Bioresource Technology xxx (2014) xxx–xxx
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Mild disintegration of the green microalgae Chlorella vulgaris using bead milling P.R. Postma a,⇑, T.L. Miron a, G. Olivieri a, M.J. Barbosa b, R.H. Wijffels a,c, M.H.M. Eppink a a
Bioprocess Engineering, AlgaePARC, Wageningen University, PO Box 16, 6700 AA Wageningen, The Netherlands Food & Biobased Research, AlgaePARC, Wageningen University and Research Centre, PO Box 17, 6700 AA Wageningen, The Netherlands c University of Nordland, Faculty of Biosciences and Aquaculture, N-8049 Bodø, Norway b
h i g h l i g h t s Bead milling can be used as a mild disintegration technique for microalgae. Full cell disintegration can be achieved in less than 1 min. Selective extraction of water soluble proteins is possible with bead milling. The specific energy consumption can be kept below 2.5 kWh kgDW
a r t i c l e
i n f o
Article history: Received 21 July 2014 Received in revised form 29 August 2014 Accepted 1 September 2014 Available online xxxx Keywords: Mild biorefinery Cell disintegration Bead mill Chlorella vulgaris Protein
1
.
a b s t r a c t In this work, the mild disintegration of the microalgae Chlorella vulgaris for the release of intracellular products has been studied. By means of bead milling the microalgae suspensions were successfully disintegrated at different biomass concentrations (25–145 gDW kg1) over a range of agitator speeds (6–12 m s1). In all cases over 97% of cell disintegration was achieved resulting in a release of water soluble proteins. A clear optimum rate of disintegration and protein release was observed at an agitator speed of 9–10 m s1 regardless of the biomass concentration. Selective extraction of water soluble proteins was observed as proteins released sooner than cell disintegration took place. Proteins could be released at 85% lower energy input than for cell disintegration resulting in specific energy consumptions well below 2.5 kWh kg1 DW. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction There is an ongoing search for new protein sources to meet the growing global food and animal feed demand (Godfray et al., 2010; Tilman et al., 2011). Microalgae can be identified and developed as novel green sources for food and/or feed (Batista et al., 2013; Becker, 2007). The microalgae Chlorella vulgaris contain 48–58% (w/w DW biomass) protein, 8–17% carbohydrates, 14–22% lipids and 6–9% ash (Spolaore et al., 2006). Besides, the amino acid composition of C. vulgaris compares favourably to other food like soybean as well as the World Health Organization reference composition (Becker, 2007). With the current estimated production costs of microalgae being € 4–10 kg1 dry weight (Norsker et al., 2011; Wijffels et al., 2010), solely production of a single product, like lipids or proteins,
⇑ Corresponding author. Tel.: +31 (0)317 48 3229. E-mail address: [email protected] (P.R. Postma).
from microalgae is not economically feasible. All valuable components such as proteins, lipids and carbohydrates must be used after extraction and separation from the microalgal biomass in order to become competitive as a source for commodities (Wijffels et al., 2010). Biorefinery comprises the downstream processing of all products produced by microalgae after cultivation. It should be taken into account, that for most biotechnological processes the costs for downstream processing can be regarded as very influential on the total costs (Delrue et al., 2012) and account up to 60% of the total product production costs (Molina Grima et al., 2003). Therefore, effort should be put in minimizing the energy requirements and capital costs (Vanthoor-Koopmans et al., 2013), not only by more energy efficient processes, but also by improving the product yield of the individual process steps with respect to downstream processing. To obtain the valuable intracellular components from the microalgae, cell disintegration is the first step to take after cell harvesting (Grimi et al., 2014; Molina Grima et al., 2003; Safi et al., 2014).
http://dx.doi.org/10.1016/j.biortech.2014.09.033 0960-8524/Ó 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Postma, P.R., et al. Mild disintegration of the green microalgae Chlorella vulgaris using bead milling. Bioresour. Technol. (2014), http://dx.doi.org/10.1016/j.biortech.2014.09.033
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Various methods for cell disintegration have been applied, e.g., enzymatic treatment, alkali treatment, high-pressure homogenization, bead milling, microwaves, pulsed electric field and ultrasonication (Doucha and Lívansky´, 2008; Gerken et al., 2013; Grimi et al., 2014; Lee et al., 2010; Safi et al., 2014; Zheng et al., 2011). Many of those disintegration treatments expose the cells and its content to harsh conditions like high pressures, high shear levels or high temperatures. To secure the solubility and functionality of water soluble proteins, temperatures above 35 °C should be avoided. Therefore, this process step needs to be performed under mild conditions i.e., avoid loss of product functionality. However, mild methods could result in low extraction yields of the products of interest (Grimi et al., 2014). On the other hand, Grimi et al. (2014) also showed that with the use of generally regarded harsh methods, selective extraction of several microalgal components, among which water soluble proteins, is possible. Recent design improvements and the possibility to control the temperature of the algae suspension, make the bead mill a suitable disintegration technique. A bead mill is a homogenizer originally designed for size reduction of paint or lacquer particles (Kula and Schütte, 1987). However, it has shown to be a useful technique for the mild disintegration of microbial cells and the release of proteins from these cells as well (Balasundaram and Pandit, 2001; Balasundaram et al., 2012; Chisti and Moo-Young, 1986; Kula and Schütte, 1987). Doucha and Lívansky´ (2008) showed that bead mills also can be used for disintegration of microalgae. Schwenzfeier et al. (2011) used a bead mill for disintegration of microalgae, which were kept frozen before use, for the release of soluble proteins. Nevertheless, to our knowledge bead mills have so far not been used for mild release of water soluble proteins from fresh microalgae. A bead mill can be operated under batch (recirculation) or continuous (single passage through milling chamber) conditions with respect to the biomass suspension. Many parameters affect the cell disruption efficiency, including: suspension feed rate for continuous operation and process time for batch operation (i.e., the residence time distribution), agitator speed, agitator design, milling chamber design, biomass concentration, bead diameter, bead density and the bead filling (Doucha and Lívansky´, 2008). From these parameters the biomass concentration (Cx) and agitator speed (us) have been identified as the most influential parameters on process time, disintegration efficiency and on the energy consumption. The aim of this work is to set the bead mill as a benchmark for mild disintegration technologies. This is achieved by evaluating the effect of biomass concentration and agitator speed during bead milling on water soluble protein extractability from C. vulgaris. In addition the process is optimized considering soluble protein yield, energy consumption and process time.
2. Methods 2.1. Microalgae and cultivation The green microalgae C. vulgaris (SAG 211-11b, EPSAG Göttingen) were grown in repeated batches in M8a-medium (Mandalam and Palsson, 1998) at 25 °C. The 12 L stirred tank photobioreactor (PBR) was stirred at 300 RPM and air was supplied at 3 L min1 with on demand CO2 supply to control the pH at 7.0. The PBR was illuminated with 50 Halogen lamps (Philips Master Line ES, 45 W, 60°), equally distributed over the reactor surface. The incident light intensity was increased from 400 to 1100 lmol m2 s1 along the course of biomass growth. For consistent biomass production, the algae of different batches were harvested during late linear growth at an optical density at 750 nm
(OD750) of 15 (corresponding to 5 g kg1). The algae suspensions were stored at 4 °C for maximal seven days until further use. Prior to disintegration the algal suspensions were concentrated by centrifugation (3500g, 20 min and 4 °C) and the supernatant was discarded. The biomass pellet was resuspended in phosphate buffered saline (PBS) (1.54 mM KH2PO4, 2.71 mM Na2HPO4.2 H2O, 155.2 mM NaCl at pH 7.0) to obtain suspensions with a Cx of 25, 87.5 or 145 g kg1 in a total volume of 185 ml. Cx is expressed as g dried biomass per kg algae suspension. 2.2. Bead mill experimental setup A Dyno-Mill Research Lab (RL) from Willy A. Bachofen AG Maschinenfabrik, Muttenz, Switzerland was used for disintegration experiments. The DYNO-mill consists of a horizontal milling chamber (Vchamber = 79.6 ml) with the engine connected over a central shaft. The algal suspension is fed to a worm screw mounted on the same shaft by a feed funnel. The beads are accelerated in a radial direction opposite to the rotational direction of the shaft by a single DYNO-accelerator (Ø 56.2 mm). Different layers of beads will be formed moving at different speeds causing the grinding effect. The major part of the energy introduced will be converted to heat due to friction. A cooling jacket integrated in the milling chamber and a cooling coil in the feed funnel were used to remove the heat and to keep the temperature below 35 °C. An external water bath was connected to the cooling jacket and coil. The algal suspension and the grinding beads are separated by a sieve plate at the outlet. After which the algal suspension is fed back to the feed funnel. Based on the advice and experience of the manufacturer Ø 1 mm ZrO2 beads (Tosoh YTZÒ) where chosen at a milling chamber filling percentage of 65% v/v. During initial trials these parameters showed promising results for algal cell disintegration (data not shown) and where therefore fixed for further experiments. 2.3. Design of experiments Modde v.9.1 Design of Experiments (DOE) software (MKS Umetrics, Sweden) was used as described by Desai et al. (2014) to study the effect of Cx and us on the cell disintegration rate kdis, soluble protein release rate kprot, pigment release rate kr, specific energy consumption E/M and soluble protein yield Yp during bead milling. For the DOE a response surface modeling (RSM) design, or more explicit a central composite face-centred design (CCF), was used. Two independent parameters (factors), us and Cx at three levels were studied, which leads to 8 experiments covering the design space. To give a good estimation of the replicate error, three replicates at the centre point values were conducted. The coded values for each parameter are shown in Table 1. 2.4. Analytical methods 2.4.1. Biomass quantification Dry weight concentration was determined as described by Lamers et al. (2010). The cells were washed using 0.2 lm filtered deionized water. For each harvest two known dilutions were
Table 1 Parameters and values used in CCF design. Parameters
Factor
Low value (1)
Centre value (0)
High value (+1)
Biomass concentration (g kg1) Agitator speed (m s1)
X1
25
87.5
145
X2
6
9
12
Please cite this article in press as: Postma, P.R., et al. Mild disintegration of the green microalgae Chlorella vulgaris using bead milling. Bioresour. Technol. (2014), http://dx.doi.org/10.1016/j.biortech.2014.09.033
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prepared of which the cell dry weight (DW) was determined as well as the OD750. DW/OD750 ratios were determined being 0.312 ± 0.022 SD. 2.4.2. Sample acquisition during bead milling During the bead milling experiments samples of a known amount of biomass (wet weight) were obtained from the feed funnel after 0, 2.5, 5.0, 7.5, 10.0, 15.0, 20.0, 25.0, 30.0, 45.0 and 60.0 min. Samples from 25, 87.5 and 145 g kg1 experiments were diluted 1, 2 and 5 times respectively prior to further analysis. For water soluble proteins, the pellet and supernatant were separated by centrifugation (20 min, 20,000g) and the supernatant was stored at 80 °C until further use. 2.4.3. Disintegration percentage analysis To determine the efficiency of the disintegration process, fresh samples of algae suspension from the bead milling experiments were analyzed using a flow cytometer (BD Accuri C6). The algae suspensions were diluted to a total volume of 1 mL using PBS buffer (pH 7.0) to acquire samples at less than 2500 events s1. A fixed volume of 15 lL was measured at a fluidics rate of 35 lL min1 and a core size of 16 lm. Forward scattering FSC, and Chlorophyll AutoFluorescence (670 nm filter) FLchl were measured. ‘‘FLchl count/lL’’ before (FLchl,0) and during (FLchl) disintegration was used to describe the disintegration degree Dis, as:
Dis ¼
FLchl;0 FLchl ðtÞ FLchl;0
ð1Þ
2.4.5. Pigment release The release rate of pigments was determined by measuring the absorbance between 400 and 750 nm of the supernatant using a microplate reader (Infinite M200, Tecan, Switzerland). Supernatant samples were diluted two times using PBS buffer to a total volume of 80 lL. The absorbance at 435 nm was used as a measure for the pigment (Chlorophyll) release. The fraction of released pigment R can be expressed by:
R¼
A435nm ðtÞ A435nm; max
in which A435nm(t) is the absorption at time t during bead milling, and A435nm,max the absorption observed at the end of the bead milling experiment. 2.4.6. Viscosity measurement The viscosity of the algae suspensions was measured using an MCR 502 rheometer (Anton Paar, Austria). For algae suspensions up to 87.5 g kg1 a double gap (DG26.7) measuring system was used, while a cone-plate (CP50-4) was used for suspension of 145 g kg1. The temperature was controlled at 12 °C. 2.5. Bead milling kinetics First order kinetics for bead milling processes (i.e., cell disintegration and cell content release) have been proposed by many authors (Chisti and Moo-Young, 1986; Doucha and Lívansky´, 2008; Kula and Schütte, 1987; Limon-Lason et al., 1979). The degree of disintegration can be described by:
Dis ¼ 1 ekdis t 2.4.4. Protein analysis For total protein content on dry weight basis, 6 mg of freeze dried biomass from start samples was re-suspended in 1.0 mL lysis buffer I (60 mM Tris, 2% SDS, pH 9.0) in lysing matrix E tubes (6914–500, MP Biomedicals Europe). The tubes were bead beaten for 3 cycles of 60 s at 6500 RPM with breaks of 120 s between cycles using a bead beater (Precellys 24, Bertin Technologies). For determination of the soluble protein content in the supernatant after disintegration, the supernatant samples were unfrozen and diluted 2 times using lysis buffer II (120 mM Tris, 4% SDS, pH 9.0). Subsequently, the samples for total and soluble protein content were incubated at 100 °C for 30 min. The protein quantitation was performed with a commercial assay (DC™ Protein assay, Bio-Rad, U.S.) using bovine serum albumin (Sigma–Aldrich A7030) as protein standard. The microplate assay protocol was followed, where 10 lL of each standard and unknown was used. Absorbance was measured at 750 nm using a microplate reader (Infinite M200, Tecan, Switzerland) in 96-well plates (Greiner bio-one 655101). The fraction of released water soluble proteins P can be expressed as:
P¼
C p ðtÞ C p;biomass
ð2Þ
in which Cp(t) is the protein concentration in the supernatant at time t, and Cp,biomass the total amount of protein inside the biomass per kg suspension. The water soluble protein yield can be described as:
Yp ¼
C p;max C p;biomass
ð3Þ
in which Cp,max is the maximal water soluble protein concentration at the end of the experiment.
ð4Þ
ð5Þ
where kdis is the kinetic rate constant for the fraction of disintegration. Similar expressions can be obtained for the fraction of released water soluble proteins P and fraction of released pigments R:
P ¼ 1 ekprot t
ð6Þ
where kprot is the rate constant for water soluble protein release.
R ¼ 1 ekr t
ð7Þ
in which kr is the rate constant for the release of pigments. 2.5.1. Location factor The value for the location factor is usually obtained by taking the ratio of one released product to the other (Balasundaram and Pandit, 2001). The same concept can be applied to express the relative release ratio of release rate of product i to the disintegration rate as:
LF i ¼
ki kdis
ð8Þ
where ki is the release rate of product i, where i can be either water soluble proteins (LFprot) or pigments (LFr). When the ratio of LFi is smaller than unity, the extraction of a product is slower than the disintegration rate, this could happen when a product is hardly water soluble or is located in intracellular organelles. Hence, when the ratio becomes larger than unity, the product is released faster than the disintegration rate. For instance cytoplasmic components can already easily be released by a single rupture of the cell wall. 2.5.2. Residence time The Dyno-Mill RL was operated in recirculation mode (i.e., batch operation). This means that only a part of the total volume Vtotal is inside the milling chamber, with volume Vchamber, at all times during recirculation. The volume ratio Vchamber/Vtotal can be used to
Please cite this article in press as: Postma, P.R., et al. Mild disintegration of the green microalgae Chlorella vulgaris using bead milling. Bioresour. Technol. (2014), http://dx.doi.org/10.1016/j.biortech.2014.09.033
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describe the residence time of the algae suspension in the milling chamber. Therefore, the time t of a single experiment was corrected by multiplying with Vchamber/Vtotal.
3. Results and discussion 3.1. Bead milling overall effect Using the chosen settings for Cx and us all algae suspensions could successfully be disintegrated at disintegration degrees over 97% and water soluble proteins were released. For one of the centre point runs (Cx: 87.5 g L1, us: 9 m s1), the effect of bead milling on kdis, kprot and kr are shown in Fig. 1. Besides, the first-order model predictions can be observed. Fig. 1 shows that more than 99% of cell disintegration can be obtained in a milling time of about 500 s. Besides, after 200–250 s, roughly 90–95% of the cells are already disintegrated. The first order model slightly underestimates this. By fitting the data to the first order model from Eq. (5) using least square error regression (LSER) a kdis of 0.016 s1 was obtained. The maximum released protein concentration in Fig. 1 is 17.5 mg mL1. However, the model estimates a maximum concentration of 17.8 mg mL1. A corresponding kprot of 0.02 s1 was obtained by LSER of Eq. (6). Over 90% of the proteins can be
Fig. 1. Fraction of cell disintegration (d), fraction of released protein (j) and fraction of released pigment (N) versus time for a Cx of 87.5 g kg1 and us of 9 m s1 (experiment 6). The solid lines represent the first order model.
extracted within 200 s, corresponding to an LFprot of 1.27, which indicates that the protein can be released faster than disintegration takes place. Fig. 1 also shows that there is a large difference between the release rate of the pigments and the disintegration rate of the cells. The estimated kr of 0.008 s1 results in an LFr of 0.52. These location factors show that different intracellular products can be selectively extracted with respect to the disintegration degree as also described by other authors (Balasundaram and Pandit, 2001; Grimi et al., 2014). 3.2. Effect of bead milling on process kinetics Using Eqs. (5–7) the release rates of the intracellular components, the disintegration rate and the maximum water soluble protein concentration were estimated. Different values for us and Cx were used to study the effect on the kinetic rate constants for disintegration, protein release and pigment release, the results are summarized in Table 2. R2 values of 0.97 or higher were obtained for all disintegration and protein release fits, R2 values for pigment release were above 0.85. First the single effects will be discussed and subsequently the interaction effects. For the first-order kinetics, the rate constant for disintegration should be independent of the initial Cx. However, it can be observed that Cx has a large impact on kdis, kprot and kr. Higher values for Cx lead to a higher release rate of intercellular components and to a higher disintegration rate up to a factor 3 and 2, respectively. The rheology of the algal suspensions most likely directly influences the rate constants. Algal slurries show Newtonian behavior at a Cx below 40 g kg1, and above this concentration the behavior becomes shear thinning non-Newtonian (pseudoplastic) (Wileman et al., 2012). This pseudoplastic behavior was also found for the algal suspensions used in this work at Cx values of 87.5 and 145 g kg1 (data not shown), while the suspension of 25 g kg1 showed Newtonian behavior as expected. The effective viscosity leff, at a shear rate c of 500 s1, for a Cx of 25, 87.5 or 145 g kg1 was found to be 1.77 103, 8.75 103 and 9.35 102 Pa s, respectively. This shows that there is a large difference between the effective viscosities of the different algal suspensions. Especially the high viscous slurries at a Cx of 145 g kg1 were more difficult to handle with respect to biomass losses and mixing properties and is therefore practically limiting. Besides, the power requirement for pumping of those slurries increases significantly (Wileman et al., 2012). The viscosity of algal slurries is species depended, and therefore it is safer to say that a slurry viscosity around 9 102 Pa s is practically limiting for the DYNOmill. The shear rates to be expected in a bead mill are in the order of 104–105 s1 (Limon-Lason et al., 1979), the differences in the applied us are expected to be negligible with respect to the high shear rates. Therefore shear rates in the same order of magnitude
Table 2 Overview of performed experimental runs and first-order model values. Experimental conditions
Model values
Location factors
Exp. no.
Cx (g kg1)
us (m s1)
Cp,max (g kg1)
kdis (s1)
kprot (s1)
kr (s1)
LFprot ()
LFr ()
1 2 3 4 5 6 7 8 9 10 11
25 25 25 87.5 87.5 87.5 87.5 87.5 145 145 145
6 9 12 6 9 9 9 12 6 9 12
4.6 4.3 4.2 14.1 17.2 17.8 13.9 17.5 22.2 26.6 25.8
0.008 0.009 0.009 0.010 0.021 0.016 0.015 0.015 0.013 0.021 0.019
0.010 0.015 0.012 0.013 0.020 0.020 0.016 0.019 0.022 0.034 0.027
0.004 0.003 0.002 0.006 0.005 0.008 0.006 0.005 0.014 0.021 0.010
1.22 1.55 1.23 1.30 0.95 1.27 1.05 1.24 1.73 1.63 1.39
0.50 0.28 0.26 0.56 0.23 0.52 0.39 0.34 1.11 1.03 0.54
Please cite this article in press as: Postma, P.R., et al. Mild disintegration of the green microalgae Chlorella vulgaris using bead milling. Bioresour. Technol. (2014), http://dx.doi.org/10.1016/j.biortech.2014.09.033
P.R. Postma et al. / Bioresource Technology xxx (2014) xxx–xxx
are assumed at us between 6 and 12 m s1. The energy dissipation S towards the suspension causing collision of the beads is related to the shear rate by S ¼ leff c_ 2 (Gers et al., 2010). At constant shear rates, this means that S, and therefore k, is directly influenced by the suspension viscosity. Gers et al. (2010) observed that the region of high energy dissipation decreased with increased viscosity (Reynolds number), while the maximum energy dissipation Smax increased. Doucha and Lívansky´ (2008) found an opposing effect of Cx on the degree of disintegration of Chlorella cells. For a Dyno-Mill KDL and a Labstar LS1 the degree decreased, while it increased for the MS 18 homogenizer. Mogren et al. (1974) found similar disintegration degrees for yeast cells at a varying Cx in a Dyno-Mill KD5. Nevertheless, they also observed a large dependence on the used strain. Limon-Lason et al. (1979) found that the disintegration rate constant decreased with an increasing Cx. Marffy and Kula (1974) found increasing rate constants for an increasing Cx using a Dyno-Mill KDL. For us, it can be seen that a higher speed resulted in higher disintegration rates. This effect can be explained by the increase of the impact forces and frequency with increasing us. However an optimum can be observed in Fig. 2a and b at 9–10 m s1 for kdis and for kprot. No significant difference (P > 0.05) could be found for kr
5
(Fig. 2c). An optimum kinetic rate constant was also observed by other authors (Currie et al., 1972; Kula and Schütte, 1987; Limon-Lason et al., 1979; Marffy and Kula, 1974). In order to break the cells, a certain stress intensity needs to be applied, however once this amount is supplied an additional increase of stress intensity cannot be utilized for faster disintegration (Bunge et al., 1992). Nevertheless, a clear decrease in the kinetic rate constant can be observed as well at us above 10 m s1. During disintegration, the cell contents are released among which DNA, causing an increase of the viscosity. On the other hand, due to the temperature increase of the suspension the viscosity is decreasing. Based on the viscosity measurements it was observed that these two effects might cancel each other out and therefore the effective viscosity is approximately constant at high shear rates in the bead mill (data not shown). Yet, Gers et al. (2010) found that for increasing Reynolds numbers (i.e., dimensionless number for flow regime characteristics), which may either be caused by an increased viscosity or increased agitation speeds, a significant shift in the flow patterns occurs. With the secondary effect that Smax decreased for Newtonian and shear thinning non-Newtonian suspensions. When looking to the interactions effects, Fig. 2a and b show that at a high Cx and at a medium to high us (9–12 m s1) higher rate constants can be achieved compared to a low Cx or a low us.
Fig. 2. Contour plots of agitator speed us [m s1] and biomass concentration Cx [g kg1] on the (a) disruption rate constant kdis [s-1], (b) protein release rate constant kprot [s1], (c) pigment release rate constant kr [s1] and (d) protein yield YP.
Please cite this article in press as: Postma, P.R., et al. Mild disintegration of the green microalgae Chlorella vulgaris using bead milling. Bioresour. Technol. (2014), http://dx.doi.org/10.1016/j.biortech.2014.09.033
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3.3. Protein yield
3.4. Energy consumption
The influence of Cx and us on the water soluble protein yield Yp is shown in Fig. 2d. No significant effect (P > 0.05) was observed for us, but it can be observed that the Yp decreases about 25% when the Cx increased from 25 to 145 g kg1. A possible cause might be the formation of insoluble protein aggregates at a high Cx due to local high protein concentrations, shear forces and interactions with other components as suggested by Wang (2005). Temperature increase was not assumed to influence protein aggregation since the temperature was kept below 35 °C in all experiments. Besides, protein stability was aimed to be enhanced by using a PBS buffer, the NaCl can increase protein solubility (Bondos and Bicknell, 2003). Nevertheless, the absolute amount of extracellular PBS buffer in the different algae suspensions is decreased with increasing algae concentration because the algae occupy more space in suspensions at a higher Cx. When the algae break during disintegration the medium composition shifts from PBS like towards the algae intracellular matrix. Especially at a high Cx the negative interactions with other components might be increased causing the formation of insoluble proteins. It was observed that these proteins are not resoluble. A similar decreasing trend in protein yield was observed by Limon-Lason et al. (1979) for disintegration of packed yeast and Schwenzfeier et al. (2011) obtained a water soluble protein concentration yield of 21% by disintegration of the green microalgae Tetraselmis sp. at a Cx of 120 g kg1. These soluble proteins showed good techno-functional properties in comparison to other food proteins. Indicating that solubility of proteins is an important characteristic for mild disintegration.
The specific energy consumption E/M of a bead mill is critical for the final process economics and sustainability. For all performed experiments, a linear increase in the specific energy consumption was observed in time. Besides, the idle energy consumption (i.e., no cells) of the Dyno-Mill accounted for 70–85% of the total energy consumption. Due to the high energy dissipation at a Cx of 25 g kg1 and us of 12 m s1, excessive temperature increases were observed which required breaks during the process to cool down the algae suspension. No temperature issues were observed at the other conditions for a total process time of 60 min. Fig. 3a shows the influence of us at a Cx of 87.5 g kg1 on the fraction of disintegration as function of the specific energy consumption, it can be seen that the specific energy consumption almost triples when increasing us from 6 to 12 m s1. Looking at a constant us of 9 m s1 for different values of Cx in Fig. 3b, it can be clearly observed that a higher Cx can be disintegrated quicker at lower energy expenses. Table 3 gives an overview of the specific energy consumption E/M, where E is the used energy (kWh) and M is the amount of disintegrated biomass (kgDW). It can be seen that the specific energy consumption decreases with increasing Cx and with decreasing us. The contour plot in Fig. 4a shows that the specific energy consumption is most energy efficient at low to moderate us and medium to high Cx. In this case, E/M is kept below 10 kWh kg1 DW which is similar to what Doucha and Lívansky´ (2008) reported for a disintegration degree of 90.6%. Nevertheless, when taking the kinetic rate constants into account as described above, selective extraction
Fig. 3. Influence of us [m s1] on (a) specific energy consumption and (b) influence of Cx [g kg1] on specific energy consumption [kWh kg1 DW].
Table 3 Overview of specific energy consumption. Experimental conditions
Energy consumption
Exp. No.
Cx (g kg1 sus)
us (m s1)
M (kgDW)
Dis (%)
E (kWh)
E/M (kWh kg1 DW)
s (sec)
E3s (kWh)
E/M3s (kWh kg1 DW)
g (%)
1 2 3 4 5 6 7 8 9 10 11
25 25 25 87.5 87.5 87.5 87.5 87.5 145 145 145
6 9 12 6 9 9 9 12 6 9 12
4.7 4.7 4.6 16.2 16.2 16.2 16.1 16.3 27.0 26.9 26.8
98.4 98.6 99.5 97.1 99.7 99.8 99.7 99.7 97.6 99.6 98.6
0.094 0.247 0.424 0.093 0.180 0.188 0.181 0.248 0.120 0.229 0.288
20.45 53.42 91.98 5.92 11.12 11.67 11.24 15.23 4.55 8.51 10.87
67.7 47.6 59.8 51.9 34.6 34.7 43.4 36.6 31.8 20.4 26.1
0.035 0.065 0.140 0.027 0.034 0.036 0.044 0.050 0.021 0.026 0.042
7.64 14.10 30.38 1.71 2.12 2.23 2.71 3.10 0.81 0.96 1.58
125.6 231.8 499.4 28.0 34.9 36.6 44.5 50.9 13.3 15.8 25.9
Please cite this article in press as: Postma, P.R., et al. Mild disintegration of the green microalgae Chlorella vulgaris using bead milling. Bioresour. Technol. (2014), http://dx.doi.org/10.1016/j.biortech.2014.09.033
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7
1 Fig. 4. Contour plots of (a) specific energy consumption E/M [kWh kg1 ] and DW] aimed on cell disintegration and (b) aimed on protein release for agitator speed us [m s biomass concentration Cx [g kg1].
of water soluble proteins is possible before reaching full disintegration of the algae suspension. The doubling time s (i.e., the time required to double the released protein concentration) can be described as:
s ¼ k1 prot lnð2Þ
ð9Þ
An overview of s for each experiment is listed in table 3. From Fig. 3 it can be observed that disintegration degrees beyond 85–90% requires excessive amounts of additional energy which is in close relation to what was found by Mogren et al. (1974). Therefore 3 doubling times 3s, corresponding to 87.5% protein release, were considered to recalculate the specific energy consumption E/M3s with:
E=M 3s ¼ E=M
3s t
ð10Þ
where t is the total time of the experiment (s). This results in an energy reduction between 60% and 88%. The contour plot with the aim on protein release is shown in Fig. 4b. The contour line corresponding to 10 kWh kg1 DW shifted drastically, opening the possibility to operate at a low Cx. For a Cx above 60 g kg1 specific energy consumptions below 3 kWh kg1 DW can be achieved. Doucha and Lívansky´ (2008) found 3.11 kWh kg1 DW at 58% disintegration and 2.82 kWh kg1 DW at 77% disintegration for suspensions of Chlorella although no values for protein release were reported. Balasundaram et al. (2012) designed a ball mill capable of disintegrating the cyanobacteria Chlorogloeopsis fritschii at an E/M of 1 1.87 kWh kg1 DW. In this study the lowest E/M3s of 0.81 kWh kgDW 1 1 was found at a Cx of 145 g kg and an us of 6 m s . Taking the energy content of algae as 21.9 kJ g1 biomass (Weyer et al., 2010), the energy consumption per energy content g of the algae is then 13.3%. For the other experiments the ratio g is shown in Table 3. Many other techniques have been proposed for cell disintegration, but only few are considered mild enough to keep the intracellular components intact. Grimi et al. (2014) used pulsed electric field, high voltage electrical discharges, ultra-sonication and high pressure homogenization in sequential mode on a 1% w/w suspension of Nannochloropsis sp. and found protein extraction values of 5.2%, 1.15%, 1.8% and 91%, respectively from a total protein yield of 70% water soluble protein (corresponding to 1.4 g protein per kg suspension). Pulsed electric field, high voltage electrical discharge, ultra-sonication and high pressure homogenization required up to 1.5, 1.5, 0.5 and 25.0 kWh kg1 DW respectively.
C. vulgaris and Nannochloropsis oculata were treated by high pressure homogenization (2% w/w suspension) by Safi et al. (2014) to release 51.7% and 49.5% water soluble proteins at a specific energy consumption of 7.5 kWh kg1 DW. The microalgae Auxenochlorella protothecoides was treated with pulsed electric field by Goettel et al. (2013) at a specific energy consumption of 0.40 kWh kg1 DW. Nevertheless, hardly any proteins were released. 3.5. Model fit and analysis In order to create the contour plots, a second order polynomial regression model was used for each output. These plots can visualize the effects of the two input parameters, us and Cx on each of the outputs (Desai et al., 2014). Using the Umetrics software, regression models were obtained for the kprot, kdis, Yp and for the E/M. Model coefficients which were insignificant at a confidence interval of 95% were removed.
kprot ¼ 1:6994 þ 0:176 X 1 þ 0:0491 X 2 0:0945 X 22 R2 ¼ 0:90
ð11Þ
kdis ¼ 1:8014 þ 0:1385 X 1 þ 0:0602 X 2 0:1126 X 22 R2 ¼ 0:77
ð12Þ
Y prot ¼ 0:4421 0:0798 X 1 R2 ¼ 0:84
ð13Þ
E=M ¼ 1:0567 0:3963 X 1 þ 0:2403 X 2 þ 0:2692 X 21 0:0822 X 22 0:0687X 1 X 2 2
R ¼ 0:99
ð14Þ
E=M3s ¼ 0:3664 0:5709 X 1 þ 0:1914 X 2 þ 0:2343 X 21 0:0773 X 1 X 2 R2 ¼ 0:98
ð15Þ
Each of the models shows a good correlation between the experimental data and the predicted data. The models were statistically evaluated using analysis of variance (ANOVA) from which was found that each of the models is significant. Two F-tests were performed in the ANOVA both assessed at a probability of 95%. The first is for the model regression, comparing the variance in the regression to the variance of the residuals and is significant when P < 0.05. The second for the lack of fit, this compares the replicate error to the model error and is significant when P > 0.05.
Please cite this article in press as: Postma, P.R., et al. Mild disintegration of the green microalgae Chlorella vulgaris using bead milling. Bioresour. Technol. (2014), http://dx.doi.org/10.1016/j.biortech.2014.09.033
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4. Conclusions A bead mill can successfully be applied for mild disintegration of microalgae slurries. Increasing biomass concentrations and agitator speeds lead to increased kinetic rate constants. Yet, an optimal agitator speed was found regardless of the biomass concentration. Water soluble protein release was found to be quicker than the biomass disintegration, while pigment release was found to be slower than the biomass disintegration. The water soluble protein yield ranged between 32% and 42%. By means of selective protein extraction, up to 85% energy reduction is realized. Specific energy consumptions as low as 0.81 kWh kg1 DW can be achieved. Acknowledgements This project is financed by the IPOP Biorefinery of Wageningen University and Research Centre. We would like to thank Willy A. Bachofen AG Maschinenfabrik, Muttenz, Switzerland for giving the opportunity to perform initial trials with the DYNO-mill RL. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.biortech.2014. 09.033. References Balasundaram, B., Pandit, A.B., 2001. Significance of location of enzymes on their release during microbial cell disruption. Biotechnol. Bioeng. 75, 607–614. Balasundaram, B., Skill, S.C., Llewellyn, C.A., 2012. A low energy process for the recovery of bioproducts from cyanobacteria using a ball mill. Biochem. Eng. J. 69, 48–56. Batista, A.P., Gouveia, L., Bandarra, N.M., Franco, J.M., Raymundo, A., 2013. Comparison of microalgal biomass profiles as novel functional ingredient for food products. Algal Res. 2, 164–173. Becker, E.W., 2007. Micro-algae as a source of protein. Biotechnol. Adv. 25, 207–210. Bondos, S.E., Bicknell, A., 2003. Detection and prevention of protein aggregation before, during, and after purification. Anal. Biochem. 316, 223–231. Bunge, F., Pietzsch, M., Müller, R., Syldatk, C., 1992. Mechanical disruption of Arthrobacter sp. DSM 3747 in stirred ball mills for the release of hydantoincleaving enzymes. Chem. Eng. Sci. 47, 225–232. Chisti, Y., Moo-Young, M., 1986. Disruption of microbial cells for intracellular products. Enzyme Microb. Technol. 8, 194–204. Currie, J.A., Dunnill, P., Lilly, M.D., 1972. Release of protein from Bakers’ yeast (Saccharomyces cerevisiae) by disruption in an industrial agitator mill. Biotechnol. Bioeng. 14, 725–736. Delrue, F., Setier, P.-A., Sahut, C., Cournac, L., Roubaud, A., Peltier, G., Froment, A.-K., 2012. An economic, sustainability, and energetic model of biodiesel production from microalgae. Bioresour. Technol. 111, 191–200. Desai, R.K., Streefland, M., Wijffels, R.H., Eppink, M.H.M., 2014. Extraction and stability of selected proteins in ionic liquid based aqueous two phase systems. Green Chem.. Doucha, J., Lívansky´, K., 2008. Influence of processing parameters on disintegration of Chlorella cells in various types of homogenizers. Appl. Microbiol. Biotechnol. 81, 431–440.
Gerken, H.G., Donohoe, B., Knoshaug, E.P., 2013. Enzymatic cell wall degradation of Chlorella vulgaris and other microalgae for biofuels production. Planta 237, 239– 253. Gers, R., Climent, E., Legendre, D., Anne-Archard, D., Frances, C., 2010. Numerical modelling of grinding in a stirred media mill: hydrodynamics and collision characteristics. Chem. Eng. Sci. 65, 2052–2064. Godfray, H.C.J., Beddington, J.R., Crute, I.R., Haddad, L., Lawrence, D., Muir, J.F., Pretty, J., Robinson, S., Thomas, S.M., Toulmin, C., 2010. Food security: the challenge of feeding 9 billion people. Science 327, 812–818. Goettel, M., Eing, C., Gusbeth, C., Straessner, R., Frey, W., 2013. Pulsed electric field assisted extraction of intracellular valuables from microalgae. Algal Res. 2, 401– 408. Grimi, N., Dubois, A., Marchal, L., Jubeau, S., Lebovka, N.I., Vorobiev, E., 2014. Selective extraction from microalgae Nannochloropsis sp. using different methods of cell disruption. Bioresour. Technol. 153, 254–259. Kula, M.-R., Schütte, H., 1987. Purification of proteins and the disruption of microbial cells. Biotechnol. Prog. 3, 31–42. Lamers, P.P., van de Laak, C.C.W., Kaasenbrood, P.S., Lorier, J., Janssen, M., De Vos, R.C.H., Bino, R.J., Wijffels, R.H., 2010. Carotenoid and fatty acid metabolism in light-stressed Dunaliella salina. Biotechnol. Bioeng. 106, 638–648. Lee, J.-Y., Yoo, C., Jun, S.-Y., Ahn, C.-Y., Oh, H.-M., 2010. Comparison of several methods for effective lipid extraction from microalgae. Bioresour. Technol. 101, S75–S77. Limon-Lason, J., Hoare, M., Orsborn, C.B., Doyle, D.J., Dunnill, P., 1979. Reactor properties of a high-speed bead mill for microbial cell rupture. Biotechnol. Bioeng. 21, 745–774. Mandalam, R.K., Palsson, B., 1998. Elemental balancing of biomass and medium composition enhances growth capacity in high-density Chlorella vulgaris cultures. Biotechnol. Bioeng. 59, 605–611. Marffy, F., Kula, M.-R., 1974. Enzyme yields from cells of brewer’s yeast disrupted by treatment in a horizontal disintegrator. Biotechnol. Bioeng. 16, 623–634. Mogren, H., Lindblom, M., Hedenskog, G., 1974. Mechanical disintegration of microorganisms in an industrial homogenizer. Biotechnol. Bioeng. 16, 261–274. Molina Grima, E., Belarbi, E.-H., Acién Fernández, F.G., Robles Medina, A., Chisti, Y., 2003. Recovery of microalgal biomass and metabolites: process options and economics. Biotechnol. Adv. 20, 491–515. Norsker, N.-H., Barbosa, M.J., Vermuë, M.H., Wijffels, R.H., 2011. Microalgal production — a close look at the economics. Biotechnol. Adv. 29, 24–27. Safi, C., Charton, M., Ursu, A.V., Laroche, C., Zebib, B., Pontalier, P.-Y., Vaca-Garcia, C., 2014. Release of hydro-soluble microalgal proteins using mechanical and chemical treatments. Algal Res. 3, 55–60. Schwenzfeier, A., Wierenga, P.A., Gruppen, H., 2011. Isolation and characterization of soluble protein from the green microalgae Tetraselmis sp.. Bioresour. Technol. 102, 9121–9127. Spolaore, P., Joannis-Cassan, C., Duran, E., Isambert, A., 2006. Commercial applications of microalgae. J. Biosci. Bioeng. 101, 87–96. Tilman, D., Balzer, C., Hill, J., Befort, B.L., 2011. Global food demand and the sustainable intensification of agriculture. Proc. Natl. Acad. Sci. 108, 20260– 20264. Vanthoor-Koopmans, M., Wijffels, R.H., Barbosa, M.J., Eppink, M.H.M., 2013. Biorefinery of microalgae for food and fuel. Bioresour. Technol. 135, 142–149. Wang, W., 2005. Protein aggregation and its inhibition in biopharmaceutics. Int. J. Pharm. 289, 1–30. Weyer, K.M., Bush, D.R., Darzins, A., Willson, B.D., 2010. Theoretical maximum algal oil production. BioEnergy Res. 3, 204–213. Wijffels, R.H., Barbosa, M.J., Eppink, M.H.M., 2010. Microalgae for the production of bulk chemicals and biofuels. Biofuels Bioprod. Biorefin. 4, 287–295. Wileman, A., Ozkan, A., Berberoglu, H., 2012. Rheological properties of algae slurries for minimizing harvesting energy requirements in biofuel production. Bioresour. Technol. 104, 432–439. Zheng, H., Yin, J., Gao, Z., Huang, H., Ji, X., Dou, C., 2011. Disruption of Chlorella vulgaris cells for the release of biodiesel-producing lipids: a comparison of grinding, ultrasonication, bead milling, enzymatic lysis, and microwaves. Appl. Biochem. Biotechnol. 164, 1215–1224.
Please cite this article in press as: Postma, P.R., et al. Mild disintegration of the green microalgae Chlorella vulgaris using bead milling. Bioresour. Technol. (2014), http://dx.doi.org/10.1016/j.biortech.2014.09.033
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Mild disintegration of the green microalgae Chlorella vulgaris using bead milling P.R. Postma a,⇑, T.L. Miron a, G. Olivieri a, M.J. Barbosa b, R.H. Wijffels a,c, M.H.M. Eppink a a
Bioprocess Engineering, AlgaePARC, Wageningen University, PO Box 16, 6700 AA Wageningen, The Netherlands Food & Biobased Research, AlgaePARC, Wageningen University and Research Centre, PO Box 17, 6700 AA Wageningen, The Netherlands c University of Nordland, Faculty of Biosciences and Aquaculture, N-8049 Bodø, Norway b
h i g h l i g h t s Bead milling can be used as a mild disintegration technique for microalgae. Full cell disintegration can be achieved in less than 1 min. Selective extraction of water soluble proteins is possible with bead milling. The specific energy consumption can be kept below 2.5 kWh kgDW
a r t i c l e
i n f o
Article history: Received 21 July 2014 Received in revised form 29 August 2014 Accepted 1 September 2014 Available online xxxx Keywords: Mild biorefinery Cell disintegration Bead mill Chlorella vulgaris Protein
1
.
a b s t r a c t In this work, the mild disintegration of the microalgae Chlorella vulgaris for the release of intracellular products has been studied. By means of bead milling the microalgae suspensions were successfully disintegrated at different biomass concentrations (25–145 gDW kg1) over a range of agitator speeds (6–12 m s1). In all cases over 97% of cell disintegration was achieved resulting in a release of water soluble proteins. A clear optimum rate of disintegration and protein release was observed at an agitator speed of 9–10 m s1 regardless of the biomass concentration. Selective extraction of water soluble proteins was observed as proteins released sooner than cell disintegration took place. Proteins could be released at 85% lower energy input than for cell disintegration resulting in specific energy consumptions well below 2.5 kWh kg1 DW. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction There is an ongoing search for new protein sources to meet the growing global food and animal feed demand (Godfray et al., 2010; Tilman et al., 2011). Microalgae can be identified and developed as novel green sources for food and/or feed (Batista et al., 2013; Becker, 2007). The microalgae Chlorella vulgaris contain 48–58% (w/w DW biomass) protein, 8–17% carbohydrates, 14–22% lipids and 6–9% ash (Spolaore et al., 2006). Besides, the amino acid composition of C. vulgaris compares favourably to other food like soybean as well as the World Health Organization reference composition (Becker, 2007). With the current estimated production costs of microalgae being € 4–10 kg1 dry weight (Norsker et al., 2011; Wijffels et al., 2010), solely production of a single product, like lipids or proteins,
⇑ Corresponding author. Tel.: +31 (0)317 48 3229. E-mail address: [email protected] (P.R. Postma).
from microalgae is not economically feasible. All valuable components such as proteins, lipids and carbohydrates must be used after extraction and separation from the microalgal biomass in order to become competitive as a source for commodities (Wijffels et al., 2010). Biorefinery comprises the downstream processing of all products produced by microalgae after cultivation. It should be taken into account, that for most biotechnological processes the costs for downstream processing can be regarded as very influential on the total costs (Delrue et al., 2012) and account up to 60% of the total product production costs (Molina Grima et al., 2003). Therefore, effort should be put in minimizing the energy requirements and capital costs (Vanthoor-Koopmans et al., 2013), not only by more energy efficient processes, but also by improving the product yield of the individual process steps with respect to downstream processing. To obtain the valuable intracellular components from the microalgae, cell disintegration is the first step to take after cell harvesting (Grimi et al., 2014; Molina Grima et al., 2003; Safi et al., 2014).
http://dx.doi.org/10.1016/j.biortech.2014.09.033 0960-8524/Ó 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Postma, P.R., et al. Mild disintegration of the green microalgae Chlorella vulgaris using bead milling. Bioresour. Technol. (2014), http://dx.doi.org/10.1016/j.biortech.2014.09.033
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Various methods for cell disintegration have been applied, e.g., enzymatic treatment, alkali treatment, high-pressure homogenization, bead milling, microwaves, pulsed electric field and ultrasonication (Doucha and Lívansky´, 2008; Gerken et al., 2013; Grimi et al., 2014; Lee et al., 2010; Safi et al., 2014; Zheng et al., 2011). Many of those disintegration treatments expose the cells and its content to harsh conditions like high pressures, high shear levels or high temperatures. To secure the solubility and functionality of water soluble proteins, temperatures above 35 °C should be avoided. Therefore, this process step needs to be performed under mild conditions i.e., avoid loss of product functionality. However, mild methods could result in low extraction yields of the products of interest (Grimi et al., 2014). On the other hand, Grimi et al. (2014) also showed that with the use of generally regarded harsh methods, selective extraction of several microalgal components, among which water soluble proteins, is possible. Recent design improvements and the possibility to control the temperature of the algae suspension, make the bead mill a suitable disintegration technique. A bead mill is a homogenizer originally designed for size reduction of paint or lacquer particles (Kula and Schütte, 1987). However, it has shown to be a useful technique for the mild disintegration of microbial cells and the release of proteins from these cells as well (Balasundaram and Pandit, 2001; Balasundaram et al., 2012; Chisti and Moo-Young, 1986; Kula and Schütte, 1987). Doucha and Lívansky´ (2008) showed that bead mills also can be used for disintegration of microalgae. Schwenzfeier et al. (2011) used a bead mill for disintegration of microalgae, which were kept frozen before use, for the release of soluble proteins. Nevertheless, to our knowledge bead mills have so far not been used for mild release of water soluble proteins from fresh microalgae. A bead mill can be operated under batch (recirculation) or continuous (single passage through milling chamber) conditions with respect to the biomass suspension. Many parameters affect the cell disruption efficiency, including: suspension feed rate for continuous operation and process time for batch operation (i.e., the residence time distribution), agitator speed, agitator design, milling chamber design, biomass concentration, bead diameter, bead density and the bead filling (Doucha and Lívansky´, 2008). From these parameters the biomass concentration (Cx) and agitator speed (us) have been identified as the most influential parameters on process time, disintegration efficiency and on the energy consumption. The aim of this work is to set the bead mill as a benchmark for mild disintegration technologies. This is achieved by evaluating the effect of biomass concentration and agitator speed during bead milling on water soluble protein extractability from C. vulgaris. In addition the process is optimized considering soluble protein yield, energy consumption and process time.
2. Methods 2.1. Microalgae and cultivation The green microalgae C. vulgaris (SAG 211-11b, EPSAG Göttingen) were grown in repeated batches in M8a-medium (Mandalam and Palsson, 1998) at 25 °C. The 12 L stirred tank photobioreactor (PBR) was stirred at 300 RPM and air was supplied at 3 L min1 with on demand CO2 supply to control the pH at 7.0. The PBR was illuminated with 50 Halogen lamps (Philips Master Line ES, 45 W, 60°), equally distributed over the reactor surface. The incident light intensity was increased from 400 to 1100 lmol m2 s1 along the course of biomass growth. For consistent biomass production, the algae of different batches were harvested during late linear growth at an optical density at 750 nm
(OD750) of 15 (corresponding to 5 g kg1). The algae suspensions were stored at 4 °C for maximal seven days until further use. Prior to disintegration the algal suspensions were concentrated by centrifugation (3500g, 20 min and 4 °C) and the supernatant was discarded. The biomass pellet was resuspended in phosphate buffered saline (PBS) (1.54 mM KH2PO4, 2.71 mM Na2HPO4.2 H2O, 155.2 mM NaCl at pH 7.0) to obtain suspensions with a Cx of 25, 87.5 or 145 g kg1 in a total volume of 185 ml. Cx is expressed as g dried biomass per kg algae suspension. 2.2. Bead mill experimental setup A Dyno-Mill Research Lab (RL) from Willy A. Bachofen AG Maschinenfabrik, Muttenz, Switzerland was used for disintegration experiments. The DYNO-mill consists of a horizontal milling chamber (Vchamber = 79.6 ml) with the engine connected over a central shaft. The algal suspension is fed to a worm screw mounted on the same shaft by a feed funnel. The beads are accelerated in a radial direction opposite to the rotational direction of the shaft by a single DYNO-accelerator (Ø 56.2 mm). Different layers of beads will be formed moving at different speeds causing the grinding effect. The major part of the energy introduced will be converted to heat due to friction. A cooling jacket integrated in the milling chamber and a cooling coil in the feed funnel were used to remove the heat and to keep the temperature below 35 °C. An external water bath was connected to the cooling jacket and coil. The algal suspension and the grinding beads are separated by a sieve plate at the outlet. After which the algal suspension is fed back to the feed funnel. Based on the advice and experience of the manufacturer Ø 1 mm ZrO2 beads (Tosoh YTZÒ) where chosen at a milling chamber filling percentage of 65% v/v. During initial trials these parameters showed promising results for algal cell disintegration (data not shown) and where therefore fixed for further experiments. 2.3. Design of experiments Modde v.9.1 Design of Experiments (DOE) software (MKS Umetrics, Sweden) was used as described by Desai et al. (2014) to study the effect of Cx and us on the cell disintegration rate kdis, soluble protein release rate kprot, pigment release rate kr, specific energy consumption E/M and soluble protein yield Yp during bead milling. For the DOE a response surface modeling (RSM) design, or more explicit a central composite face-centred design (CCF), was used. Two independent parameters (factors), us and Cx at three levels were studied, which leads to 8 experiments covering the design space. To give a good estimation of the replicate error, three replicates at the centre point values were conducted. The coded values for each parameter are shown in Table 1. 2.4. Analytical methods 2.4.1. Biomass quantification Dry weight concentration was determined as described by Lamers et al. (2010). The cells were washed using 0.2 lm filtered deionized water. For each harvest two known dilutions were
Table 1 Parameters and values used in CCF design. Parameters
Factor
Low value (1)
Centre value (0)
High value (+1)
Biomass concentration (g kg1) Agitator speed (m s1)
X1
25
87.5
145
X2
6
9
12
Please cite this article in press as: Postma, P.R., et al. Mild disintegration of the green microalgae Chlorella vulgaris using bead milling. Bioresour. Technol. (2014), http://dx.doi.org/10.1016/j.biortech.2014.09.033
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prepared of which the cell dry weight (DW) was determined as well as the OD750. DW/OD750 ratios were determined being 0.312 ± 0.022 SD. 2.4.2. Sample acquisition during bead milling During the bead milling experiments samples of a known amount of biomass (wet weight) were obtained from the feed funnel after 0, 2.5, 5.0, 7.5, 10.0, 15.0, 20.0, 25.0, 30.0, 45.0 and 60.0 min. Samples from 25, 87.5 and 145 g kg1 experiments were diluted 1, 2 and 5 times respectively prior to further analysis. For water soluble proteins, the pellet and supernatant were separated by centrifugation (20 min, 20,000g) and the supernatant was stored at 80 °C until further use. 2.4.3. Disintegration percentage analysis To determine the efficiency of the disintegration process, fresh samples of algae suspension from the bead milling experiments were analyzed using a flow cytometer (BD Accuri C6). The algae suspensions were diluted to a total volume of 1 mL using PBS buffer (pH 7.0) to acquire samples at less than 2500 events s1. A fixed volume of 15 lL was measured at a fluidics rate of 35 lL min1 and a core size of 16 lm. Forward scattering FSC, and Chlorophyll AutoFluorescence (670 nm filter) FLchl were measured. ‘‘FLchl count/lL’’ before (FLchl,0) and during (FLchl) disintegration was used to describe the disintegration degree Dis, as:
Dis ¼
FLchl;0 FLchl ðtÞ FLchl;0
ð1Þ
2.4.5. Pigment release The release rate of pigments was determined by measuring the absorbance between 400 and 750 nm of the supernatant using a microplate reader (Infinite M200, Tecan, Switzerland). Supernatant samples were diluted two times using PBS buffer to a total volume of 80 lL. The absorbance at 435 nm was used as a measure for the pigment (Chlorophyll) release. The fraction of released pigment R can be expressed by:
R¼
A435nm ðtÞ A435nm; max
in which A435nm(t) is the absorption at time t during bead milling, and A435nm,max the absorption observed at the end of the bead milling experiment. 2.4.6. Viscosity measurement The viscosity of the algae suspensions was measured using an MCR 502 rheometer (Anton Paar, Austria). For algae suspensions up to 87.5 g kg1 a double gap (DG26.7) measuring system was used, while a cone-plate (CP50-4) was used for suspension of 145 g kg1. The temperature was controlled at 12 °C. 2.5. Bead milling kinetics First order kinetics for bead milling processes (i.e., cell disintegration and cell content release) have been proposed by many authors (Chisti and Moo-Young, 1986; Doucha and Lívansky´, 2008; Kula and Schütte, 1987; Limon-Lason et al., 1979). The degree of disintegration can be described by:
Dis ¼ 1 ekdis t 2.4.4. Protein analysis For total protein content on dry weight basis, 6 mg of freeze dried biomass from start samples was re-suspended in 1.0 mL lysis buffer I (60 mM Tris, 2% SDS, pH 9.0) in lysing matrix E tubes (6914–500, MP Biomedicals Europe). The tubes were bead beaten for 3 cycles of 60 s at 6500 RPM with breaks of 120 s between cycles using a bead beater (Precellys 24, Bertin Technologies). For determination of the soluble protein content in the supernatant after disintegration, the supernatant samples were unfrozen and diluted 2 times using lysis buffer II (120 mM Tris, 4% SDS, pH 9.0). Subsequently, the samples for total and soluble protein content were incubated at 100 °C for 30 min. The protein quantitation was performed with a commercial assay (DC™ Protein assay, Bio-Rad, U.S.) using bovine serum albumin (Sigma–Aldrich A7030) as protein standard. The microplate assay protocol was followed, where 10 lL of each standard and unknown was used. Absorbance was measured at 750 nm using a microplate reader (Infinite M200, Tecan, Switzerland) in 96-well plates (Greiner bio-one 655101). The fraction of released water soluble proteins P can be expressed as:
P¼
C p ðtÞ C p;biomass
ð2Þ
in which Cp(t) is the protein concentration in the supernatant at time t, and Cp,biomass the total amount of protein inside the biomass per kg suspension. The water soluble protein yield can be described as:
Yp ¼
C p;max C p;biomass
ð3Þ
in which Cp,max is the maximal water soluble protein concentration at the end of the experiment.
ð4Þ
ð5Þ
where kdis is the kinetic rate constant for the fraction of disintegration. Similar expressions can be obtained for the fraction of released water soluble proteins P and fraction of released pigments R:
P ¼ 1 ekprot t
ð6Þ
where kprot is the rate constant for water soluble protein release.
R ¼ 1 ekr t
ð7Þ
in which kr is the rate constant for the release of pigments. 2.5.1. Location factor The value for the location factor is usually obtained by taking the ratio of one released product to the other (Balasundaram and Pandit, 2001). The same concept can be applied to express the relative release ratio of release rate of product i to the disintegration rate as:
LF i ¼
ki kdis
ð8Þ
where ki is the release rate of product i, where i can be either water soluble proteins (LFprot) or pigments (LFr). When the ratio of LFi is smaller than unity, the extraction of a product is slower than the disintegration rate, this could happen when a product is hardly water soluble or is located in intracellular organelles. Hence, when the ratio becomes larger than unity, the product is released faster than the disintegration rate. For instance cytoplasmic components can already easily be released by a single rupture of the cell wall. 2.5.2. Residence time The Dyno-Mill RL was operated in recirculation mode (i.e., batch operation). This means that only a part of the total volume Vtotal is inside the milling chamber, with volume Vchamber, at all times during recirculation. The volume ratio Vchamber/Vtotal can be used to
Please cite this article in press as: Postma, P.R., et al. Mild disintegration of the green microalgae Chlorella vulgaris using bead milling. Bioresour. Technol. (2014), http://dx.doi.org/10.1016/j.biortech.2014.09.033
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describe the residence time of the algae suspension in the milling chamber. Therefore, the time t of a single experiment was corrected by multiplying with Vchamber/Vtotal.
3. Results and discussion 3.1. Bead milling overall effect Using the chosen settings for Cx and us all algae suspensions could successfully be disintegrated at disintegration degrees over 97% and water soluble proteins were released. For one of the centre point runs (Cx: 87.5 g L1, us: 9 m s1), the effect of bead milling on kdis, kprot and kr are shown in Fig. 1. Besides, the first-order model predictions can be observed. Fig. 1 shows that more than 99% of cell disintegration can be obtained in a milling time of about 500 s. Besides, after 200–250 s, roughly 90–95% of the cells are already disintegrated. The first order model slightly underestimates this. By fitting the data to the first order model from Eq. (5) using least square error regression (LSER) a kdis of 0.016 s1 was obtained. The maximum released protein concentration in Fig. 1 is 17.5 mg mL1. However, the model estimates a maximum concentration of 17.8 mg mL1. A corresponding kprot of 0.02 s1 was obtained by LSER of Eq. (6). Over 90% of the proteins can be
Fig. 1. Fraction of cell disintegration (d), fraction of released protein (j) and fraction of released pigment (N) versus time for a Cx of 87.5 g kg1 and us of 9 m s1 (experiment 6). The solid lines represent the first order model.
extracted within 200 s, corresponding to an LFprot of 1.27, which indicates that the protein can be released faster than disintegration takes place. Fig. 1 also shows that there is a large difference between the release rate of the pigments and the disintegration rate of the cells. The estimated kr of 0.008 s1 results in an LFr of 0.52. These location factors show that different intracellular products can be selectively extracted with respect to the disintegration degree as also described by other authors (Balasundaram and Pandit, 2001; Grimi et al., 2014). 3.2. Effect of bead milling on process kinetics Using Eqs. (5–7) the release rates of the intracellular components, the disintegration rate and the maximum water soluble protein concentration were estimated. Different values for us and Cx were used to study the effect on the kinetic rate constants for disintegration, protein release and pigment release, the results are summarized in Table 2. R2 values of 0.97 or higher were obtained for all disintegration and protein release fits, R2 values for pigment release were above 0.85. First the single effects will be discussed and subsequently the interaction effects. For the first-order kinetics, the rate constant for disintegration should be independent of the initial Cx. However, it can be observed that Cx has a large impact on kdis, kprot and kr. Higher values for Cx lead to a higher release rate of intercellular components and to a higher disintegration rate up to a factor 3 and 2, respectively. The rheology of the algal suspensions most likely directly influences the rate constants. Algal slurries show Newtonian behavior at a Cx below 40 g kg1, and above this concentration the behavior becomes shear thinning non-Newtonian (pseudoplastic) (Wileman et al., 2012). This pseudoplastic behavior was also found for the algal suspensions used in this work at Cx values of 87.5 and 145 g kg1 (data not shown), while the suspension of 25 g kg1 showed Newtonian behavior as expected. The effective viscosity leff, at a shear rate c of 500 s1, for a Cx of 25, 87.5 or 145 g kg1 was found to be 1.77 103, 8.75 103 and 9.35 102 Pa s, respectively. This shows that there is a large difference between the effective viscosities of the different algal suspensions. Especially the high viscous slurries at a Cx of 145 g kg1 were more difficult to handle with respect to biomass losses and mixing properties and is therefore practically limiting. Besides, the power requirement for pumping of those slurries increases significantly (Wileman et al., 2012). The viscosity of algal slurries is species depended, and therefore it is safer to say that a slurry viscosity around 9 102 Pa s is practically limiting for the DYNOmill. The shear rates to be expected in a bead mill are in the order of 104–105 s1 (Limon-Lason et al., 1979), the differences in the applied us are expected to be negligible with respect to the high shear rates. Therefore shear rates in the same order of magnitude
Table 2 Overview of performed experimental runs and first-order model values. Experimental conditions
Model values
Location factors
Exp. no.
Cx (g kg1)
us (m s1)
Cp,max (g kg1)
kdis (s1)
kprot (s1)
kr (s1)
LFprot ()
LFr ()
1 2 3 4 5 6 7 8 9 10 11
25 25 25 87.5 87.5 87.5 87.5 87.5 145 145 145
6 9 12 6 9 9 9 12 6 9 12
4.6 4.3 4.2 14.1 17.2 17.8 13.9 17.5 22.2 26.6 25.8
0.008 0.009 0.009 0.010 0.021 0.016 0.015 0.015 0.013 0.021 0.019
0.010 0.015 0.012 0.013 0.020 0.020 0.016 0.019 0.022 0.034 0.027
0.004 0.003 0.002 0.006 0.005 0.008 0.006 0.005 0.014 0.021 0.010
1.22 1.55 1.23 1.30 0.95 1.27 1.05 1.24 1.73 1.63 1.39
0.50 0.28 0.26 0.56 0.23 0.52 0.39 0.34 1.11 1.03 0.54
Please cite this article in press as: Postma, P.R., et al. Mild disintegration of the green microalgae Chlorella vulgaris using bead milling. Bioresour. Technol. (2014), http://dx.doi.org/10.1016/j.biortech.2014.09.033
P.R. Postma et al. / Bioresource Technology xxx (2014) xxx–xxx
are assumed at us between 6 and 12 m s1. The energy dissipation S towards the suspension causing collision of the beads is related to the shear rate by S ¼ leff c_ 2 (Gers et al., 2010). At constant shear rates, this means that S, and therefore k, is directly influenced by the suspension viscosity. Gers et al. (2010) observed that the region of high energy dissipation decreased with increased viscosity (Reynolds number), while the maximum energy dissipation Smax increased. Doucha and Lívansky´ (2008) found an opposing effect of Cx on the degree of disintegration of Chlorella cells. For a Dyno-Mill KDL and a Labstar LS1 the degree decreased, while it increased for the MS 18 homogenizer. Mogren et al. (1974) found similar disintegration degrees for yeast cells at a varying Cx in a Dyno-Mill KD5. Nevertheless, they also observed a large dependence on the used strain. Limon-Lason et al. (1979) found that the disintegration rate constant decreased with an increasing Cx. Marffy and Kula (1974) found increasing rate constants for an increasing Cx using a Dyno-Mill KDL. For us, it can be seen that a higher speed resulted in higher disintegration rates. This effect can be explained by the increase of the impact forces and frequency with increasing us. However an optimum can be observed in Fig. 2a and b at 9–10 m s1 for kdis and for kprot. No significant difference (P > 0.05) could be found for kr
5
(Fig. 2c). An optimum kinetic rate constant was also observed by other authors (Currie et al., 1972; Kula and Schütte, 1987; Limon-Lason et al., 1979; Marffy and Kula, 1974). In order to break the cells, a certain stress intensity needs to be applied, however once this amount is supplied an additional increase of stress intensity cannot be utilized for faster disintegration (Bunge et al., 1992). Nevertheless, a clear decrease in the kinetic rate constant can be observed as well at us above 10 m s1. During disintegration, the cell contents are released among which DNA, causing an increase of the viscosity. On the other hand, due to the temperature increase of the suspension the viscosity is decreasing. Based on the viscosity measurements it was observed that these two effects might cancel each other out and therefore the effective viscosity is approximately constant at high shear rates in the bead mill (data not shown). Yet, Gers et al. (2010) found that for increasing Reynolds numbers (i.e., dimensionless number for flow regime characteristics), which may either be caused by an increased viscosity or increased agitation speeds, a significant shift in the flow patterns occurs. With the secondary effect that Smax decreased for Newtonian and shear thinning non-Newtonian suspensions. When looking to the interactions effects, Fig. 2a and b show that at a high Cx and at a medium to high us (9–12 m s1) higher rate constants can be achieved compared to a low Cx or a low us.
Fig. 2. Contour plots of agitator speed us [m s1] and biomass concentration Cx [g kg1] on the (a) disruption rate constant kdis [s-1], (b) protein release rate constant kprot [s1], (c) pigment release rate constant kr [s1] and (d) protein yield YP.
Please cite this article in press as: Postma, P.R., et al. Mild disintegration of the green microalgae Chlorella vulgaris using bead milling. Bioresour. Technol. (2014), http://dx.doi.org/10.1016/j.biortech.2014.09.033
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3.3. Protein yield
3.4. Energy consumption
The influence of Cx and us on the water soluble protein yield Yp is shown in Fig. 2d. No significant effect (P > 0.05) was observed for us, but it can be observed that the Yp decreases about 25% when the Cx increased from 25 to 145 g kg1. A possible cause might be the formation of insoluble protein aggregates at a high Cx due to local high protein concentrations, shear forces and interactions with other components as suggested by Wang (2005). Temperature increase was not assumed to influence protein aggregation since the temperature was kept below 35 °C in all experiments. Besides, protein stability was aimed to be enhanced by using a PBS buffer, the NaCl can increase protein solubility (Bondos and Bicknell, 2003). Nevertheless, the absolute amount of extracellular PBS buffer in the different algae suspensions is decreased with increasing algae concentration because the algae occupy more space in suspensions at a higher Cx. When the algae break during disintegration the medium composition shifts from PBS like towards the algae intracellular matrix. Especially at a high Cx the negative interactions with other components might be increased causing the formation of insoluble proteins. It was observed that these proteins are not resoluble. A similar decreasing trend in protein yield was observed by Limon-Lason et al. (1979) for disintegration of packed yeast and Schwenzfeier et al. (2011) obtained a water soluble protein concentration yield of 21% by disintegration of the green microalgae Tetraselmis sp. at a Cx of 120 g kg1. These soluble proteins showed good techno-functional properties in comparison to other food proteins. Indicating that solubility of proteins is an important characteristic for mild disintegration.
The specific energy consumption E/M of a bead mill is critical for the final process economics and sustainability. For all performed experiments, a linear increase in the specific energy consumption was observed in time. Besides, the idle energy consumption (i.e., no cells) of the Dyno-Mill accounted for 70–85% of the total energy consumption. Due to the high energy dissipation at a Cx of 25 g kg1 and us of 12 m s1, excessive temperature increases were observed which required breaks during the process to cool down the algae suspension. No temperature issues were observed at the other conditions for a total process time of 60 min. Fig. 3a shows the influence of us at a Cx of 87.5 g kg1 on the fraction of disintegration as function of the specific energy consumption, it can be seen that the specific energy consumption almost triples when increasing us from 6 to 12 m s1. Looking at a constant us of 9 m s1 for different values of Cx in Fig. 3b, it can be clearly observed that a higher Cx can be disintegrated quicker at lower energy expenses. Table 3 gives an overview of the specific energy consumption E/M, where E is the used energy (kWh) and M is the amount of disintegrated biomass (kgDW). It can be seen that the specific energy consumption decreases with increasing Cx and with decreasing us. The contour plot in Fig. 4a shows that the specific energy consumption is most energy efficient at low to moderate us and medium to high Cx. In this case, E/M is kept below 10 kWh kg1 DW which is similar to what Doucha and Lívansky´ (2008) reported for a disintegration degree of 90.6%. Nevertheless, when taking the kinetic rate constants into account as described above, selective extraction
Fig. 3. Influence of us [m s1] on (a) specific energy consumption and (b) influence of Cx [g kg1] on specific energy consumption [kWh kg1 DW].
Table 3 Overview of specific energy consumption. Experimental conditions
Energy consumption
Exp. No.
Cx (g kg1 sus)
us (m s1)
M (kgDW)
Dis (%)
E (kWh)
E/M (kWh kg1 DW)
s (sec)
E3s (kWh)
E/M3s (kWh kg1 DW)
g (%)
1 2 3 4 5 6 7 8 9 10 11
25 25 25 87.5 87.5 87.5 87.5 87.5 145 145 145
6 9 12 6 9 9 9 12 6 9 12
4.7 4.7 4.6 16.2 16.2 16.2 16.1 16.3 27.0 26.9 26.8
98.4 98.6 99.5 97.1 99.7 99.8 99.7 99.7 97.6 99.6 98.6
0.094 0.247 0.424 0.093 0.180 0.188 0.181 0.248 0.120 0.229 0.288
20.45 53.42 91.98 5.92 11.12 11.67 11.24 15.23 4.55 8.51 10.87
67.7 47.6 59.8 51.9 34.6 34.7 43.4 36.6 31.8 20.4 26.1
0.035 0.065 0.140 0.027 0.034 0.036 0.044 0.050 0.021 0.026 0.042
7.64 14.10 30.38 1.71 2.12 2.23 2.71 3.10 0.81 0.96 1.58
125.6 231.8 499.4 28.0 34.9 36.6 44.5 50.9 13.3 15.8 25.9
Please cite this article in press as: Postma, P.R., et al. Mild disintegration of the green microalgae Chlorella vulgaris using bead milling. Bioresour. Technol. (2014), http://dx.doi.org/10.1016/j.biortech.2014.09.033
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7
1 Fig. 4. Contour plots of (a) specific energy consumption E/M [kWh kg1 ] and DW] aimed on cell disintegration and (b) aimed on protein release for agitator speed us [m s biomass concentration Cx [g kg1].
of water soluble proteins is possible before reaching full disintegration of the algae suspension. The doubling time s (i.e., the time required to double the released protein concentration) can be described as:
s ¼ k1 prot lnð2Þ
ð9Þ
An overview of s for each experiment is listed in table 3. From Fig. 3 it can be observed that disintegration degrees beyond 85–90% requires excessive amounts of additional energy which is in close relation to what was found by Mogren et al. (1974). Therefore 3 doubling times 3s, corresponding to 87.5% protein release, were considered to recalculate the specific energy consumption E/M3s with:
E=M 3s ¼ E=M
3s t
ð10Þ
where t is the total time of the experiment (s). This results in an energy reduction between 60% and 88%. The contour plot with the aim on protein release is shown in Fig. 4b. The contour line corresponding to 10 kWh kg1 DW shifted drastically, opening the possibility to operate at a low Cx. For a Cx above 60 g kg1 specific energy consumptions below 3 kWh kg1 DW can be achieved. Doucha and Lívansky´ (2008) found 3.11 kWh kg1 DW at 58% disintegration and 2.82 kWh kg1 DW at 77% disintegration for suspensions of Chlorella although no values for protein release were reported. Balasundaram et al. (2012) designed a ball mill capable of disintegrating the cyanobacteria Chlorogloeopsis fritschii at an E/M of 1 1.87 kWh kg1 DW. In this study the lowest E/M3s of 0.81 kWh kgDW 1 1 was found at a Cx of 145 g kg and an us of 6 m s . Taking the energy content of algae as 21.9 kJ g1 biomass (Weyer et al., 2010), the energy consumption per energy content g of the algae is then 13.3%. For the other experiments the ratio g is shown in Table 3. Many other techniques have been proposed for cell disintegration, but only few are considered mild enough to keep the intracellular components intact. Grimi et al. (2014) used pulsed electric field, high voltage electrical discharges, ultra-sonication and high pressure homogenization in sequential mode on a 1% w/w suspension of Nannochloropsis sp. and found protein extraction values of 5.2%, 1.15%, 1.8% and 91%, respectively from a total protein yield of 70% water soluble protein (corresponding to 1.4 g protein per kg suspension). Pulsed electric field, high voltage electrical discharge, ultra-sonication and high pressure homogenization required up to 1.5, 1.5, 0.5 and 25.0 kWh kg1 DW respectively.
C. vulgaris and Nannochloropsis oculata were treated by high pressure homogenization (2% w/w suspension) by Safi et al. (2014) to release 51.7% and 49.5% water soluble proteins at a specific energy consumption of 7.5 kWh kg1 DW. The microalgae Auxenochlorella protothecoides was treated with pulsed electric field by Goettel et al. (2013) at a specific energy consumption of 0.40 kWh kg1 DW. Nevertheless, hardly any proteins were released. 3.5. Model fit and analysis In order to create the contour plots, a second order polynomial regression model was used for each output. These plots can visualize the effects of the two input parameters, us and Cx on each of the outputs (Desai et al., 2014). Using the Umetrics software, regression models were obtained for the kprot, kdis, Yp and for the E/M. Model coefficients which were insignificant at a confidence interval of 95% were removed.
kprot ¼ 1:6994 þ 0:176 X 1 þ 0:0491 X 2 0:0945 X 22 R2 ¼ 0:90
ð11Þ
kdis ¼ 1:8014 þ 0:1385 X 1 þ 0:0602 X 2 0:1126 X 22 R2 ¼ 0:77
ð12Þ
Y prot ¼ 0:4421 0:0798 X 1 R2 ¼ 0:84
ð13Þ
E=M ¼ 1:0567 0:3963 X 1 þ 0:2403 X 2 þ 0:2692 X 21 0:0822 X 22 0:0687X 1 X 2 2
R ¼ 0:99
ð14Þ
E=M3s ¼ 0:3664 0:5709 X 1 þ 0:1914 X 2 þ 0:2343 X 21 0:0773 X 1 X 2 R2 ¼ 0:98
ð15Þ
Each of the models shows a good correlation between the experimental data and the predicted data. The models were statistically evaluated using analysis of variance (ANOVA) from which was found that each of the models is significant. Two F-tests were performed in the ANOVA both assessed at a probability of 95%. The first is for the model regression, comparing the variance in the regression to the variance of the residuals and is significant when P < 0.05. The second for the lack of fit, this compares the replicate error to the model error and is significant when P > 0.05.
Please cite this article in press as: Postma, P.R., et al. Mild disintegration of the green microalgae Chlorella vulgaris using bead milling. Bioresour. Technol. (2014), http://dx.doi.org/10.1016/j.biortech.2014.09.033
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P.R. Postma et al. / Bioresource Technology xxx (2014) xxx–xxx
4. Conclusions A bead mill can successfully be applied for mild disintegration of microalgae slurries. Increasing biomass concentrations and agitator speeds lead to increased kinetic rate constants. Yet, an optimal agitator speed was found regardless of the biomass concentration. Water soluble protein release was found to be quicker than the biomass disintegration, while pigment release was found to be slower than the biomass disintegration. The water soluble protein yield ranged between 32% and 42%. By means of selective protein extraction, up to 85% energy reduction is realized. Specific energy consumptions as low as 0.81 kWh kg1 DW can be achieved. Acknowledgements This project is financed by the IPOP Biorefinery of Wageningen University and Research Centre. We would like to thank Willy A. Bachofen AG Maschinenfabrik, Muttenz, Switzerland for giving the opportunity to perform initial trials with the DYNO-mill RL. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.biortech.2014. 09.033. References Balasundaram, B., Pandit, A.B., 2001. Significance of location of enzymes on their release during microbial cell disruption. Biotechnol. Bioeng. 75, 607–614. Balasundaram, B., Skill, S.C., Llewellyn, C.A., 2012. A low energy process for the recovery of bioproducts from cyanobacteria using a ball mill. Biochem. Eng. J. 69, 48–56. Batista, A.P., Gouveia, L., Bandarra, N.M., Franco, J.M., Raymundo, A., 2013. Comparison of microalgal biomass profiles as novel functional ingredient for food products. Algal Res. 2, 164–173. Becker, E.W., 2007. Micro-algae as a source of protein. Biotechnol. Adv. 25, 207–210. Bondos, S.E., Bicknell, A., 2003. Detection and prevention of protein aggregation before, during, and after purification. Anal. Biochem. 316, 223–231. Bunge, F., Pietzsch, M., Müller, R., Syldatk, C., 1992. Mechanical disruption of Arthrobacter sp. DSM 3747 in stirred ball mills for the release of hydantoincleaving enzymes. Chem. Eng. Sci. 47, 225–232. Chisti, Y., Moo-Young, M., 1986. Disruption of microbial cells for intracellular products. Enzyme Microb. Technol. 8, 194–204. Currie, J.A., Dunnill, P., Lilly, M.D., 1972. Release of protein from Bakers’ yeast (Saccharomyces cerevisiae) by disruption in an industrial agitator mill. Biotechnol. Bioeng. 14, 725–736. Delrue, F., Setier, P.-A., Sahut, C., Cournac, L., Roubaud, A., Peltier, G., Froment, A.-K., 2012. An economic, sustainability, and energetic model of biodiesel production from microalgae. Bioresour. Technol. 111, 191–200. Desai, R.K., Streefland, M., Wijffels, R.H., Eppink, M.H.M., 2014. Extraction and stability of selected proteins in ionic liquid based aqueous two phase systems. Green Chem.. Doucha, J., Lívansky´, K., 2008. Influence of processing parameters on disintegration of Chlorella cells in various types of homogenizers. Appl. Microbiol. Biotechnol. 81, 431–440.
Gerken, H.G., Donohoe, B., Knoshaug, E.P., 2013. Enzymatic cell wall degradation of Chlorella vulgaris and other microalgae for biofuels production. Planta 237, 239– 253. Gers, R., Climent, E., Legendre, D., Anne-Archard, D., Frances, C., 2010. Numerical modelling of grinding in a stirred media mill: hydrodynamics and collision characteristics. Chem. Eng. Sci. 65, 2052–2064. Godfray, H.C.J., Beddington, J.R., Crute, I.R., Haddad, L., Lawrence, D., Muir, J.F., Pretty, J., Robinson, S., Thomas, S.M., Toulmin, C., 2010. Food security: the challenge of feeding 9 billion people. Science 327, 812–818. Goettel, M., Eing, C., Gusbeth, C., Straessner, R., Frey, W., 2013. Pulsed electric field assisted extraction of intracellular valuables from microalgae. Algal Res. 2, 401– 408. Grimi, N., Dubois, A., Marchal, L., Jubeau, S., Lebovka, N.I., Vorobiev, E., 2014. Selective extraction from microalgae Nannochloropsis sp. using different methods of cell disruption. Bioresour. Technol. 153, 254–259. Kula, M.-R., Schütte, H., 1987. Purification of proteins and the disruption of microbial cells. Biotechnol. Prog. 3, 31–42. Lamers, P.P., van de Laak, C.C.W., Kaasenbrood, P.S., Lorier, J., Janssen, M., De Vos, R.C.H., Bino, R.J., Wijffels, R.H., 2010. Carotenoid and fatty acid metabolism in light-stressed Dunaliella salina. Biotechnol. Bioeng. 106, 638–648. Lee, J.-Y., Yoo, C., Jun, S.-Y., Ahn, C.-Y., Oh, H.-M., 2010. Comparison of several methods for effective lipid extraction from microalgae. Bioresour. Technol. 101, S75–S77. Limon-Lason, J., Hoare, M., Orsborn, C.B., Doyle, D.J., Dunnill, P., 1979. Reactor properties of a high-speed bead mill for microbial cell rupture. Biotechnol. Bioeng. 21, 745–774. Mandalam, R.K., Palsson, B., 1998. Elemental balancing of biomass and medium composition enhances growth capacity in high-density Chlorella vulgaris cultures. Biotechnol. Bioeng. 59, 605–611. Marffy, F., Kula, M.-R., 1974. Enzyme yields from cells of brewer’s yeast disrupted by treatment in a horizontal disintegrator. Biotechnol. Bioeng. 16, 623–634. Mogren, H., Lindblom, M., Hedenskog, G., 1974. Mechanical disintegration of microorganisms in an industrial homogenizer. Biotechnol. Bioeng. 16, 261–274. Molina Grima, E., Belarbi, E.-H., Acién Fernández, F.G., Robles Medina, A., Chisti, Y., 2003. Recovery of microalgal biomass and metabolites: process options and economics. Biotechnol. Adv. 20, 491–515. Norsker, N.-H., Barbosa, M.J., Vermuë, M.H., Wijffels, R.H., 2011. Microalgal production — a close look at the economics. Biotechnol. Adv. 29, 24–27. Safi, C., Charton, M., Ursu, A.V., Laroche, C., Zebib, B., Pontalier, P.-Y., Vaca-Garcia, C., 2014. Release of hydro-soluble microalgal proteins using mechanical and chemical treatments. Algal Res. 3, 55–60. Schwenzfeier, A., Wierenga, P.A., Gruppen, H., 2011. Isolation and characterization of soluble protein from the green microalgae Tetraselmis sp.. Bioresour. Technol. 102, 9121–9127. Spolaore, P., Joannis-Cassan, C., Duran, E., Isambert, A., 2006. Commercial applications of microalgae. J. Biosci. Bioeng. 101, 87–96. Tilman, D., Balzer, C., Hill, J., Befort, B.L., 2011. Global food demand and the sustainable intensification of agriculture. Proc. Natl. Acad. Sci. 108, 20260– 20264. Vanthoor-Koopmans, M., Wijffels, R.H., Barbosa, M.J., Eppink, M.H.M., 2013. Biorefinery of microalgae for food and fuel. Bioresour. Technol. 135, 142–149. Wang, W., 2005. Protein aggregation and its inhibition in biopharmaceutics. Int. J. Pharm. 289, 1–30. Weyer, K.M., Bush, D.R., Darzins, A., Willson, B.D., 2010. Theoretical maximum algal oil production. BioEnergy Res. 3, 204–213. Wijffels, R.H., Barbosa, M.J., Eppink, M.H.M., 2010. Microalgae for the production of bulk chemicals and biofuels. Biofuels Bioprod. Biorefin. 4, 287–295. Wileman, A., Ozkan, A., Berberoglu, H., 2012. Rheological properties of algae slurries for minimizing harvesting energy requirements in biofuel production. Bioresour. Technol. 104, 432–439. Zheng, H., Yin, J., Gao, Z., Huang, H., Ji, X., Dou, C., 2011. Disruption of Chlorella vulgaris cells for the release of biodiesel-producing lipids: a comparison of grinding, ultrasonication, bead milling, enzymatic lysis, and microwaves. Appl. Biochem. Biotechnol. 164, 1215–1224.
Please cite this article in press as: Postma, P.R., et al. Mild disintegration of the green microalgae Chlorella vulgaris using bead milling. Bioresour. Technol. (2014), http://dx.doi.org/10.1016/j.biortech.2014.09.033
