent yeast represents a major waste of brewing industry, which is usually sold for minimal price, can be obtained for free (if transportation costs are paid by the user) or it has to be disposed of according to regulations. The focus of the presented paper is to develop a novel flocculation agent based on spent brewer’s yeast (Saccharomyces pastorianus) and test its harvesting efficiency in culture media of various compositions for freshwater microalgae Chlorella vulgaris. 2. Methods 2.1. Microorganism, cultivation and preparation of algal suspension C. vulgaris Beijerinck strain P12 was maintained and cultivated in batch mode in 3 L photobioreactor according to previously described procedures (Prochazkova et al., 2013a), i.e. glass tubes were situated in a water bath (30 °C) under continuous illumination 100 lmol/m2/s (PAR sensor QSL-2101, Biospherical Instruments Inc., USA) and feeding of a mixture of air with 2% CO2 (v/v) at 15 L/h per tube. Each tube contained 300 mL of mineral medium, having the initial composition (mg/L): 1100 (NH2)2CO, 238 KH2PO4, 204 MgSO47H2O, 40 C10H12O8N2NaFe, 88 CaCl2, 0.832 H3BO3, 0.946 CuSO45H2O, 3.294 MnCl24H2O, 0.172 (NH4)6 Mo7O244H2O, 2.678 ZnSO47H2O, 0.616 CoSO47H2O, and 0.0014 (NH4)VO3. The pH value was adjusted to 6.5–7.0 using 1 M KOH prior to inoculation from an agar plate. The medium was treated as for outdoor culture so it was not sterilized, but distilled water was used nevertheless. After 144 h of cultivation ensuring linear growth a biomass concentration of 5 g/L was obtained. Subsequently, the microalgal cells were centrifuged and washed twice with distilled water (7800g, 5 min) and used to prepare algal suspensions of a defined concentration for subsequent flocculation experiments and/or zeta potential measurements. 2.2. Preparation of modified spent yeast (MSY) flocculant Spent brewer’s yeast (S. pastorianus) slurry (solid content 15% w/v) was collected at the end of beer maturation (2 °C). The yeast slurry was exposed to cell autolysis for 24 h at 50 °C and subsequently the solid fraction was separated from the yeast extract by centrifugation (Saksinchai et al., 2001). Further treatment was carried out on the solid fraction (20 g/L) at 80 °C using 1 M NaOH for one hour. Then the mixture was cooled, centrifuged and washed with water. The obtained spent yeast microparticles rich in b-glucan were used for chemical modification. The modification of spent yeast microparticles was performed according to the literature (Branyik et al., 2001). Firstly, 3.5 g of dry spent yeast microparticles were placed in a beaker containing 30 g Na2SO4, 4.3 g NaOH and 100 mL distilled water. Under continuous stirring the mixture was heated to 37 °C and then in the interval of 2 h under the same conditions 15 g of 50% (w/v)
29
2-chloro-N,N-diethylethylamine hydrochloride (DEAE, 99% purity, Sigma Aldrich) was added drop-wise to the mixture. Secondly, the suspension was heated to 60 °C for 30 min and then cooled to room temperature with adding more distilled water. Upon centrifugation (7800g, 5 min) and thrice washing with distilled water, the modified spent yeast (MSY) microparticles, used in the subsequent studies as flocculant, were lyophilized (Heto Power Dry LL3000, Thermo Fisher Scientific, USA) and stored at 4 °C until used for flocculation tests and surface analyses. 2.3. X-ray photoelectron spectroscopy (XPS) of modified spent yeast (MSY) flocculant Successful surface modification of prepared and lyophilized unmodified spent yeast (USY) and modified spent yeast (MSY) microparticles was tested using X-ray photoelectron spectroscopy (ESCAProbeP, Omicron Nanotechnology GmbH, Germany). The elemental content of nitrogen, oxygen, carbon and silicon present on the studied surface of USY and MSY was determined. Obtained results are expressed as atomic percent (at.%). In order to quantify the extent of cationization by the amine group of DEAE, the degree of modification (DM) was calculated as follows: DM = (N1 N0)/N0, where N0 is the nitrogen content (at.%) of the USY microparticle surface and N1 the nitrogen content (at.%) of the MSY microparticle surface. 2.4. Zeta potential measurements and particle size determination The zeta potentials of C. vulgaris cells and MSY microparticles were measured at 25 °C using the Zetasizer Nano-ZS (Malvern, UK) and calculated according to the Smoluchowski equation. The surface charge of C. vulgaris cells (10–50 mg/L) or MSY microparticles (10–50 mg/L) were measured in (i) model environments (10 mM KCl, pH 2–12), (ii) used mineral medium (UMM) at the end of cultivation (after 144 h), (iii) fresh complete mineral medium (CMM) and (iv) fresh mineral medium (MM) lacking the following components: sulfur (MM-S, MgSO47H2O), nitrogen (MM-N, (NH2)2CO), iron (MM-Fe, C10H12O8N2NaFe) or phosphorus (MM-P, KH2PO4). The pH of CMM, UMM and MM lacking some components was adjusted to 6.8. All samples were measured six times. Presented results are mean values ± standard deviation. Zetasizer Nano-ZS was also used for determining the size of microalgae and MSY microparticles. 2.5. Flocculation tests The flocculation tests were carried out in a mechanically mixed system composed of a cylindrical glass vessel stirred with a top-driven (overhead stirrer DLH, VELP Scientifica) simple straight-blade impeller with the following dimensions: internal vessel diameter 75 mm, height of liquid 75 mm (corresponds to 250 mL volume), width of four 7.5 mm wide agitation baffles, distance of stirrer from vessel bottom 15 mm, impeller diameter 33 mm and height 10 mm. The separation of microalgae with MSY flocculant based on modified spent brewer’s yeast was tested in different environments, where microalgal suspensions (250 mL, 0.15–1.05 g/L) were contacted with MSY under mechanical agitation 500 rpm for 10 s followed by 60 rpm for 30 min. After 30 min of mixing the suspension was allowed to stand for 10 min during which the formed flocs settled. The absorbance of the supernatant (3 mL) taken 20 mm below the liquid surface was then measured at 750 nm and the harvesting efficiency (HE,%) was calculated as follows: HE = [(A0 A1)/A0] 100, where A0 is the initial absorbance of the microalgal suspension before separation and A1 is the absorbance of the supernatant after flocculation. Due to the small cell size of C. vulgaris and long settling time, the self-sedimentation
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of microalgal cells was neglected. Concentration factors were calculated according to Granados et al. (2012). The harvesting efficiency (HE) of the MSY flocculant was tested in previously defined model environments UMM, CMM, MM-N, MM-Fe and MM-P, the pH of which was adjusted to 6.8. All experiments were performed in triplicate and the presented results are mean values ± standard deviation. 2.6. Algogenic organic matter analysis The used mineral medium (UMM) at the end of cultivation (after 144 h) was tested for the presence of algogenic organic matter (AOM). As the majority of AOM is composed of polysaccharides, the quantification was performed using phenol–sulfuric method and glucose as standard (Dubois et al., 1956). All experiments were performed in triplicate and the presented results are mean values ± standard deviation.
40 30
Zeta potenal (mV)
30
20 10 0 -10 -20 -30 -40
0
2
6
8
pH
10
12
14
A B C
Fig. 1. Zeta potentials of modified spent yeast (MSY) flocculant (A), unmodified spent yeast microparticles (B) and Chlorella vulgaris cells (C) in 10 mM KCl of different pH.
3. Results and discussion
30
A B
25
3.1. Modified spent yeast microparticles (MSY) as flocculant
20 Zeta potenal (mV)
The novel flocculation agent was prepared from spent brewer’s yeast, a cheap by-product of the brewing industry. At the beginning, spent brewer’s yeast were exposed to cell autolysis resulting in a soluble spent yeast lysate (usually used for yeast extract production) and solid cell debris fraction (Saksinchai et al., 2001). This solid fraction was further treated with hydrolysis in alkaline environment (Ryu et al., 2013) to further purify the yeast cell debris. Subsequently the obtained spent yeast microparticles were modified with diethylaminoethyl hydrochloride (DEAE). The treatment resulted in introducing anion exchange functional groups onto the surface of spent yeast microparticles. Anion exchangers with this functional group (DEAE-cellulose) are typically applied in chromatography (Venkatachalam et al., 2013). The granularity of the resulting MSY was determined using a laser interferometric method via Zetasizer ZS Nano. It was found that 31% of the particles had its diameter in the range 97 ± 11 nm, while 69% were significantly larger with an average diameter of 1211 ± 750 nm. XPS analysis of unmodified spent yeast (USY) and modified spent yeast (MSY) microparticles proved successful change in surface composition (Table 1). According to the increase in nitrogen content after modification it can be assumed that the introduction of DEAE functional groups onto the surface of spent brewer’s yeast was accomplished. The degree of modification (DM) was calculated and found to be equal to 2.33. The findings are furthermore supported by zeta potential (ZP) measurements under model conditions in 10 mM KCl (Fig. 1). C. vulgaris cells maintained a negative surface charge in almost entire pH range, whereas due to chemical modification, the ZP of the unmodified spent yeast microparticles shifted significantly towards net positive surface charge at all tested pH values (Fig. 1). The obtained results also indicate that MSY possess positive surface charge in all tested growth media (Fig. 2). However, the value of the surface charge (zeta potential) is a function of both pH and medium composition (Figs. 1 and 2). Compared to other microparticles that have been tested for their surface charge dependency on pH value, e.g. magnetic beads (Prochazkova et al., 2013a), synthetic iron oxide microparticles
4
15 10 5 0 -5 -10 -15 -20 CMM
UMM
MM-N
MM-P
MM-S
MM-Fe
Fig. 2. Zeta potentials of modified spent yeast (MSY) flocculant (A) and C. vulgaris cells (B) in fresh complete mineral medium (CMM), used mineral medium (UMM) and fresh mineral media lacking nitrogen (MM-N), phosphorus (MM-P), sulfur (MM-S) and iron (MM-Fe).
(Prochazkova et al., 2013b), chitosan microparticles (Wu et al., 2006) or alumina particles (Yu and Somasundaran, 1996), MSY do not display an isoelectric point in the tested situations but only a gradual decrease in surface charge as individual DEAE surface groups become less protonated with increasing pH (Fig. 1). A similar trend has been observed in the case of cationic corn and potato starch (Anthony and Sims, 2013). Thus, the potential availability of MSY as flocculants under various conditions is quite broad. Especially attractive is the continuous positive surface charge of MSY in different culture media, whereas C. vulgaris maintained a negative surface charge in all tested situations (Fig. 2). Thus, both studied surfaces display charge independence upon the presence/ absence of certain ions and/or culture media components, broadening the possibilities of MSY application. Nevertheless, differences may be observed in individual values of zeta potentials between algae and MSY particles. The highest difference (40 mV) occurred when excluding KH2PO4 from the culture medium and the lowest (27 mV) in the case of UMM. This can lead to stronger
Table 1 XPS surface analysis of unmodified spent yeast (USY) and modified spent yeast (MSY) microparticles and the degree of surface modification (DM). Material
Oxygen (at.%)
Carbon (at.%)
Silicon (at.%)
Nitrogen (at.%)
DM
USY MSY
28.99 25.36
68.33 70.30
1.46 0.28
1.22 4.06
2.33
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or weaker electrostatic interactions which can subsequently influence harvesting efficiency (see Section 3.3). 3.2. Flocculation in model environment
HE (%)
A model environment consisting of a symmetrical electrolyte (10 mM KCl) was chosen for testing the sole effect of pH on flocculation of C. vulgaris with MSY. The solution had an ionic strength of freshwater microalgae culture media and simultaneously contained no multivalent ions (e.g. Mg2+, Ca2+) known to cause inorganic precipitate formation with subsequent cell flocculation at higher pH values (Uduman et al., 2010; Vandamme et al., 2012a). The effect of pH on harvesting efficiency (HE) of microalgae at different MSY to microalgae (CV) mass ratios (MSY:CV) is depicted on Fig. 3. The most effective cell harvesting (HE > 90%) was achieved at pH 7 and 10 at a MSY:CV ratio equal to 30 mg/g, while at pH 4 the initially high HE (80% at 12 mg/g) slightly decreased with the increasing MSY:CV ratio (Fig. 3). These findings are in a slight discrepancy to the observed zeta potential differences between C. vulgaris cells and MSY microparticles (Fig. 1), since at pH 4 the electrostatic driving force should lead to similarly effective harvesting as at pH 7 and 10. The observed phenomenon is possibly caused by structural changes and/or reorganization of MSY particles at acidic pH values. Potential algal binding sites (positively charged surface niches) may thus be unapproachable for the cells of such size as Chlorella. According to obtained results (Fig. 3) it can be expected that the C. vulgaris vs. MSY microparticles interaction involves not only electrostatic forces but also other types of interactions (e.g. attractive van der Waals forces and hydrogen bonds, repulsive acid–base interactions), the magnitude of which is under the influence of pH. Similar observations, where flocculation occurred due to several surface interactions including prominent but non-dominant (electrostatic) charge neutralization, have been reported in the case of harvesting various Chlorella strains with chitosan (Cheng et al., 2011). For moderate pH values (pH 4–10) in model environment (10 mM KCl) MSY microparticles can be considered as a very effective flocculant (Table 2). The concentration factors achieved by MSY microparticles under optimal dosage reached up to 35, which is comparable with the results given for polyelectrolytes by Granados et al. (2012). Similarly to other polyelectrolyte flocculants, MSY microparticles show maximum HE followed by a slight decrease with increasing MSY:CV ratio (Fig. 3). As in the case of polyelectrolyte flocculants, the explanation of the observed phenomenon can be mutual steric hindrance and/or electrostatic repulsion of the flocculant’s functional groups upon reaching a certain concentration under given experimental conditions (Vandamme et al., 2010). 100 90 80 70 60 50 40 30 20 10 0
pH 7 pH 4 pH 10
0
20
40
60
80
100
120
140
MSY:CV (mg/g) Fig. 3. Microalgal harvesting efficiencies (HE) at different modified spent yeast (MSY) flocculant to microalgae (CV) mass ratios at initial biomass concentration of 0.2 g/L in 10 mM KCl of different pH.
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3.3. Flocculation in culture medium Culture medium composition greatly effects the formation of MSY-microalgae aggregates and the amount of flocculating agent needed for efficient biomass separation (Fig. 4). The required MSY:CV ratios for reaching harvesting efficiencies above 90% were in the range from 0.4 to 27.6 mg/g, thus in some cases even lower than under model environment (Fig. 3). This increased HE can be ascribed to the absence of interfering phosphates, which has already been reported (Prochazkova et al., 2013b). The highest HEs from all tested situations at minimum dose of MSY:CV, i.e. 0.4 mg/g for 91% (Fig. 4) and 2.1 mg/g for 96% (data not shown), were achieved when excluding the main source of phosphorus (KH2PO4) from the mineral medium (MM-P). In CMM the dose increased to 12 mg/g for HE 93% (Fig. 4). At the absence of nitrogen and iron (MM-N, MM-Fe) the doses of MSY flocculant were comparable with CMM, while in MM-S the required dose was somewhat higher (Fig. 4). However, these doses are significantly lower when compared to cationic starch (Greenfloc 120) or chitosan acting at the absence of algogenic organic matter (Table 2). Furthermore, the harvesting of C. vulgaris by MSY was tested in used mineral medium (UMM) to monitor the effect of algogenic organic matter (AOM) as it has been reported to negatively affect flocculation (Henderson et al., 2008; Shelef et al., 1984; Vandamme et al., 2012b). Firstly, the culture media used were tested for the presence of AOM. Fresh complete mineral medium (CMM) was found to have a content of 1.04 ± 0.01 mg glucose eq. per liter, while used mineral medium (UMM) had 37.56 ± 0.26 mg glucose eq. per liter. These findings suggest that the strain of C. vulgaris used in this work releases AOM into the medium during cultivation, which also reflected in increased MSY dose from 12 mg/g in CMM (HE 93%) to 110 mg/g in UMM (HE 91%) at microalgal biomass concentrations 0.2 and 0.15 g/L, respectively (Figs. 4 and 5). Secondly, various mass ratios of MSY to microalgal biomass (CV) have been tested in UMM with microalgal biomass concentrations ranging from 0.15 to 1.05 g/L (Fig. 5). It can be seen that the MSY:CV ratio is not constant over the studied biomass concentration range. The increasing MSY dose observed at lower biomass concentrations (0.15–0.33 g/L) dropped to approximately 51 mg/g for biomass concentration 0.65 g/L and then slightly increased to 63 mg/g at 1.05 g/L. The initial increase in required dose of cationic starch was observed also for Parachlorella at biomass concentrations from 0.04 to 0.3 g/L, but this study did not deal with higher biomass concentrations (Vandamme et al., 2010). Given to the surface modification of the MSY with DEAE groups, the observed flocculation mechanism can be considered similar to that of polyelectrolytes. The flocculation by polyelectrolytes is considered to be governed by two major mechanisms, the electrical double layer compression or charge neutralization and interparticle polymer bridging (Yu and Somasundaran, 1996). Thus, a possible explanation of the decreasing flocculate dosage at higher biomass concentrations (Fig. 5) can be the occurrence of more frequent cell–flocculant interactions at higher microalgal biomass concentrations leading to more extensive microalgae-MSY contact and surface polymer bridging. The same trend, i.e. the denser the cell suspension, the less flocculant needed per cell, has been observed also in the case of microalgal flocculation by magnesium and calcium under alkaline conditions (Schlesinger et al., 2012) or during flocculation of Muriellopsis sp. cultures using polyelectrolyte EM16 (Granados et al., 2012). When harvesting Chlorella cells suspended in UMM with MSY, the required flocculant doses were significantly lower compared to aluminum sulfate, cationic starch, magnetic particles or chitosan flocculation under culture media conditions (AOM presence), but similar to the cationic biopolymer cassia gum (Table 2). Nevertheless when considering the application of a particular flocculant, the dependency upon algal species
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Table 2 Effect of different flocculants, medium composition and presence of algogenic organic matter (AOM) on flocculant dosage and harvesting efficiency during harvesting of Chlorella sp.
a b c d
Flocculant
FA:MAa (mg/g); Xc (g/L)
HEb (%)
Medium; pH
AOMd
References
Cationic cassia gum Moringa oleifera seed flour Aluminum sulfate Magnetic particles MagSilica 50–85 Uncoated magnetite microparticles Chitosan Chitosan Cationic starch Greenfloc 120 Cationic starch Greenfloc 120 Aluminum sulfate Chitosan–Fe3O4 nanoparticle composites Modified spent yeast (MSY) Modified spent yeast (MSY)
46.6; 0.75 1000; 1 1000; 1 3000; 0.65 380; 0.8 300; 0.25 32; 0.25 360; 0.25
93 88 94 90 90 85 85 85
TAP; 7.6 WC; 9.3 WC; 8.5 IGV; 8 Chu 13; 7–9 WC; 7.5 WC; 7.5 WC; 8
Yes Yes Yes Yes Yes Yes No Yes
Banerjee et al. (2014) Teixeira et al. (2012) Teixeira et al. (2012) Cerff et al. (2012) Xu et al. (2011) Vandamme et al. (2012b) Vandamme et al. (2012b) Vandamme et al. (2012b)
80; 0.25
85
WC; 8
No
Vandamme et al. (2012b)
460; 0.25 1400; 1 51; 0.65 0.4; 0.2
85 99 90 91
WC; 5.5 Modified N8; 6.5 UMM; 6.8 MM-P; 6.8
Yes Yes Yes No
Vandamme et al. (2012b) Lee et al. (2013) This work This work
Flocculant (FA) to microalgae (MA) mass ratio. Harvesting efficiency (%). Dry microalgal biomass concentration (g/L). Presence of AOM during flocculation experiments.
35
MSY:CV (mg/g)
30 25 20 15 10 5 0
CMM
MM-P MM-N MM-S Culture medium composion
MM-Fe
Fig. 4. Mass ratios of modified spent yeast (MSY) flocculant to microalgae (CV) tested in fresh complete mineral medium (CMM) and fresh mineral media lacking phosphorus (MM-P), nitrogen (MM-N), sulfur (MM-S) or iron (MM-Fe) for harvesting efficiencies over 90%. Initial biomass concentrations were 0.2 g/L.
MSY:CV (mg/g)
150
100
As in the case of flocculation under model environment (10 mM KCl), the concentration factors in CMM, MM-P, MM-N and MM-Fe reached up to 36, whereas the concentration factor in UMM was only 15. The observed concentration factor decrease can be explained by the presence of AOM, which can either sterically hinder the MSY-microalgae interaction and/or cause repulsive electrostatic interactions (Henderson et al., 2008). Nevertheless, the concentration factor of 15 achieved for C. vulgaris in this work with MSY (51 mg/L) is higher than the concentration factor 10 when harvesting Chlorella with 120 mg/L chitosan (Rashid et al., 2013). An economically viable production of microalgal biomass is a practical challenge. When pre-selecting the most appropriate flocculants, various aspects of the process (microalgal species, required concentration factor, reuse of media, presence of AOM, etc.) and nature of the product (food, feed, cosmetic, fuel, etc.) have to be taken into account. Finally the most decisive selection criteria are efficiency, dosage and overall cost of separation. Several cost estimates have been published (Banerjee et al., 2014; Farid et al., 2013; Granados et al., 2012) and today there is a trend to use cheap polymeric flocculants. The MSY flocculant prepared from spent brewer’s yeast is in accordance with these requirements and in addition it represents a beneficial use for a significant brewery by-product with environmental impact. 4. Conclusions
50
0
0
0.2
0.4 0.6 0.8 1 Biomass concentraon (g/L)
1.2
Fig. 5. Mass ratios of modified spent yeast (MSY) flocculant to microalgae (CV) at different Chlorella biomass concentrations in used mineral medium (UMM) for harvesting efficiencies over 90%.
must be also taken into account. For instance when applying cationic starch only 33 mg/g were required for Scenedesmus as compared to 80 mg/L for Chlorella (Vandamme et al., 2010). It seems that larger microalgal cells such as Scenedesmus sp. and Muriellopsis sp. (Granados et al., 2012) require lower flocculant doses. Simultaneously it was found that that the HE of each polyelectrolyte varied with studied strain, thus a single polyelectrolyte cannot be selected for all microalgae (Granados et al., 2012).
Successful introduction of positively charged functional groups (DEAE) onto microparticles prepared from spent brewer’s yeast led to development of novel, highly efficient cationic flocculant (MSY) for harvesting C. vulgaris. Harvesting efficiencies above 90% were achieved for MSY doses of 0.4 mg/g in fresh mineral medium lacking phosphates and 51 mg/g in used mineral medium directly after cultivation. Results showed the importance of culture medium composition and effect of algogenic organic matter on harvesting. MSY can be regarded as one of the most dose–effective flocculants and it represents an interesting application for spent brewer’s yeast, a considerable industrial by-product. Acknowledgements The financial support by the Ministry of Education, Youth and Sports of the Czech Republic through Grants MSM6046137305 and LJ12002 (realized in program GESHER/MOST, MSMT-10317/ 2012-36) is gratefully acknowledged.
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References Anthony, R., Sims, R., 2013. Cationic starch for microalgae and total phosphorus removal from wastewater. J. Appl. Polym. Sci. 130, 2572–2578. Banerjee, C., Ghosh, S., Sen, G., Mishra, S., Shukla, P., Bandopadhyay, R., 2014. Study of algal biomass harvesting through cationic cassia gum, a natural plant based biopolymer. Bioresour. Technol. 151, 6–11. Branyik, T., Vicente, A.A., Machado Cruz, J.M., Teixeira, J.A., 2001. Spent grains – a new support for brewing yeast immobilisation. Biotechnol. Lett. 23, 1073–1078. Cerff, M., Morweiser, M., Dillschneider, R., Michel, A., Menzel, K., Posten, C., 2012. Harvesting fresh water and marine algae by magnetic separation: screening of separation parameters and high gradient magnetic filtration. Bioresour. Technol. 118, 289–295. Dubois, M., Gilles, K.A., Hamilton, J.K., Rebers, P.A., Smith, F., 1956. Colorimetric method for determination of sugars and related substances. Anal. Chem. 28, 350–356. Farid, M.S., Shariati, A., Badakhshan, A., Anvaripour, B., 2013. Using nano-chitosan for harvesting microalga Nannochloropsis sp. Bioresour. Technol. 131, 555–559. Granados, M.R., Acién, F.G., Gómez, C., Fernández-Sevilla, J.M., Molina Grima, E., 2012. Evaluation of flocculants for the recovery of freshwater microalgae. Bioresour. Technol. 118, 102–110. Harun, R., Singh, M., Forde, G.M., Danquah, M.K., 2010. Bioprocess engineering of microalgae to produce a variety of consumer products. Renew. Sustain. Energy Rev. 14, 1037–1047. Henderson, R.K., Baker, A., Parsons, S.A., Jefferson, B., 2008. Characterisation of algogenic organic matter extracted from cyanobacteria, green algae and diatoms. Water Res. 42, 3435–3445. Huige, N.J., 2006. Brewery by-products and effluents. In: Priest, F.G., Stewart, G.G. (Eds.), Handbook of Brewing, second ed. CRC Press, USA, pp. 655–713. Chae, H.J., Joo, H., In, M.-J., 2001. Utilization of brewer’s yeast cells for the production of food-grade yeast extract. Part 1: effects of different enzymatic treatments on solid and protein recovery and flavor characteristics. Bioresour. Technol. 76, 253–258. Cheng, Y.-S., Zheng, Y., Labavitch, J.M., VanderGheynst, J.S., 2011. The impact of cell wall carbohydrate composition on the chitosan flocculation of Chlorella. Process Biochem. 46, 1927–1933. Lee, K., Lee, S.Y., Na, J.-G., Jeon, S.G., Praveenkumar, R., Kim, D.-M., Chang, W.-S., Oh, Y.-K., 2013. Magnetophoretic harvesting of oleaginous Chlorella sp. by using biocompatible chitosan/magnetic nanoparticle composites. Bioresour. Technol. 149, 575–578. Marsalkova, B., Sirmerova, M., Kurec, M., Branyik, T., Branyikova, I., Melzoch, K., Zachleder, V., 2010. Microalgae Chlorella sp. as an alternative source of fermentable sugars. Chem. Eng. Trans. 21, 1279–1284. Milledge, J., 2011. Commercial application of microalgae other than as biofuels: a brief review. Rev. Environ. Sci. Biotechnol. 10, 31–41. Pragya, N., Pandey, K.K., Sahoo, P.K., 2013. A review on harvesting, oil extraction and biofuels production technologies from microalgae. Renew. Sustain. Energy Rev. 24, 159–171. Prochazkova, G., Podolova, N., Safarik, I., Zachleder, V., Branyik, T., 2013a. Physicochemical approach to freshwater microalgae harvesting with magnetic particles. Colloids Surf. B 112, 213–218.
33
Prochazkova, G., Safarik, I., Branyik, T., 2013b. Harvesting microalgae with microwave synthesized magnetic microparticles. Bioresour. Technol. 130, 472–477. Rashid, N., Rehman, S.U., Han, J.-I., 2013. Rapid harvesting of freshwater microalgae using chitosan. Process Biochem. 48, 1107–1110. Ryu, B.-G., Kim, K., Kim, J., Han, J.-I., Yang, J.-W., 2013. Use of organic waste from the brewery industry for high-density cultivation of the docosahexaenoic acid-rich microalga, Aurantiochytrium sp. KRS101. Bioresour. Technol. 129, 351–359. Saksinchai, S., Suphantharika, M., Verduyn, C., 2001. Application of a simple yeast extract from spent brewer’s yeast for growth and sporulation of Bacillus thuringiensis subsp. kurstaki: a physiological study. World J. Microbiol. Biotechnol. 17, 307–316. Shelef, G., Sukenik, A., Green, M., 1984. Microalgae Harvesting and Processing: A Literature Review. SERI/STR-231-2396. Solar Energy Research Institute, Golden Colorado. Schlesinger, A., Eisenstadt, D., Bar-Gil, A., Carmely, H., Einbinder, S., Gressel, J., 2012. Inexpensive non-toxic flocculation of microalgae contradicts theories; overcoming a major hurdle to bulk algal production. Biotechnol. Adv. 30, 1023–1030. Sukenik, A., Shelef, G., 1984. Algal autoflocculation—verification and proposed mechanism. Biotechnol. Bioeng. 26, 142–147. Teixeira, C., Kirsten, F., Teixeira, P., 2012. Evaluation of Moringa oleifera seed flour as a flocculating agent for potential biodiesel producer microalgae. J. Appl. Phycol. 24, 557–563. Uduman, N., Qi, Y., Danquah, M.K., Forde, G.M., Hoadley, A., 2010. Dewatering of microalgal cultures: a major bottleneck to algae-based fuels. J. Renew. Sustain. Energy 2, 1–15. Vandamme, D., Foubert, I., Muylaert, K., 2013. Flocculation as a low-cost method for harvesting microalgae for bulk biomass production. Trends Biotechnol. 31, 233– 239. Vandamme, D., Foubert, I., Fraeye, I., Meesschaert, B., Muylaert, K., 2012a. Flocculation of Chlorella vulgaris induced by high pH: role of magnesium and calcium and practical implications. Bioresour. Technol. 105, 114–119. Vandamme, D., Foubert, I., Fraeye, I., Muylaert, K., 2012b. Influence of organic matter generated by Chlorella vulgaris on five different modes of flocculation. Bioresour. Technol. 124, 508–511. Vandamme, D., Foubert, I., Meesschaert, B., Muylaert, K., 2010. Flocculation of microalgae using cationic starch. J. Appl. Phycol. 22, 525–530. Venkatachalam, G., Gummadi, S., Doble, M., 2013. Extraction and purification of cyclic b-glucan. In: Cyclic b-Glucans from Microorganisms. Springer, Berlin, pp. 63–69. Wu, Y., Guo, J., Yang, W., Wang, C., Fu, S., 2006. Preparation and characterization of chitosan–poly(acrylic acid) polymer magnetic microspheres. Polymer 47, 5287– 5294. Xu, L., Guo, C., Wang, F., Zheng, S., Liu, C.-Z., 2011. A simple and rapid harvesting method for microalgae by in situ magnetic separation. Bioresour. Technol. 102, 10047–10051. Yu, X., Somasundaran, P., 1996. Role of polymer conformation in interparticlebridging dominated flocculation. J. Colloid Interface Sci. 177, 283–287. Zhang, J., Hu, B., 2012. A novel method to harvest microalgae via co-culture of filamentous fungi to form cell pellets. Bioresour. Technol. 114, 529–535.
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Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Harvesting freshwater Chlorella vulgaris with flocculant derived from spent brewer’s yeast Gita Prochazkova a, Petr Kastanek b, Tomas Branyik a,⇑ a b
Department of Biotechnology, Institute of Chemical Technology Prague, Technicka 5, 166 28 Prague, Czech Republic EcoFuel Laboratories Ltd., Ocelarska 392/9, 190 00 Prague, Czech Republic
h i g h l i g h t s Successful flocculation with novel agent derived from spent brewer’s yeast. Over 90% harvesting efficiency achieved at dosage P0.4 mg flocculant/g biomass. Dosage increase required when phosphates or algogenic organic matter are present.
a r t i c l e
i n f o
Article history: Received 2 September 2014 Received in revised form 10 November 2014 Accepted 12 November 2014 Available online 18 November 2014 Keywords: Microalgae harvesting Chlorella Flocculation Spent brewer’s yeast Surface modification
a b s t r a c t One of the key bottlenecks of the economically viable production of low added value microalgal products (food supplements, feed, biofuels) is the harvesting of cells from diluted culture medium. The main goals of this work were to prepare a novel flocculation agent based on spent brewer’s yeast, a brewery by-product, and to test its harvesting efficiency on freshwater Chlorella vulgaris in different environments. The yeast was first autolyzed/hydrolyzed and subsequently chemically modified with 2-chloro-N, N-diethylethylamine hydrochloride (DEAE). Second, optimal dosage of modified spent yeast (MSY) flocculant for harvesting C. vulgaris was determined in culture media of various compositions. It was found that the absence of phosphorus ions decreased (0.4 mg MSY/g biomass), while the presence of algogenic organic matter (AOM) increased (51 mg MSY/g biomass) the required dosage of flocculant as compared to complete mineral medium with phosphorus and without AOM (12 mg MSY/g biomass). Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Microalgae represent a vast group of microorganisms which can produce a range of various biotechnologically attractive products (e.g. polysaccharides, polyunsaturated fatty acids, phytohormones, vitamins). Due to a continuously rising demand worldwide on cheap and sustainable energy resources, many studies also focus on microalgal biomass rich in lipids or starch as an alternative source for biofuel production (Harun et al., 2010; Marsalkova et al., 2010; Milledge, 2011). For this purpose large-scale production systems are a necessity, but the current microalgal production cannot prove cost-efficient as it is hindered by various bottlenecks, one of them being the harvesting of microalgal biomass. The reasons for difficult harvesting are the low sedimentation velocity of microalgae given by their small cell sizes (typically 5–10 lm), colloidal character with repulsive negative surface ⇑ Corresponding author. Tel.: +420 220 444 126; fax: +420 224 311 082. E-mail address: [email protected] (T. Branyik). http://dx.doi.org/10.1016/j.biortech.2014.11.056 0960-8524/Ó 2014 Elsevier Ltd. All rights reserved.
charges and low biomass concentrations (Shelef et al., 1984). Before applying an energy consuming process that requires costly equipment (e.g. filtration), it is beneficial to pre-concentrate the microalgal culture using a less expensive technique such as flocculation (Granados et al., 2012; Vandamme et al., 2012a). Via flocculation concentration factors of 20–100 can be reached, thus a dilute suspension of e.g. 0.5 g/L dry matter is concentrated to a slurry of 10–50 g/L. An algal paste of 25% dry matter content can be obtained by further dewatering using a mechanical method such as centrifugation (Vandamme et al., 2013). Various (in)organic flocculation agents can be applied for this purpose e.g. synthetic and natural polymers/polyelectrolytes, polyvalent metal ions such as Al3+ or Fe3+ (Shelef et al., 1984) or magnetic particles (Cerff et al., 2012; Prochazkova et al., 2013a,b). Nevertheless, their use is frequently accompanied by high price of the agent, microalgal growth inhibition by alum (Lee et al., 2013) or risk of biomass contamination by metals. Some of these drawbacks could be eliminated by applying biodegradable and non-toxic agents based on positively charged starch (Vandamme
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et al., 2010) or cationic cassia gum (Banerjee et al., 2014). Other opportunities are presented by autoflocculation under alkaline conditions or bio-flocculation, i.e. the introduction of a flocculating microbial species (e.g. bacteria, fungi, diatoms or microalgae) to a non-flocculating one (Sukenik and Shelef, 1984; Zhang and Hu, 2012). But these phenomena cannot be applied upon all biotechnological processes as the appropriate algal species may be sensitive to high pH values or addition of other microbial species is undesirable. Further disadvantages of commonly applied flocculants have been reviewed (Pragya et al., 2013; Schlesinger et al., 2012). Spent brewer’s yeast represent a by-product which is currently used mainly as feed for livestock, or to a lesser extent the cells are also used to produce yeast extract or dried, compressed and sold as a food supplement (Huige, 2006; Chae et al., 2001). However, spent yeast represents a major waste of brewing industry, which is usually sold for minimal price, can be obtained for free (if transportation costs are paid by the user) or it has to be disposed of according to regulations. The focus of the presented paper is to develop a novel flocculation agent based on spent brewer’s yeast (Saccharomyces pastorianus) and test its harvesting efficiency in culture media of various compositions for freshwater microalgae Chlorella vulgaris. 2. Methods 2.1. Microorganism, cultivation and preparation of algal suspension C. vulgaris Beijerinck strain P12 was maintained and cultivated in batch mode in 3 L photobioreactor according to previously described procedures (Prochazkova et al., 2013a), i.e. glass tubes were situated in a water bath (30 °C) under continuous illumination 100 lmol/m2/s (PAR sensor QSL-2101, Biospherical Instruments Inc., USA) and feeding of a mixture of air with 2% CO2 (v/v) at 15 L/h per tube. Each tube contained 300 mL of mineral medium, having the initial composition (mg/L): 1100 (NH2)2CO, 238 KH2PO4, 204 MgSO47H2O, 40 C10H12O8N2NaFe, 88 CaCl2, 0.832 H3BO3, 0.946 CuSO45H2O, 3.294 MnCl24H2O, 0.172 (NH4)6 Mo7O244H2O, 2.678 ZnSO47H2O, 0.616 CoSO47H2O, and 0.0014 (NH4)VO3. The pH value was adjusted to 6.5–7.0 using 1 M KOH prior to inoculation from an agar plate. The medium was treated as for outdoor culture so it was not sterilized, but distilled water was used nevertheless. After 144 h of cultivation ensuring linear growth a biomass concentration of 5 g/L was obtained. Subsequently, the microalgal cells were centrifuged and washed twice with distilled water (7800g, 5 min) and used to prepare algal suspensions of a defined concentration for subsequent flocculation experiments and/or zeta potential measurements. 2.2. Preparation of modified spent yeast (MSY) flocculant Spent brewer’s yeast (S. pastorianus) slurry (solid content 15% w/v) was collected at the end of beer maturation (2 °C). The yeast slurry was exposed to cell autolysis for 24 h at 50 °C and subsequently the solid fraction was separated from the yeast extract by centrifugation (Saksinchai et al., 2001). Further treatment was carried out on the solid fraction (20 g/L) at 80 °C using 1 M NaOH for one hour. Then the mixture was cooled, centrifuged and washed with water. The obtained spent yeast microparticles rich in b-glucan were used for chemical modification. The modification of spent yeast microparticles was performed according to the literature (Branyik et al., 2001). Firstly, 3.5 g of dry spent yeast microparticles were placed in a beaker containing 30 g Na2SO4, 4.3 g NaOH and 100 mL distilled water. Under continuous stirring the mixture was heated to 37 °C and then in the interval of 2 h under the same conditions 15 g of 50% (w/v)
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2-chloro-N,N-diethylethylamine hydrochloride (DEAE, 99% purity, Sigma Aldrich) was added drop-wise to the mixture. Secondly, the suspension was heated to 60 °C for 30 min and then cooled to room temperature with adding more distilled water. Upon centrifugation (7800g, 5 min) and thrice washing with distilled water, the modified spent yeast (MSY) microparticles, used in the subsequent studies as flocculant, were lyophilized (Heto Power Dry LL3000, Thermo Fisher Scientific, USA) and stored at 4 °C until used for flocculation tests and surface analyses. 2.3. X-ray photoelectron spectroscopy (XPS) of modified spent yeast (MSY) flocculant Successful surface modification of prepared and lyophilized unmodified spent yeast (USY) and modified spent yeast (MSY) microparticles was tested using X-ray photoelectron spectroscopy (ESCAProbeP, Omicron Nanotechnology GmbH, Germany). The elemental content of nitrogen, oxygen, carbon and silicon present on the studied surface of USY and MSY was determined. Obtained results are expressed as atomic percent (at.%). In order to quantify the extent of cationization by the amine group of DEAE, the degree of modification (DM) was calculated as follows: DM = (N1 N0)/N0, where N0 is the nitrogen content (at.%) of the USY microparticle surface and N1 the nitrogen content (at.%) of the MSY microparticle surface. 2.4. Zeta potential measurements and particle size determination The zeta potentials of C. vulgaris cells and MSY microparticles were measured at 25 °C using the Zetasizer Nano-ZS (Malvern, UK) and calculated according to the Smoluchowski equation. The surface charge of C. vulgaris cells (10–50 mg/L) or MSY microparticles (10–50 mg/L) were measured in (i) model environments (10 mM KCl, pH 2–12), (ii) used mineral medium (UMM) at the end of cultivation (after 144 h), (iii) fresh complete mineral medium (CMM) and (iv) fresh mineral medium (MM) lacking the following components: sulfur (MM-S, MgSO47H2O), nitrogen (MM-N, (NH2)2CO), iron (MM-Fe, C10H12O8N2NaFe) or phosphorus (MM-P, KH2PO4). The pH of CMM, UMM and MM lacking some components was adjusted to 6.8. All samples were measured six times. Presented results are mean values ± standard deviation. Zetasizer Nano-ZS was also used for determining the size of microalgae and MSY microparticles. 2.5. Flocculation tests The flocculation tests were carried out in a mechanically mixed system composed of a cylindrical glass vessel stirred with a top-driven (overhead stirrer DLH, VELP Scientifica) simple straight-blade impeller with the following dimensions: internal vessel diameter 75 mm, height of liquid 75 mm (corresponds to 250 mL volume), width of four 7.5 mm wide agitation baffles, distance of stirrer from vessel bottom 15 mm, impeller diameter 33 mm and height 10 mm. The separation of microalgae with MSY flocculant based on modified spent brewer’s yeast was tested in different environments, where microalgal suspensions (250 mL, 0.15–1.05 g/L) were contacted with MSY under mechanical agitation 500 rpm for 10 s followed by 60 rpm for 30 min. After 30 min of mixing the suspension was allowed to stand for 10 min during which the formed flocs settled. The absorbance of the supernatant (3 mL) taken 20 mm below the liquid surface was then measured at 750 nm and the harvesting efficiency (HE,%) was calculated as follows: HE = [(A0 A1)/A0] 100, where A0 is the initial absorbance of the microalgal suspension before separation and A1 is the absorbance of the supernatant after flocculation. Due to the small cell size of C. vulgaris and long settling time, the self-sedimentation
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of microalgal cells was neglected. Concentration factors were calculated according to Granados et al. (2012). The harvesting efficiency (HE) of the MSY flocculant was tested in previously defined model environments UMM, CMM, MM-N, MM-Fe and MM-P, the pH of which was adjusted to 6.8. All experiments were performed in triplicate and the presented results are mean values ± standard deviation. 2.6. Algogenic organic matter analysis The used mineral medium (UMM) at the end of cultivation (after 144 h) was tested for the presence of algogenic organic matter (AOM). As the majority of AOM is composed of polysaccharides, the quantification was performed using phenol–sulfuric method and glucose as standard (Dubois et al., 1956). All experiments were performed in triplicate and the presented results are mean values ± standard deviation.
40 30
Zeta potenal (mV)
30
20 10 0 -10 -20 -30 -40
0
2
6
8
pH
10
12
14
A B C
Fig. 1. Zeta potentials of modified spent yeast (MSY) flocculant (A), unmodified spent yeast microparticles (B) and Chlorella vulgaris cells (C) in 10 mM KCl of different pH.
3. Results and discussion
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A B
25
3.1. Modified spent yeast microparticles (MSY) as flocculant
20 Zeta potenal (mV)
The novel flocculation agent was prepared from spent brewer’s yeast, a cheap by-product of the brewing industry. At the beginning, spent brewer’s yeast were exposed to cell autolysis resulting in a soluble spent yeast lysate (usually used for yeast extract production) and solid cell debris fraction (Saksinchai et al., 2001). This solid fraction was further treated with hydrolysis in alkaline environment (Ryu et al., 2013) to further purify the yeast cell debris. Subsequently the obtained spent yeast microparticles were modified with diethylaminoethyl hydrochloride (DEAE). The treatment resulted in introducing anion exchange functional groups onto the surface of spent yeast microparticles. Anion exchangers with this functional group (DEAE-cellulose) are typically applied in chromatography (Venkatachalam et al., 2013). The granularity of the resulting MSY was determined using a laser interferometric method via Zetasizer ZS Nano. It was found that 31% of the particles had its diameter in the range 97 ± 11 nm, while 69% were significantly larger with an average diameter of 1211 ± 750 nm. XPS analysis of unmodified spent yeast (USY) and modified spent yeast (MSY) microparticles proved successful change in surface composition (Table 1). According to the increase in nitrogen content after modification it can be assumed that the introduction of DEAE functional groups onto the surface of spent brewer’s yeast was accomplished. The degree of modification (DM) was calculated and found to be equal to 2.33. The findings are furthermore supported by zeta potential (ZP) measurements under model conditions in 10 mM KCl (Fig. 1). C. vulgaris cells maintained a negative surface charge in almost entire pH range, whereas due to chemical modification, the ZP of the unmodified spent yeast microparticles shifted significantly towards net positive surface charge at all tested pH values (Fig. 1). The obtained results also indicate that MSY possess positive surface charge in all tested growth media (Fig. 2). However, the value of the surface charge (zeta potential) is a function of both pH and medium composition (Figs. 1 and 2). Compared to other microparticles that have been tested for their surface charge dependency on pH value, e.g. magnetic beads (Prochazkova et al., 2013a), synthetic iron oxide microparticles
4
15 10 5 0 -5 -10 -15 -20 CMM
UMM
MM-N
MM-P
MM-S
MM-Fe
Fig. 2. Zeta potentials of modified spent yeast (MSY) flocculant (A) and C. vulgaris cells (B) in fresh complete mineral medium (CMM), used mineral medium (UMM) and fresh mineral media lacking nitrogen (MM-N), phosphorus (MM-P), sulfur (MM-S) and iron (MM-Fe).
(Prochazkova et al., 2013b), chitosan microparticles (Wu et al., 2006) or alumina particles (Yu and Somasundaran, 1996), MSY do not display an isoelectric point in the tested situations but only a gradual decrease in surface charge as individual DEAE surface groups become less protonated with increasing pH (Fig. 1). A similar trend has been observed in the case of cationic corn and potato starch (Anthony and Sims, 2013). Thus, the potential availability of MSY as flocculants under various conditions is quite broad. Especially attractive is the continuous positive surface charge of MSY in different culture media, whereas C. vulgaris maintained a negative surface charge in all tested situations (Fig. 2). Thus, both studied surfaces display charge independence upon the presence/ absence of certain ions and/or culture media components, broadening the possibilities of MSY application. Nevertheless, differences may be observed in individual values of zeta potentials between algae and MSY particles. The highest difference (40 mV) occurred when excluding KH2PO4 from the culture medium and the lowest (27 mV) in the case of UMM. This can lead to stronger
Table 1 XPS surface analysis of unmodified spent yeast (USY) and modified spent yeast (MSY) microparticles and the degree of surface modification (DM). Material
Oxygen (at.%)
Carbon (at.%)
Silicon (at.%)
Nitrogen (at.%)
DM
USY MSY
28.99 25.36
68.33 70.30
1.46 0.28
1.22 4.06
2.33
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or weaker electrostatic interactions which can subsequently influence harvesting efficiency (see Section 3.3). 3.2. Flocculation in model environment
HE (%)
A model environment consisting of a symmetrical electrolyte (10 mM KCl) was chosen for testing the sole effect of pH on flocculation of C. vulgaris with MSY. The solution had an ionic strength of freshwater microalgae culture media and simultaneously contained no multivalent ions (e.g. Mg2+, Ca2+) known to cause inorganic precipitate formation with subsequent cell flocculation at higher pH values (Uduman et al., 2010; Vandamme et al., 2012a). The effect of pH on harvesting efficiency (HE) of microalgae at different MSY to microalgae (CV) mass ratios (MSY:CV) is depicted on Fig. 3. The most effective cell harvesting (HE > 90%) was achieved at pH 7 and 10 at a MSY:CV ratio equal to 30 mg/g, while at pH 4 the initially high HE (80% at 12 mg/g) slightly decreased with the increasing MSY:CV ratio (Fig. 3). These findings are in a slight discrepancy to the observed zeta potential differences between C. vulgaris cells and MSY microparticles (Fig. 1), since at pH 4 the electrostatic driving force should lead to similarly effective harvesting as at pH 7 and 10. The observed phenomenon is possibly caused by structural changes and/or reorganization of MSY particles at acidic pH values. Potential algal binding sites (positively charged surface niches) may thus be unapproachable for the cells of such size as Chlorella. According to obtained results (Fig. 3) it can be expected that the C. vulgaris vs. MSY microparticles interaction involves not only electrostatic forces but also other types of interactions (e.g. attractive van der Waals forces and hydrogen bonds, repulsive acid–base interactions), the magnitude of which is under the influence of pH. Similar observations, where flocculation occurred due to several surface interactions including prominent but non-dominant (electrostatic) charge neutralization, have been reported in the case of harvesting various Chlorella strains with chitosan (Cheng et al., 2011). For moderate pH values (pH 4–10) in model environment (10 mM KCl) MSY microparticles can be considered as a very effective flocculant (Table 2). The concentration factors achieved by MSY microparticles under optimal dosage reached up to 35, which is comparable with the results given for polyelectrolytes by Granados et al. (2012). Similarly to other polyelectrolyte flocculants, MSY microparticles show maximum HE followed by a slight decrease with increasing MSY:CV ratio (Fig. 3). As in the case of polyelectrolyte flocculants, the explanation of the observed phenomenon can be mutual steric hindrance and/or electrostatic repulsion of the flocculant’s functional groups upon reaching a certain concentration under given experimental conditions (Vandamme et al., 2010). 100 90 80 70 60 50 40 30 20 10 0
pH 7 pH 4 pH 10
0
20
40
60
80
100
120
140
MSY:CV (mg/g) Fig. 3. Microalgal harvesting efficiencies (HE) at different modified spent yeast (MSY) flocculant to microalgae (CV) mass ratios at initial biomass concentration of 0.2 g/L in 10 mM KCl of different pH.
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3.3. Flocculation in culture medium Culture medium composition greatly effects the formation of MSY-microalgae aggregates and the amount of flocculating agent needed for efficient biomass separation (Fig. 4). The required MSY:CV ratios for reaching harvesting efficiencies above 90% were in the range from 0.4 to 27.6 mg/g, thus in some cases even lower than under model environment (Fig. 3). This increased HE can be ascribed to the absence of interfering phosphates, which has already been reported (Prochazkova et al., 2013b). The highest HEs from all tested situations at minimum dose of MSY:CV, i.e. 0.4 mg/g for 91% (Fig. 4) and 2.1 mg/g for 96% (data not shown), were achieved when excluding the main source of phosphorus (KH2PO4) from the mineral medium (MM-P). In CMM the dose increased to 12 mg/g for HE 93% (Fig. 4). At the absence of nitrogen and iron (MM-N, MM-Fe) the doses of MSY flocculant were comparable with CMM, while in MM-S the required dose was somewhat higher (Fig. 4). However, these doses are significantly lower when compared to cationic starch (Greenfloc 120) or chitosan acting at the absence of algogenic organic matter (Table 2). Furthermore, the harvesting of C. vulgaris by MSY was tested in used mineral medium (UMM) to monitor the effect of algogenic organic matter (AOM) as it has been reported to negatively affect flocculation (Henderson et al., 2008; Shelef et al., 1984; Vandamme et al., 2012b). Firstly, the culture media used were tested for the presence of AOM. Fresh complete mineral medium (CMM) was found to have a content of 1.04 ± 0.01 mg glucose eq. per liter, while used mineral medium (UMM) had 37.56 ± 0.26 mg glucose eq. per liter. These findings suggest that the strain of C. vulgaris used in this work releases AOM into the medium during cultivation, which also reflected in increased MSY dose from 12 mg/g in CMM (HE 93%) to 110 mg/g in UMM (HE 91%) at microalgal biomass concentrations 0.2 and 0.15 g/L, respectively (Figs. 4 and 5). Secondly, various mass ratios of MSY to microalgal biomass (CV) have been tested in UMM with microalgal biomass concentrations ranging from 0.15 to 1.05 g/L (Fig. 5). It can be seen that the MSY:CV ratio is not constant over the studied biomass concentration range. The increasing MSY dose observed at lower biomass concentrations (0.15–0.33 g/L) dropped to approximately 51 mg/g for biomass concentration 0.65 g/L and then slightly increased to 63 mg/g at 1.05 g/L. The initial increase in required dose of cationic starch was observed also for Parachlorella at biomass concentrations from 0.04 to 0.3 g/L, but this study did not deal with higher biomass concentrations (Vandamme et al., 2010). Given to the surface modification of the MSY with DEAE groups, the observed flocculation mechanism can be considered similar to that of polyelectrolytes. The flocculation by polyelectrolytes is considered to be governed by two major mechanisms, the electrical double layer compression or charge neutralization and interparticle polymer bridging (Yu and Somasundaran, 1996). Thus, a possible explanation of the decreasing flocculate dosage at higher biomass concentrations (Fig. 5) can be the occurrence of more frequent cell–flocculant interactions at higher microalgal biomass concentrations leading to more extensive microalgae-MSY contact and surface polymer bridging. The same trend, i.e. the denser the cell suspension, the less flocculant needed per cell, has been observed also in the case of microalgal flocculation by magnesium and calcium under alkaline conditions (Schlesinger et al., 2012) or during flocculation of Muriellopsis sp. cultures using polyelectrolyte EM16 (Granados et al., 2012). When harvesting Chlorella cells suspended in UMM with MSY, the required flocculant doses were significantly lower compared to aluminum sulfate, cationic starch, magnetic particles or chitosan flocculation under culture media conditions (AOM presence), but similar to the cationic biopolymer cassia gum (Table 2). Nevertheless when considering the application of a particular flocculant, the dependency upon algal species
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G. Prochazkova et al. / Bioresource Technology 177 (2015) 28–33
Table 2 Effect of different flocculants, medium composition and presence of algogenic organic matter (AOM) on flocculant dosage and harvesting efficiency during harvesting of Chlorella sp.
a b c d
Flocculant
FA:MAa (mg/g); Xc (g/L)
HEb (%)
Medium; pH
AOMd
References
Cationic cassia gum Moringa oleifera seed flour Aluminum sulfate Magnetic particles MagSilica 50–85 Uncoated magnetite microparticles Chitosan Chitosan Cationic starch Greenfloc 120 Cationic starch Greenfloc 120 Aluminum sulfate Chitosan–Fe3O4 nanoparticle composites Modified spent yeast (MSY) Modified spent yeast (MSY)
46.6; 0.75 1000; 1 1000; 1 3000; 0.65 380; 0.8 300; 0.25 32; 0.25 360; 0.25
93 88 94 90 90 85 85 85
TAP; 7.6 WC; 9.3 WC; 8.5 IGV; 8 Chu 13; 7–9 WC; 7.5 WC; 7.5 WC; 8
Yes Yes Yes Yes Yes Yes No Yes
Banerjee et al. (2014) Teixeira et al. (2012) Teixeira et al. (2012) Cerff et al. (2012) Xu et al. (2011) Vandamme et al. (2012b) Vandamme et al. (2012b) Vandamme et al. (2012b)
80; 0.25
85
WC; 8
No
Vandamme et al. (2012b)
460; 0.25 1400; 1 51; 0.65 0.4; 0.2
85 99 90 91
WC; 5.5 Modified N8; 6.5 UMM; 6.8 MM-P; 6.8
Yes Yes Yes No
Vandamme et al. (2012b) Lee et al. (2013) This work This work
Flocculant (FA) to microalgae (MA) mass ratio. Harvesting efficiency (%). Dry microalgal biomass concentration (g/L). Presence of AOM during flocculation experiments.
35
MSY:CV (mg/g)
30 25 20 15 10 5 0
CMM
MM-P MM-N MM-S Culture medium composion
MM-Fe
Fig. 4. Mass ratios of modified spent yeast (MSY) flocculant to microalgae (CV) tested in fresh complete mineral medium (CMM) and fresh mineral media lacking phosphorus (MM-P), nitrogen (MM-N), sulfur (MM-S) or iron (MM-Fe) for harvesting efficiencies over 90%. Initial biomass concentrations were 0.2 g/L.
MSY:CV (mg/g)
150
100
As in the case of flocculation under model environment (10 mM KCl), the concentration factors in CMM, MM-P, MM-N and MM-Fe reached up to 36, whereas the concentration factor in UMM was only 15. The observed concentration factor decrease can be explained by the presence of AOM, which can either sterically hinder the MSY-microalgae interaction and/or cause repulsive electrostatic interactions (Henderson et al., 2008). Nevertheless, the concentration factor of 15 achieved for C. vulgaris in this work with MSY (51 mg/L) is higher than the concentration factor 10 when harvesting Chlorella with 120 mg/L chitosan (Rashid et al., 2013). An economically viable production of microalgal biomass is a practical challenge. When pre-selecting the most appropriate flocculants, various aspects of the process (microalgal species, required concentration factor, reuse of media, presence of AOM, etc.) and nature of the product (food, feed, cosmetic, fuel, etc.) have to be taken into account. Finally the most decisive selection criteria are efficiency, dosage and overall cost of separation. Several cost estimates have been published (Banerjee et al., 2014; Farid et al., 2013; Granados et al., 2012) and today there is a trend to use cheap polymeric flocculants. The MSY flocculant prepared from spent brewer’s yeast is in accordance with these requirements and in addition it represents a beneficial use for a significant brewery by-product with environmental impact. 4. Conclusions
50
0
0
0.2
0.4 0.6 0.8 1 Biomass concentraon (g/L)
1.2
Fig. 5. Mass ratios of modified spent yeast (MSY) flocculant to microalgae (CV) at different Chlorella biomass concentrations in used mineral medium (UMM) for harvesting efficiencies over 90%.
must be also taken into account. For instance when applying cationic starch only 33 mg/g were required for Scenedesmus as compared to 80 mg/L for Chlorella (Vandamme et al., 2010). It seems that larger microalgal cells such as Scenedesmus sp. and Muriellopsis sp. (Granados et al., 2012) require lower flocculant doses. Simultaneously it was found that that the HE of each polyelectrolyte varied with studied strain, thus a single polyelectrolyte cannot be selected for all microalgae (Granados et al., 2012).
Successful introduction of positively charged functional groups (DEAE) onto microparticles prepared from spent brewer’s yeast led to development of novel, highly efficient cationic flocculant (MSY) for harvesting C. vulgaris. Harvesting efficiencies above 90% were achieved for MSY doses of 0.4 mg/g in fresh mineral medium lacking phosphates and 51 mg/g in used mineral medium directly after cultivation. Results showed the importance of culture medium composition and effect of algogenic organic matter on harvesting. MSY can be regarded as one of the most dose–effective flocculants and it represents an interesting application for spent brewer’s yeast, a considerable industrial by-product. Acknowledgements The financial support by the Ministry of Education, Youth and Sports of the Czech Republic through Grants MSM6046137305 and LJ12002 (realized in program GESHER/MOST, MSMT-10317/ 2012-36) is gratefully acknowledged.
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References Anthony, R., Sims, R., 2013. Cationic starch for microalgae and total phosphorus removal from wastewater. J. Appl. Polym. Sci. 130, 2572–2578. Banerjee, C., Ghosh, S., Sen, G., Mishra, S., Shukla, P., Bandopadhyay, R., 2014. Study of algal biomass harvesting through cationic cassia gum, a natural plant based biopolymer. Bioresour. Technol. 151, 6–11. Branyik, T., Vicente, A.A., Machado Cruz, J.M., Teixeira, J.A., 2001. Spent grains – a new support for brewing yeast immobilisation. Biotechnol. Lett. 23, 1073–1078. Cerff, M., Morweiser, M., Dillschneider, R., Michel, A., Menzel, K., Posten, C., 2012. Harvesting fresh water and marine algae by magnetic separation: screening of separation parameters and high gradient magnetic filtration. Bioresour. Technol. 118, 289–295. Dubois, M., Gilles, K.A., Hamilton, J.K., Rebers, P.A., Smith, F., 1956. Colorimetric method for determination of sugars and related substances. Anal. Chem. 28, 350–356. Farid, M.S., Shariati, A., Badakhshan, A., Anvaripour, B., 2013. Using nano-chitosan for harvesting microalga Nannochloropsis sp. Bioresour. Technol. 131, 555–559. Granados, M.R., Acién, F.G., Gómez, C., Fernández-Sevilla, J.M., Molina Grima, E., 2012. Evaluation of flocculants for the recovery of freshwater microalgae. Bioresour. Technol. 118, 102–110. Harun, R., Singh, M., Forde, G.M., Danquah, M.K., 2010. Bioprocess engineering of microalgae to produce a variety of consumer products. Renew. Sustain. Energy Rev. 14, 1037–1047. Henderson, R.K., Baker, A., Parsons, S.A., Jefferson, B., 2008. Characterisation of algogenic organic matter extracted from cyanobacteria, green algae and diatoms. Water Res. 42, 3435–3445. Huige, N.J., 2006. Brewery by-products and effluents. In: Priest, F.G., Stewart, G.G. (Eds.), Handbook of Brewing, second ed. CRC Press, USA, pp. 655–713. Chae, H.J., Joo, H., In, M.-J., 2001. Utilization of brewer’s yeast cells for the production of food-grade yeast extract. Part 1: effects of different enzymatic treatments on solid and protein recovery and flavor characteristics. Bioresour. Technol. 76, 253–258. Cheng, Y.-S., Zheng, Y., Labavitch, J.M., VanderGheynst, J.S., 2011. The impact of cell wall carbohydrate composition on the chitosan flocculation of Chlorella. Process Biochem. 46, 1927–1933. Lee, K., Lee, S.Y., Na, J.-G., Jeon, S.G., Praveenkumar, R., Kim, D.-M., Chang, W.-S., Oh, Y.-K., 2013. Magnetophoretic harvesting of oleaginous Chlorella sp. by using biocompatible chitosan/magnetic nanoparticle composites. Bioresour. Technol. 149, 575–578. Marsalkova, B., Sirmerova, M., Kurec, M., Branyik, T., Branyikova, I., Melzoch, K., Zachleder, V., 2010. Microalgae Chlorella sp. as an alternative source of fermentable sugars. Chem. Eng. Trans. 21, 1279–1284. Milledge, J., 2011. Commercial application of microalgae other than as biofuels: a brief review. Rev. Environ. Sci. Biotechnol. 10, 31–41. Pragya, N., Pandey, K.K., Sahoo, P.K., 2013. A review on harvesting, oil extraction and biofuels production technologies from microalgae. Renew. Sustain. Energy Rev. 24, 159–171. Prochazkova, G., Podolova, N., Safarik, I., Zachleder, V., Branyik, T., 2013a. Physicochemical approach to freshwater microalgae harvesting with magnetic particles. Colloids Surf. B 112, 213–218.
33
Prochazkova, G., Safarik, I., Branyik, T., 2013b. Harvesting microalgae with microwave synthesized magnetic microparticles. Bioresour. Technol. 130, 472–477. Rashid, N., Rehman, S.U., Han, J.-I., 2013. Rapid harvesting of freshwater microalgae using chitosan. Process Biochem. 48, 1107–1110. Ryu, B.-G., Kim, K., Kim, J., Han, J.-I., Yang, J.-W., 2013. Use of organic waste from the brewery industry for high-density cultivation of the docosahexaenoic acid-rich microalga, Aurantiochytrium sp. KRS101. Bioresour. Technol. 129, 351–359. Saksinchai, S., Suphantharika, M., Verduyn, C., 2001. Application of a simple yeast extract from spent brewer’s yeast for growth and sporulation of Bacillus thuringiensis subsp. kurstaki: a physiological study. World J. Microbiol. Biotechnol. 17, 307–316. Shelef, G., Sukenik, A., Green, M., 1984. Microalgae Harvesting and Processing: A Literature Review. SERI/STR-231-2396. Solar Energy Research Institute, Golden Colorado. Schlesinger, A., Eisenstadt, D., Bar-Gil, A., Carmely, H., Einbinder, S., Gressel, J., 2012. Inexpensive non-toxic flocculation of microalgae contradicts theories; overcoming a major hurdle to bulk algal production. Biotechnol. Adv. 30, 1023–1030. Sukenik, A., Shelef, G., 1984. Algal autoflocculation—verification and proposed mechanism. Biotechnol. Bioeng. 26, 142–147. Teixeira, C., Kirsten, F., Teixeira, P., 2012. Evaluation of Moringa oleifera seed flour as a flocculating agent for potential biodiesel producer microalgae. J. Appl. Phycol. 24, 557–563. Uduman, N., Qi, Y., Danquah, M.K., Forde, G.M., Hoadley, A., 2010. Dewatering of microalgal cultures: a major bottleneck to algae-based fuels. J. Renew. Sustain. Energy 2, 1–15. Vandamme, D., Foubert, I., Muylaert, K., 2013. Flocculation as a low-cost method for harvesting microalgae for bulk biomass production. Trends Biotechnol. 31, 233– 239. Vandamme, D., Foubert, I., Fraeye, I., Meesschaert, B., Muylaert, K., 2012a. Flocculation of Chlorella vulgaris induced by high pH: role of magnesium and calcium and practical implications. Bioresour. Technol. 105, 114–119. Vandamme, D., Foubert, I., Fraeye, I., Muylaert, K., 2012b. Influence of organic matter generated by Chlorella vulgaris on five different modes of flocculation. Bioresour. Technol. 124, 508–511. Vandamme, D., Foubert, I., Meesschaert, B., Muylaert, K., 2010. Flocculation of microalgae using cationic starch. J. Appl. Phycol. 22, 525–530. Venkatachalam, G., Gummadi, S., Doble, M., 2013. Extraction and purification of cyclic b-glucan. In: Cyclic b-Glucans from Microorganisms. Springer, Berlin, pp. 63–69. Wu, Y., Guo, J., Yang, W., Wang, C., Fu, S., 2006. Preparation and characterization of chitosan–poly(acrylic acid) polymer magnetic microspheres. Polymer 47, 5287– 5294. Xu, L., Guo, C., Wang, F., Zheng, S., Liu, C.-Z., 2011. A simple and rapid harvesting method for microalgae by in situ magnetic separation. Bioresour. Technol. 102, 10047–10051. Yu, X., Somasundaran, P., 1996. Role of polymer conformation in interparticlebridging dominated flocculation. J. Colloid Interface Sci. 177, 283–287. Zhang, J., Hu, B., 2012. A novel method to harvest microalgae via co-culture of filamentous fungi to form cell pellets. Bioresour. Technol. 114, 529–535.
