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Influence of cell properties on rheological characterization of microalgae suspensions
Bioresource Technology 139 (2013) 209–213
Contents lists available at SciVerse ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Influence of cell properties on rheological characterization of microalgae suspensions Xinru Zhang a, Zeyi Jiang b,⇑, Liang Chen a, Aihui Chou a, Hai Yan c, Yi Y. Zuo d, Xinxin Zhang e a
School of Mechanical Engineering, University of Science and Technology Beijing, Beijing 100083, China Engineering Research Center for Energy Saving and Environmental Protection, University of Science and Technology Beijing, Beijing 100083, China c Department of Biological Science and Technology, University of Science and Technology Beijing, Beijing 100083, China d Department of Mechanical Engineering, University of Hawaii at Manoa, Honolulu, HI, USA e Beijing Key Laboratory for Energy Saving and Emission Reduction of Metallurgical Industry, University of Science and Technology Beijing, Beijing 100083, China b
h i g h l i g h t s Rheological properties of two algal strains suspension were reported. Algal suspensions displayed a shear thinning non-Newtonian behavior. Smaller algal cells caused higher effective viscosity of microalgae suspensions. Cell charge played a negligible role in affecting effective viscosity.
a r t i c l e
i n f o
Article history: Received 5 February 2013 Received in revised form 26 March 2013 Accepted 29 March 2013 Available online 6 April 2013 Keywords: Microalgae suspension Suspension viscosity Algae cell property Biofuel
a b s t r a c t The influences of algal cell size and surface charge on rheological properties of microalgae suspensions were investigated. The effective viscosity of two microalgae suspensions, i.e., the freshwater Chlorella sp. and the marine Chlorella sp., was measured as a function of their volume fractions in the range of 0.70–4.31%. The hydrodynamic diameters of the freshwater Chlorella sp. and the marine Chlorella sp. were measured to be 3.13 and 6.00 lm, respectively. The Zeta potentials of these two algal cells were measured to be 23.73 and 81.81 mV, respectively. The intrinsic viscosities of these two microalgae suspensions were further determined to be 24.7 and 16.1, respectively. Combining with theoretical models, these results indicated that the algal cell size has a predominant effect over cell surface charge in affecting rheological properties of microalgae suspensions. Smaller algal cells result in a higher effective viscosity of the microalgae suspension. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction Safe sustainable liquid biofuels are imminently needed to displace fossil oil in the near future, which contributes to global warming and is of limited availability (Amaro et al., 2011). The biofuels produced from renewable resources such as oil crops, animal fat, waste cooking oil and microalgae could help to reduce the world’s reliance on petroleum fuels and CO2 emissions (Naik et al., 2010). Compared with other oil crops and animal fats, microalgae have many advantages, such as high photosynthetic efficiency and oil productivity, short life cycles, easier to scale up, and less labor required for production (Li et al., 2008). Previous study (Chisti, 2007) suggests that biofuels produced from microalgae appear to be the only renewable energy source that has the potential to completely displace petroleum-derived transport fuels ⇑ Corresponding author. Tel.: +86 10 62334971. E-mail address: [email protected] (Z. Jiang). 0960-8524/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biortech.2013.03.195
without adversely affecting the food supply and other crop products. Despite the fact that microalgae have some clear advantages over conventional biofuel sources, broad commercialization of microalgae biofuel has been restrained due to large energy requirements and high costs in the process of cultivation, harvesting and oil extraction, which is mainly owing to the dilute nature of microalgae cultures (Beal et al., 2012; Laurent et al., 2009; Schenk et al., 2008). The rheological properties of microalgae suspensions affect not only the transport phenomena in bioreactors but also the downstream bioprocessing technologies such as harvesting and dewatering, thus directly impact the energy requirements and costs of algae biofuel production. For example, at present, the biomass concentration of microalgae suspension is only 0.5 g/L in open raceway ponds and 5 g/L in photobioreactors (Pulz, 2001). The harvesting culture suspensions need to be dewatered as much as possible to simplify the subsequent lipid extraction steps. Generally, in the primary dewatering step, the suspensions are
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concentrated by 100–200 times and become algal slurries (Uduman et al., 2010). And then these concentrated microalgae slurries will be pumped to the next biomass processing unit. Consequently, it is important and necessary to understand the rheological properties of microalgae suspensions in a broad concentration range from dilute suspensions to concentrated algal slurries for designing effective photobioreactors and enhancing downstream processes toward a large scale production of biofuel. Microalgae suspensions are complex fluids that consist of water, dissolved salts, polymeric substances, and algal cells. Rheological properties of microalgae suspensions are in large dependent on the biomass concentration, physical properties of the algal cells and the rheological properties of liquid phase. Previous study found that, below a critical concentration, most microalgae suspensions displayed a Newtonian behavior (Sirin et al., 2013; Wilemana et al., 2012), and above a certain concentration, some microalgae suspensions exhibited non-Newtonian fluid behavior (Al-Asheh et al., 2002; Chen et al., 1997; Fernandes et al., 1991; Wayne et al., 1993; Wilemana et al., 2012). Compared to the effect of algal cell concentration, the influence of cell properties on rheological behavior of microalgae suspensions is still poorly understood. The objective of this paper is to study the influence of algal cell size and surface charge on the rheological properties of microalgae suspensions. Two widely-used algal strains for biofuel production, i.e., the freshwater Chlorella sp. and the marine Chlorella sp., were used in this study. As will be shown later, these two algal cells share the same shape but different sizes and surface charges. By combining experimental data of algal characterization, rheological measurement and theoretical colloidal models, the effects of cell size and surface charge of these two algal strains on the effective viscosity of the microalgae suspensions were studied. 2. Materials and methods 2.1. Algal strains and sample preparation The algal strains used in this study were freshwater Chlorella sp. and marine Chlorella sp. Freshwater Chlorella sp. was provided by the Department of Biological Science and Technology at the University of Science and Technology Beijing (Jia et al., 2011). Marine Chlorella sp. was obtained from the Center for Collections of Marine Algae (CCMA) at the Xiamen University. The freshwater Chlorella sp. was cultivated in the Bold’s Basal medium and the marine Chlorella sp. was cultivated in the f/2 medium. Both culture media were prepared according the recipes described by Andersen (Andersen, 2005). Both of the microalgae were cultivated in a 5 L photobioreactor at 25 ± 1 °C under continuous irradiance of 100 lmol m2 s and continuous aeration rate of 15 mL/min by a pump. The suspensions were cultured for 7 days before cells were harvested for experiments. Samples used in this study were prepared from actively growing cultures during their exponential growth phase. The cultivated algal cells were centrifuged at 4000 rpm (1070g) for 4 min at room temperature followed by washing with the 0.1% NaCl solutions. After three rounds of centrifugation and washing, the suspensions were resuspended in fresh nutrient media and mixed by a stirrer to prepare concentrated homogeneous samples which would be used for dilution in the experiments.
Furthermore, the measurements of the cell hydrodynamic size and surface charges were conducted. The hydrodynamic size distribution and mean volume diameters DMVD of microalgae cells which were used to determine the volume fractions of microalgae suspensions were measured by a laser particle sizer (Microtrac Inc., USA, MicrotracS3500SI). Surface charges of algal cells were obtained from the Zeta potential measurements (Microtrac Inc., USA, Nanotrac wave). Before the measurements of DMVD and Zeta potential, algal suspensions were filtered through a polyether sulfone filter paper with a pore size 15 lm to remove large algal flocculations and to have a single cell algal suspension for measurements. The volume fractions of microalgae suspensions u were derived by,
u ¼ NpD3MVD =6
ð1Þ
where u is the volume fraction of the cell suspensions, DMVD is the mean volume diameter which can be obtained from the measurement of the size distribution, N is the cell number concentration per cubic meter liquid. N was measured using a Neubauer counting chamber. In order to improve the precision and accuracy of the cell number concentration, the numbers of microalgae within all small cells were obtained, and the error of N is less than 1.5% (Hua, 1986). For the convenience and accuracy of volume fractions of microalgae suspensions, the relationships between N and the optical density OD of microalgae suspensions were measured using a 1 cm pathlength cuvette visible spectrophotometer. Firstly, the characteristic spectra for the freshwater Chlorella sp. and the marine Chlorella sp. were determined. Then the calibration curves of N as a function of OD at the characteristic spectrum were developed. 2.3. Rheological measurement The effective viscosity of microalgae suspension was determined at 25 °C using a Brookfield programmable LVDVII + digital viscometer fitted with a ultralow adaptor (Brookfield Engineering Laboratories Inc., USA). To assess the accuracy of the rheological measurement, the effective viscosity of polystyrene microbead suspensions (Aladding Reagent Inc., Shanghai, China) with the same size distributions as the freshwater Chlorella sp. suspension were measured. Effective viscosities of polystyrene microbead suspensions with a volume fraction of 4.77%, 3.53%, 2.35%, 1.18% were measured. The results indicated that the relative viscosity of microbeads suspensions lrel as a function of volume fractions matched to the Einstein equation well (lrel = leff/l0, where leff is the effective viscosity of suspensions and l0 is the viscosity of suspending medium), which indicates that the rheological measurements in this study were reliable and the precision was enough. During the rheological measurements of microalgae suspensions, experiments were conducted for shear rate c from 5.0 to 100 s1, which correspond to values expected in field applications (Mitsuhashi et al., 1995). All measurements were performed three times and the arithmetic averages of the results were reported. The maximum standard errors for the shear rate and shear stress were 3.7% and 2.6%, respectively. 3. Results and discussion 3.1. Algal cell properties and microalgae suspension concentration
2.2. Sample calibration The shapes of the algal cells were firstly observed by scanning electron microscopy (SEM). And then the cell shape and mean physical dry diameters DMD were derived using the image processing software Image J1.44p basing on the SEM pictures.
Fig. 1 shows the hydrodynamic size distribution of freshwater Chlorella sp. and marine Chlorella sp. determined by a laser particle sizer. The mean volume diameter of these two algal cells was measured to be 3.13 ± 0.80 and 6.00 ± 0.95 lm, respectively. Morphology of dehydrated cells of these two microalgae was determined
X. Zhang et al. / Bioresource Technology 139 (2013) 209–213
Fig. 1. Hydrodynamic size distributions for (a) freshwater Chlorella sp. (b) marine Chlorella sp. It indicated that the mean volume diameters of freshwater Chlorella sp. and marine Chlorella sp. were measured to be 3.13 ± 0.80 lm and 6.00 ± 0.95 lm, respectively.
with SEM. Scanning electron micrographs indicated that both microalgae have a nearly spherical shape and dry cells of these two algae have a similar diameter (2.89 ± 0.60 lm vs. 2.93 ± 0.70 lm). It should be noted that the particle size characterized in colloidal suspensions (i.e., the hydrodynamic size) is always larger than the size of dehydrated particles (Hiemenz and Rajagopalan, 1997). Zeta potential of the freshwater Chlorella sp. and the marine Chlorella sp. was measured to be 23.73 mV and 81.81 mV, respectively. Fig. 2 shows the calibration curves of concentrations for freshwater Chlorella sp. and marine Chlorella sp. suspensions. Fig. 2a indicates the absorption spectra of microalgae suspensions at various cell concentrations. There was one peak around the red (near 690 nm) light region for both algal strains. Therefore, in the experiments, OD was measured at 690 nm and was related to the number of cells per unit volume of liquid, and OD was defined as,
OD ¼ log 10 ðT 690 =T 690 fresh media Þ
ð2Þ
where T690 and T690 fresh media are the transmittance at 690 nm of the microalgae cells in fresh media and fresh media alone, respectively. Fig. 2b shows N versus OD at 690 nm for freshwater Chlorella sp. and marine Chlorella sp. It indicates that one unit of optical density at 690 nm corresponds to freshwater Chlorella sp. and marine Chlorella sp. cells number concentrations of 8.61 1012 and 4.689 1012 cells m3, respectively. Based on Eq. (1), the volume fractions u of the freshwater Chlorella sp. and the marine Chlorella sp. as a function of OD at 690 nm were described as u = 1.38 104 OD and u = 5.27 104OD, respectively. 3.2. Rheological properties of algae suspensions Fig. 3 shows the effective viscosity leff as a function of shear rate c for the freshwater Chlorella sp. suspensions with a volume
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Fig. 2. (a) Absorption spectra of microalgae suspensions with different cell number concentrations measured using a 1 cm pathlength cuvette visible spectrophotometer. There was one peak around the red (near 690 nm) light regions. In the experiments, the optical density OD was measured at 690 nm. (b) Calibration curves of cell number concentration N as a function of optical density OD at 690 nm for microalgae. N was measured using a Neubauer counting chamber. OD at 690 nm was measured using a 1 cm pathlength cuvette visible spectrophotometer.
fraction of 4.00%, 2.50%, 1.50%, 0.70%, and for the marine Chlorella sp. suspensions with a volume fraction of 4.31%, 3.38%, 2.47%, 1.31%. It indicates that the effective viscosity of freshwater Chlorella sp. suspensions with the volume fractions below 0.7% was 1.1–1.2 mPa s, which is almost the same as the effective viscosity of the suspending medium. Above the volume fraction of 0.7%, the freshwater Chlorella sp. suspensions showed an increase in the effective viscosity and displayed a shear thinning non-Newtonian behavior. Whereas, for the marine Chlorella sp., the effective viscosity of suspensions with the volume fractions below 1.31% was 1.1 to 1.2 mPa.s, almost the same as the effective viscosity of the suspending medium. Above the volume fraction of 1.31%, the marine Chlorella sp. suspensions also showed an increase in the effective viscosity and displayed a shear thinning non-Newtonian behavior. Our results agree well with previous study on rheological properties of microalgae suspensions. (Sirin et al., 2013) found that below a certain concentration, most algae suspensions exhibit the Newtonian fluid behavior. However, above a threshold concentration, algae suspensions displayed a non-Newtonian behavior (Adesanya et al., 2012; Rafai et al., 2010; Wilemana et al., 2012). Furthermore, this shear thinning behavior is consistent with many results reported for different microalgae suspensions and is attributed to mechanisms in which the shear stress, transmitted through the continuous medium, deforms or orients the suspended cells in opposition to the randomizing effects of Brownian motion (Jan and Wagner, 2009; Mueller et al., 2010). In general, the non-Newtonian behavior of condensed microalgae suspensions has a significant impact on the microalgae pumping effectiveness and its total bioenergy harvest effectiveness (Wilemana et al., 2012).
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Fig. 4. Relative viscosities lrel of the freshwater Chlorella sp. and the marine Chlorella sp. suspensions as a function of volume fractions (measured at a shear rate of 6 s1). Measurements are fitted to Krieger and Dougherty’s semi-empirical law using um = 0.62 and [l] = 24.7 and 16.1.
dispersed phase particles (Berg, 2009), u is the volume fraction of the suspension, k is a hydrodynamic interaction coefficient which increases as the particle size decreases according to the empirical equation (Matsumoto and Sherman, 1969),
k ¼ 1:079 þ expð0:1008=DÞ þ expð0:00290=D2 Þ
Fig. 3. Effective viscosity leff as a function of shear rate c for (a) freshwater Chlorella sp. suspensions and (b) marine Chlorella sp. suspensions at various volume fractions. The measurements were determined at 25 °C using a Bokfield programmable LVDVII + digital viscometer fitted with a ultralow adaptor.
Moreover, previous study has shown that, at lower concentrations, the morphology of algal cell has no significant impact on the effective viscosity of microalgae suspensions (Sirin et al., 2013). However, at increasing concentrations, the effective viscosity of the freshwater Chlorella sp. suspension was found to be more than 1.25 times higher than that of the marine Chlorella sp. suspension at the same volume fractions (Fig. 3). It should be noted that although the suspending medium of these two algal strains was different, their effective viscosities were almost the same. Moreover, the effective viscosities of the suspending medium for these two algal strains suggest that no viscous substances such as extracellular polysaccharide were excreted by these two algal strains (Sirin et al., 2013). Therefore, the different rheological behaviors of these two microalgae suspensions can be only due to their different algal cell properties. Considering that these two algal strains have a nearly same spherical shape but differ in the hydrodynamic size (3.13 ± 0.80 lm vs. 6.00 ± 0.95 lm) and the surface Zeta potential (23.73 mV vs. 81.81 mV), the influences of these two physical properties on the effective viscosity of microalgae suspensions will therefore be discussed. 3.3. Influence of cell sizes on suspension rheology As shown in Fig. 3, the effective viscosities of these two algal strains suspensions with the same volume fractions were different. To assess the influence of cell sizes on algal suspensions rheology, the above results were discussed based on the classical Mooney equation (Mooney, 1951),
lrel ¼ expf½lu=ð1 kuÞg
ð3Þ
where lrel is the relative viscosity of suspension, [l] is the intrinsic viscosity which yields valuable information on the nature of
ð4Þ
where D is the particle diameter. Therefore, the Mooney equation predicts that the relative viscosity of particle suspensions decreases with the increase of particle diameters. This is consistent with our experimental results. In order to quantity the influence of cell size on microalgae suspensions, the intrinsic viscosity [l], which was one of important rheological parameters for the freshwater Chlorella sp. and the marine Chlorella sp., was obtained basing on the Krieger and Dougherty’s semi-empirical law (Krieger and Dougherty, 1959),
leff ¼ l0 ð1 u=um Þ½lum
ð5Þ
where um is the maximal packing volume fraction. To quantify the intrinsic viscosity [l] of microalgae suspension, the effective viscosities (measured at a shear rate of 6 s1) versus volume fractions were fitted using Krieger and Dougherty’s semiempirical law and the maximal packing volume fraction was set to 0.62 (Rafai et al., 2010). And thus the intrinsic viscosity [l] of microalgae suspension was left as a free parameter. As shown in Fig. 4, this result in the intrinsic viscosity [l] of the freshwater Chlorella sp. and the marine Chlorella sp. suspensions were fitted to be 24.7 and 16.1, respectively. In other words, the intrinsic viscosity of the freshwater Chlorella sp. was more than 50% larger than that of the marine Chlorella sp.. This analysis showed that the hydrodynamic size of algal cell has a significant influence on the effective viscosity of microalgae suspension. 3.4. Influence of cell charges on suspension rheology Generally, particle charges affect the rheological behavior of particle suspensions. The effective viscosity of electrically charged particle suspension can be increased through the enhancement of the effective diameter of charged particles, as described by the Smoluchowski model (Bird et al., 2006),
leff =l0 ¼ 1 þ 2:5u½1 þ ðer e0 n=2pRÞ2 =l0 ke
ð6Þ
where er is the relative permittivity; e0 is the permittivity of free space; ke is the conductivity of suspension medium; R is the radius of the particle; n is the Zeta potential of the microalgae cell. The Zeta potentials of the freshwater Chlorella sp. and the marine Chlorella sp. were measured to be 23.73 mV and 81.81 mV, respectively. Furthermore, er, e0 and ke were assumed to be 81, 8.85 1012 F/m and
X. Zhang et al. / Bioresource Technology 139 (2013) 209–213
0.1 to 1 S/m, respectively. The term of (ere0n/2pR)2/l0ke in the Smoluchowski model was estimated far less than 1, which was negligible. By combining the above analysis about the influence of cell size on effective viscosity of microalgae suspensions, it was safely concluded that the influence of microalgae cell charges on the effective viscosity is much less than that of the algal cell size. 4. Conclusions In conclusion, the experiments showed that the intrinsic viscosity of the freshwater Chlorella sp. was more than 50% larger than that of the marine Chlorella sp. By comparing experimental data with the classical Mooney model, Krieger–Dougherty’s equation and Smoluchowski model, it was concluded that the algal cell size has a predominant effect over its surface charge in affecting rheological properties of microalgae suspensions. Smaller algal cells cause a higher effective viscosity of the microalgae suspension. These results may have important implications for the design of microalgae reactors, agitation and pumping systems, and for microalgae production, harvesting, and oil extraction. Acknowledgements This research was supported by the Fundamental Research Funds for the Central Universities (FRF-AS-09-003A), the Doctoral Scientific Fund Project of the Ministry of Education of China (20110006130002). References Adesanya, V.O., Vadillo, D.C., Mackley, M.R., 2012. The rheological characterization of algae suspensions for the production of biofuels. J. Rheol. 56 (4), 925. Al-Asheh, S., Abu-Jdayil, B., Abunasser, N., Barakat, A., 2002. Rheological characteristics of microbial suspensions of Pseudomonas aeruginosa and Bacillus cereus. Int. J. Biol. Macromol. 30 (2), 67–74. Amaro, H.M., Guedes, A.C., Malcat, F.X., 2011. Advances and perspectives in using microalgae to produce biodiesel. Appl. Energy 88 (10), 3402–3410. Andersen, R.A., 2005. Algal Culturing Techniques. Academic Press, Burlington, MA, USA. Beal, C.M., Hebner, R.E., Webber, M.E., Ruoff, R.S., Seibert, A.F., 2012. The energy return on investment for algal biocurde: results for a research production facility. Bioenergy Res. 5 (2), 341–362. Berg, J.C., 2009. An Introduction to Interfaces and Colloids: The Bridge to Nanoscience. World Scientific Publishing Co. Pte. Ltd., Singapore. Bird, R.B., Stewart, W.E., Lightfoot, E.N., 2006. Transport Phenomena, second ed. John Wiley & Sons, USA.
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Chen, F., Chen, H., Gong, X., 1997. Mixotrophic and heterotrophic growth of Haematococcus Lacustris and rheological behaviour of the cell suspensions. Bioresour. Technol. 62 (1–2), 19–24. Chisti, Y., 2007. Biodiesel from microalgae. Biotechnol. Adv. 25 (3), 294–306. Fernandes, H.L., Lupi, F., Tomé, M.M., Sá-Correia, I., Novais, J.M., 1991. Rheological behaviour of the culture medium during growth of the microalgal Botryococcus braunii. Bioresour. Technol. 38, 133–136. Hiemenz, P.C., Rajagopalan, R., 1997. Principles of Colloid and Surface Chemistry, third & expanded ed. Dekker, New York, USA. Hua, R.C., 1986. Unicellular Algal Culture and Utilization. China Agriculture Press, Beijing. Jan, M., Wagner, N.J., 2009. Current trends in suspension rheology. J. Nonnewton. Fluid Mech. 157 (3), 147–150. Jia, X., Yan, H., Wang, Z., He, H., Xu, Q., Wang, H., Yin, C., Liu, L., 2011. Carbon dioxide fixation by Chlorella sp. USTB-01 with a fermentor-helical combined photobioreactor. Front. Environ. Sci. Engin. China 5 (3), 402–408. Krieger, I.M., Dougherty, T.J., 1959. A mechanism for non-Newtonian flow in suspensions of rigid spheres. Trans. Soc. Rheol. 3, 137–152. Laurent, L., Arnaud, H., Bruno, S., Jean-Philippe, S., Olivier, B., 2009. Life-cycle assessment of biodiesel production from microalgae. Environ. Sci. Technol. 43 (17), 6475–6481. Li, Q., Du, W., Liu, D., 2008. Perspectives of microbial oils for biodiesel production. Appl. Microbiol. Biotechnol. 80 (5), 749–756. Matsumoto, S., Sherman, P., 1969. The viscosity of microemulsions. J. Colloid Interface Sci. 30 (4), 525–536. Mitsuhashi, S., Hosaka, K., Tomonaga, E., Muramatsu, H., Tanishita, K., 1995. Effects of shear flow on photosynthesis in a dilute suspension of microalgae. Appl. Microbiol. Biotechnol. 42, 744–749. Mooney, M., 1951. The viscosity of a concentrated suspension of spherical particles. J. Colloid Sci. 6 (6), 162–170. Mueller, S., Llewellin, E.W., Mader, H.M., 2010. The rheology of suspensions of solid particles. Proc. R. Soc. A 466, 1201–1208. Naik, S.N., Goud, V.V., Rout, P.K., Dalai, A.K., 2010. Production of first and second generation biofuels: a comprehensive review. Renew. Sustain. Energy Rev. 14 (2), 578–597. Pulz, O., 2001. Photobioreactors: production systems for phototrophic microorganisms. Appl. Microbiol. Biotechnol. 57, 287–293. Rafai, S., Jibuti, L., Peyla, P., 2010. Effective viscosity of microswimmer suspensions. Phys. Rev. Lett. 104 (9), 098102. Schenk, P.M., Skye, R., Thomas-Hall, Evan., Evan, S., Ute, C., Marx jan, H., Mussgnug Clemens, P., Olaf, K., Ben, H., 2008. Second generation biofuels: high-efficiency microalgae for biodiesel production. Bioenergy Res. 1 (1), 20–43. Sirin, S., Clavero, E., Salvadó, J., 2013. Potential pre-concentration methods for Nannochloropsis gaditana and a comparative study of pre-concentrated sample properties. Bioresour. Technol. 132, 293–304. Uduman, N., Ying, Q., Michael, K., Danquah, M.K., Forde, G.M., Andrew, H., 2010. Dewatering of microalgal cultures: a major bottleneck to algae-based fuels. J. Renew. Sust. Energy 2 (1), 012701. Wayne, R., Alden, C., Emery, H., 1993. Plant cell suspension culture rheology. Biotechnol. Bioeng. 42 (4), 520–526. Wilemana, A., Ozkanb, A., Berberoglu, H., 2012. Rheological properties of algae slurries for minimizing harvesting energy requirements in biofuel production. Bioresour. Technol. 104, 432–439.
Contents lists available at SciVerse ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Influence of cell properties on rheological characterization of microalgae suspensions Xinru Zhang a, Zeyi Jiang b,⇑, Liang Chen a, Aihui Chou a, Hai Yan c, Yi Y. Zuo d, Xinxin Zhang e a
School of Mechanical Engineering, University of Science and Technology Beijing, Beijing 100083, China Engineering Research Center for Energy Saving and Environmental Protection, University of Science and Technology Beijing, Beijing 100083, China c Department of Biological Science and Technology, University of Science and Technology Beijing, Beijing 100083, China d Department of Mechanical Engineering, University of Hawaii at Manoa, Honolulu, HI, USA e Beijing Key Laboratory for Energy Saving and Emission Reduction of Metallurgical Industry, University of Science and Technology Beijing, Beijing 100083, China b
h i g h l i g h t s Rheological properties of two algal strains suspension were reported. Algal suspensions displayed a shear thinning non-Newtonian behavior. Smaller algal cells caused higher effective viscosity of microalgae suspensions. Cell charge played a negligible role in affecting effective viscosity.
a r t i c l e
i n f o
Article history: Received 5 February 2013 Received in revised form 26 March 2013 Accepted 29 March 2013 Available online 6 April 2013 Keywords: Microalgae suspension Suspension viscosity Algae cell property Biofuel
a b s t r a c t The influences of algal cell size and surface charge on rheological properties of microalgae suspensions were investigated. The effective viscosity of two microalgae suspensions, i.e., the freshwater Chlorella sp. and the marine Chlorella sp., was measured as a function of their volume fractions in the range of 0.70–4.31%. The hydrodynamic diameters of the freshwater Chlorella sp. and the marine Chlorella sp. were measured to be 3.13 and 6.00 lm, respectively. The Zeta potentials of these two algal cells were measured to be 23.73 and 81.81 mV, respectively. The intrinsic viscosities of these two microalgae suspensions were further determined to be 24.7 and 16.1, respectively. Combining with theoretical models, these results indicated that the algal cell size has a predominant effect over cell surface charge in affecting rheological properties of microalgae suspensions. Smaller algal cells result in a higher effective viscosity of the microalgae suspension. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction Safe sustainable liquid biofuels are imminently needed to displace fossil oil in the near future, which contributes to global warming and is of limited availability (Amaro et al., 2011). The biofuels produced from renewable resources such as oil crops, animal fat, waste cooking oil and microalgae could help to reduce the world’s reliance on petroleum fuels and CO2 emissions (Naik et al., 2010). Compared with other oil crops and animal fats, microalgae have many advantages, such as high photosynthetic efficiency and oil productivity, short life cycles, easier to scale up, and less labor required for production (Li et al., 2008). Previous study (Chisti, 2007) suggests that biofuels produced from microalgae appear to be the only renewable energy source that has the potential to completely displace petroleum-derived transport fuels ⇑ Corresponding author. Tel.: +86 10 62334971. E-mail address: [email protected] (Z. Jiang). 0960-8524/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biortech.2013.03.195
without adversely affecting the food supply and other crop products. Despite the fact that microalgae have some clear advantages over conventional biofuel sources, broad commercialization of microalgae biofuel has been restrained due to large energy requirements and high costs in the process of cultivation, harvesting and oil extraction, which is mainly owing to the dilute nature of microalgae cultures (Beal et al., 2012; Laurent et al., 2009; Schenk et al., 2008). The rheological properties of microalgae suspensions affect not only the transport phenomena in bioreactors but also the downstream bioprocessing technologies such as harvesting and dewatering, thus directly impact the energy requirements and costs of algae biofuel production. For example, at present, the biomass concentration of microalgae suspension is only 0.5 g/L in open raceway ponds and 5 g/L in photobioreactors (Pulz, 2001). The harvesting culture suspensions need to be dewatered as much as possible to simplify the subsequent lipid extraction steps. Generally, in the primary dewatering step, the suspensions are
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concentrated by 100–200 times and become algal slurries (Uduman et al., 2010). And then these concentrated microalgae slurries will be pumped to the next biomass processing unit. Consequently, it is important and necessary to understand the rheological properties of microalgae suspensions in a broad concentration range from dilute suspensions to concentrated algal slurries for designing effective photobioreactors and enhancing downstream processes toward a large scale production of biofuel. Microalgae suspensions are complex fluids that consist of water, dissolved salts, polymeric substances, and algal cells. Rheological properties of microalgae suspensions are in large dependent on the biomass concentration, physical properties of the algal cells and the rheological properties of liquid phase. Previous study found that, below a critical concentration, most microalgae suspensions displayed a Newtonian behavior (Sirin et al., 2013; Wilemana et al., 2012), and above a certain concentration, some microalgae suspensions exhibited non-Newtonian fluid behavior (Al-Asheh et al., 2002; Chen et al., 1997; Fernandes et al., 1991; Wayne et al., 1993; Wilemana et al., 2012). Compared to the effect of algal cell concentration, the influence of cell properties on rheological behavior of microalgae suspensions is still poorly understood. The objective of this paper is to study the influence of algal cell size and surface charge on the rheological properties of microalgae suspensions. Two widely-used algal strains for biofuel production, i.e., the freshwater Chlorella sp. and the marine Chlorella sp., were used in this study. As will be shown later, these two algal cells share the same shape but different sizes and surface charges. By combining experimental data of algal characterization, rheological measurement and theoretical colloidal models, the effects of cell size and surface charge of these two algal strains on the effective viscosity of the microalgae suspensions were studied. 2. Materials and methods 2.1. Algal strains and sample preparation The algal strains used in this study were freshwater Chlorella sp. and marine Chlorella sp. Freshwater Chlorella sp. was provided by the Department of Biological Science and Technology at the University of Science and Technology Beijing (Jia et al., 2011). Marine Chlorella sp. was obtained from the Center for Collections of Marine Algae (CCMA) at the Xiamen University. The freshwater Chlorella sp. was cultivated in the Bold’s Basal medium and the marine Chlorella sp. was cultivated in the f/2 medium. Both culture media were prepared according the recipes described by Andersen (Andersen, 2005). Both of the microalgae were cultivated in a 5 L photobioreactor at 25 ± 1 °C under continuous irradiance of 100 lmol m2 s and continuous aeration rate of 15 mL/min by a pump. The suspensions were cultured for 7 days before cells were harvested for experiments. Samples used in this study were prepared from actively growing cultures during their exponential growth phase. The cultivated algal cells were centrifuged at 4000 rpm (1070g) for 4 min at room temperature followed by washing with the 0.1% NaCl solutions. After three rounds of centrifugation and washing, the suspensions were resuspended in fresh nutrient media and mixed by a stirrer to prepare concentrated homogeneous samples which would be used for dilution in the experiments.
Furthermore, the measurements of the cell hydrodynamic size and surface charges were conducted. The hydrodynamic size distribution and mean volume diameters DMVD of microalgae cells which were used to determine the volume fractions of microalgae suspensions were measured by a laser particle sizer (Microtrac Inc., USA, MicrotracS3500SI). Surface charges of algal cells were obtained from the Zeta potential measurements (Microtrac Inc., USA, Nanotrac wave). Before the measurements of DMVD and Zeta potential, algal suspensions were filtered through a polyether sulfone filter paper with a pore size 15 lm to remove large algal flocculations and to have a single cell algal suspension for measurements. The volume fractions of microalgae suspensions u were derived by,
u ¼ NpD3MVD =6
ð1Þ
where u is the volume fraction of the cell suspensions, DMVD is the mean volume diameter which can be obtained from the measurement of the size distribution, N is the cell number concentration per cubic meter liquid. N was measured using a Neubauer counting chamber. In order to improve the precision and accuracy of the cell number concentration, the numbers of microalgae within all small cells were obtained, and the error of N is less than 1.5% (Hua, 1986). For the convenience and accuracy of volume fractions of microalgae suspensions, the relationships between N and the optical density OD of microalgae suspensions were measured using a 1 cm pathlength cuvette visible spectrophotometer. Firstly, the characteristic spectra for the freshwater Chlorella sp. and the marine Chlorella sp. were determined. Then the calibration curves of N as a function of OD at the characteristic spectrum were developed. 2.3. Rheological measurement The effective viscosity of microalgae suspension was determined at 25 °C using a Brookfield programmable LVDVII + digital viscometer fitted with a ultralow adaptor (Brookfield Engineering Laboratories Inc., USA). To assess the accuracy of the rheological measurement, the effective viscosity of polystyrene microbead suspensions (Aladding Reagent Inc., Shanghai, China) with the same size distributions as the freshwater Chlorella sp. suspension were measured. Effective viscosities of polystyrene microbead suspensions with a volume fraction of 4.77%, 3.53%, 2.35%, 1.18% were measured. The results indicated that the relative viscosity of microbeads suspensions lrel as a function of volume fractions matched to the Einstein equation well (lrel = leff/l0, where leff is the effective viscosity of suspensions and l0 is the viscosity of suspending medium), which indicates that the rheological measurements in this study were reliable and the precision was enough. During the rheological measurements of microalgae suspensions, experiments were conducted for shear rate c from 5.0 to 100 s1, which correspond to values expected in field applications (Mitsuhashi et al., 1995). All measurements were performed three times and the arithmetic averages of the results were reported. The maximum standard errors for the shear rate and shear stress were 3.7% and 2.6%, respectively. 3. Results and discussion 3.1. Algal cell properties and microalgae suspension concentration
2.2. Sample calibration The shapes of the algal cells were firstly observed by scanning electron microscopy (SEM). And then the cell shape and mean physical dry diameters DMD were derived using the image processing software Image J1.44p basing on the SEM pictures.
Fig. 1 shows the hydrodynamic size distribution of freshwater Chlorella sp. and marine Chlorella sp. determined by a laser particle sizer. The mean volume diameter of these two algal cells was measured to be 3.13 ± 0.80 and 6.00 ± 0.95 lm, respectively. Morphology of dehydrated cells of these two microalgae was determined
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Fig. 1. Hydrodynamic size distributions for (a) freshwater Chlorella sp. (b) marine Chlorella sp. It indicated that the mean volume diameters of freshwater Chlorella sp. and marine Chlorella sp. were measured to be 3.13 ± 0.80 lm and 6.00 ± 0.95 lm, respectively.
with SEM. Scanning electron micrographs indicated that both microalgae have a nearly spherical shape and dry cells of these two algae have a similar diameter (2.89 ± 0.60 lm vs. 2.93 ± 0.70 lm). It should be noted that the particle size characterized in colloidal suspensions (i.e., the hydrodynamic size) is always larger than the size of dehydrated particles (Hiemenz and Rajagopalan, 1997). Zeta potential of the freshwater Chlorella sp. and the marine Chlorella sp. was measured to be 23.73 mV and 81.81 mV, respectively. Fig. 2 shows the calibration curves of concentrations for freshwater Chlorella sp. and marine Chlorella sp. suspensions. Fig. 2a indicates the absorption spectra of microalgae suspensions at various cell concentrations. There was one peak around the red (near 690 nm) light region for both algal strains. Therefore, in the experiments, OD was measured at 690 nm and was related to the number of cells per unit volume of liquid, and OD was defined as,
OD ¼ log 10 ðT 690 =T 690 fresh media Þ
ð2Þ
where T690 and T690 fresh media are the transmittance at 690 nm of the microalgae cells in fresh media and fresh media alone, respectively. Fig. 2b shows N versus OD at 690 nm for freshwater Chlorella sp. and marine Chlorella sp. It indicates that one unit of optical density at 690 nm corresponds to freshwater Chlorella sp. and marine Chlorella sp. cells number concentrations of 8.61 1012 and 4.689 1012 cells m3, respectively. Based on Eq. (1), the volume fractions u of the freshwater Chlorella sp. and the marine Chlorella sp. as a function of OD at 690 nm were described as u = 1.38 104 OD and u = 5.27 104OD, respectively. 3.2. Rheological properties of algae suspensions Fig. 3 shows the effective viscosity leff as a function of shear rate c for the freshwater Chlorella sp. suspensions with a volume
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Fig. 2. (a) Absorption spectra of microalgae suspensions with different cell number concentrations measured using a 1 cm pathlength cuvette visible spectrophotometer. There was one peak around the red (near 690 nm) light regions. In the experiments, the optical density OD was measured at 690 nm. (b) Calibration curves of cell number concentration N as a function of optical density OD at 690 nm for microalgae. N was measured using a Neubauer counting chamber. OD at 690 nm was measured using a 1 cm pathlength cuvette visible spectrophotometer.
fraction of 4.00%, 2.50%, 1.50%, 0.70%, and for the marine Chlorella sp. suspensions with a volume fraction of 4.31%, 3.38%, 2.47%, 1.31%. It indicates that the effective viscosity of freshwater Chlorella sp. suspensions with the volume fractions below 0.7% was 1.1–1.2 mPa s, which is almost the same as the effective viscosity of the suspending medium. Above the volume fraction of 0.7%, the freshwater Chlorella sp. suspensions showed an increase in the effective viscosity and displayed a shear thinning non-Newtonian behavior. Whereas, for the marine Chlorella sp., the effective viscosity of suspensions with the volume fractions below 1.31% was 1.1 to 1.2 mPa.s, almost the same as the effective viscosity of the suspending medium. Above the volume fraction of 1.31%, the marine Chlorella sp. suspensions also showed an increase in the effective viscosity and displayed a shear thinning non-Newtonian behavior. Our results agree well with previous study on rheological properties of microalgae suspensions. (Sirin et al., 2013) found that below a certain concentration, most algae suspensions exhibit the Newtonian fluid behavior. However, above a threshold concentration, algae suspensions displayed a non-Newtonian behavior (Adesanya et al., 2012; Rafai et al., 2010; Wilemana et al., 2012). Furthermore, this shear thinning behavior is consistent with many results reported for different microalgae suspensions and is attributed to mechanisms in which the shear stress, transmitted through the continuous medium, deforms or orients the suspended cells in opposition to the randomizing effects of Brownian motion (Jan and Wagner, 2009; Mueller et al., 2010). In general, the non-Newtonian behavior of condensed microalgae suspensions has a significant impact on the microalgae pumping effectiveness and its total bioenergy harvest effectiveness (Wilemana et al., 2012).
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Fig. 4. Relative viscosities lrel of the freshwater Chlorella sp. and the marine Chlorella sp. suspensions as a function of volume fractions (measured at a shear rate of 6 s1). Measurements are fitted to Krieger and Dougherty’s semi-empirical law using um = 0.62 and [l] = 24.7 and 16.1.
dispersed phase particles (Berg, 2009), u is the volume fraction of the suspension, k is a hydrodynamic interaction coefficient which increases as the particle size decreases according to the empirical equation (Matsumoto and Sherman, 1969),
k ¼ 1:079 þ expð0:1008=DÞ þ expð0:00290=D2 Þ
Fig. 3. Effective viscosity leff as a function of shear rate c for (a) freshwater Chlorella sp. suspensions and (b) marine Chlorella sp. suspensions at various volume fractions. The measurements were determined at 25 °C using a Bokfield programmable LVDVII + digital viscometer fitted with a ultralow adaptor.
Moreover, previous study has shown that, at lower concentrations, the morphology of algal cell has no significant impact on the effective viscosity of microalgae suspensions (Sirin et al., 2013). However, at increasing concentrations, the effective viscosity of the freshwater Chlorella sp. suspension was found to be more than 1.25 times higher than that of the marine Chlorella sp. suspension at the same volume fractions (Fig. 3). It should be noted that although the suspending medium of these two algal strains was different, their effective viscosities were almost the same. Moreover, the effective viscosities of the suspending medium for these two algal strains suggest that no viscous substances such as extracellular polysaccharide were excreted by these two algal strains (Sirin et al., 2013). Therefore, the different rheological behaviors of these two microalgae suspensions can be only due to their different algal cell properties. Considering that these two algal strains have a nearly same spherical shape but differ in the hydrodynamic size (3.13 ± 0.80 lm vs. 6.00 ± 0.95 lm) and the surface Zeta potential (23.73 mV vs. 81.81 mV), the influences of these two physical properties on the effective viscosity of microalgae suspensions will therefore be discussed. 3.3. Influence of cell sizes on suspension rheology As shown in Fig. 3, the effective viscosities of these two algal strains suspensions with the same volume fractions were different. To assess the influence of cell sizes on algal suspensions rheology, the above results were discussed based on the classical Mooney equation (Mooney, 1951),
lrel ¼ expf½lu=ð1 kuÞg
ð3Þ
where lrel is the relative viscosity of suspension, [l] is the intrinsic viscosity which yields valuable information on the nature of
ð4Þ
where D is the particle diameter. Therefore, the Mooney equation predicts that the relative viscosity of particle suspensions decreases with the increase of particle diameters. This is consistent with our experimental results. In order to quantity the influence of cell size on microalgae suspensions, the intrinsic viscosity [l], which was one of important rheological parameters for the freshwater Chlorella sp. and the marine Chlorella sp., was obtained basing on the Krieger and Dougherty’s semi-empirical law (Krieger and Dougherty, 1959),
leff ¼ l0 ð1 u=um Þ½lum
ð5Þ
where um is the maximal packing volume fraction. To quantify the intrinsic viscosity [l] of microalgae suspension, the effective viscosities (measured at a shear rate of 6 s1) versus volume fractions were fitted using Krieger and Dougherty’s semiempirical law and the maximal packing volume fraction was set to 0.62 (Rafai et al., 2010). And thus the intrinsic viscosity [l] of microalgae suspension was left as a free parameter. As shown in Fig. 4, this result in the intrinsic viscosity [l] of the freshwater Chlorella sp. and the marine Chlorella sp. suspensions were fitted to be 24.7 and 16.1, respectively. In other words, the intrinsic viscosity of the freshwater Chlorella sp. was more than 50% larger than that of the marine Chlorella sp.. This analysis showed that the hydrodynamic size of algal cell has a significant influence on the effective viscosity of microalgae suspension. 3.4. Influence of cell charges on suspension rheology Generally, particle charges affect the rheological behavior of particle suspensions. The effective viscosity of electrically charged particle suspension can be increased through the enhancement of the effective diameter of charged particles, as described by the Smoluchowski model (Bird et al., 2006),
leff =l0 ¼ 1 þ 2:5u½1 þ ðer e0 n=2pRÞ2 =l0 ke
ð6Þ
where er is the relative permittivity; e0 is the permittivity of free space; ke is the conductivity of suspension medium; R is the radius of the particle; n is the Zeta potential of the microalgae cell. The Zeta potentials of the freshwater Chlorella sp. and the marine Chlorella sp. were measured to be 23.73 mV and 81.81 mV, respectively. Furthermore, er, e0 and ke were assumed to be 81, 8.85 1012 F/m and
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0.1 to 1 S/m, respectively. The term of (ere0n/2pR)2/l0ke in the Smoluchowski model was estimated far less than 1, which was negligible. By combining the above analysis about the influence of cell size on effective viscosity of microalgae suspensions, it was safely concluded that the influence of microalgae cell charges on the effective viscosity is much less than that of the algal cell size. 4. Conclusions In conclusion, the experiments showed that the intrinsic viscosity of the freshwater Chlorella sp. was more than 50% larger than that of the marine Chlorella sp. By comparing experimental data with the classical Mooney model, Krieger–Dougherty’s equation and Smoluchowski model, it was concluded that the algal cell size has a predominant effect over its surface charge in affecting rheological properties of microalgae suspensions. Smaller algal cells cause a higher effective viscosity of the microalgae suspension. These results may have important implications for the design of microalgae reactors, agitation and pumping systems, and for microalgae production, harvesting, and oil extraction. Acknowledgements This research was supported by the Fundamental Research Funds for the Central Universities (FRF-AS-09-003A), the Doctoral Scientific Fund Project of the Ministry of Education of China (20110006130002). References Adesanya, V.O., Vadillo, D.C., Mackley, M.R., 2012. The rheological characterization of algae suspensions for the production of biofuels. J. Rheol. 56 (4), 925. Al-Asheh, S., Abu-Jdayil, B., Abunasser, N., Barakat, A., 2002. Rheological characteristics of microbial suspensions of Pseudomonas aeruginosa and Bacillus cereus. Int. J. Biol. Macromol. 30 (2), 67–74. Amaro, H.M., Guedes, A.C., Malcat, F.X., 2011. Advances and perspectives in using microalgae to produce biodiesel. Appl. Energy 88 (10), 3402–3410. Andersen, R.A., 2005. Algal Culturing Techniques. Academic Press, Burlington, MA, USA. Beal, C.M., Hebner, R.E., Webber, M.E., Ruoff, R.S., Seibert, A.F., 2012. The energy return on investment for algal biocurde: results for a research production facility. Bioenergy Res. 5 (2), 341–362. Berg, J.C., 2009. An Introduction to Interfaces and Colloids: The Bridge to Nanoscience. World Scientific Publishing Co. Pte. Ltd., Singapore. Bird, R.B., Stewart, W.E., Lightfoot, E.N., 2006. Transport Phenomena, second ed. John Wiley & Sons, USA.
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