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Comparison of Four Quantitative Techniques for Monitoring Microalgae Disruption by Low-frequency Ultrasound and Acoustic Energy Efficiency Xiao TAN, Danfeng ZHANG, Keshab PARAJULI, Sanjina UPADHYAY, Yuji JIANG, and Zhipeng Duan Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.7b05896 • Publication Date (Web): 12 Feb 2018 Downloaded from http://pubs.acs.org on February 14, 2018
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Comparison of Four Quantitative Techniques for Monitoring Microalgae Disruption by
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Low-frequency Ultrasound and Acoustic Energy Efficiency
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Authors: Xiao Tan1, Danfeng Zhang1, Keshab Parajuli2, Sanjina Upadhyay3, Yuji Jiang4, Zhipeng Duan1*
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Affiliations:
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1
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Education, College of Environment, Hohai University, Nanjing 210098, China
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2
Origin Energy Limited, Adelaide, SA 5000, Australia
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3
Water Research Centre, School of Biological Sciences, The University of Adelaide, Adelaide, SA 5005,
Key Laboratory of Integrated Regulation and Resource Development on Shallow Lakes, Ministry of
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Australia
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4
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Sciences, Nanjing 210008, China
State Key Laboratory of Soil and Sustainable Agriculture, Institute of Soil Science, Chinese Academy of
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*Correspondence to: Zhipeng Duan, Key Laboratory of Integrated Regulation and Resource
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Development on Shallow Lake of Ministry of Education, College of Environment, Hohai University, 1
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Xikang Road, Nanjing, China.
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Tel.: +86 25-83786897; E-mail:
[email protected] 1
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Abstract
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Ultrasound has been regarded as an environmental friendly technology to utilize microalgae
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biomass and control algal blooms. In this study, four quantitative techniques, including cell counting,
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optical density of algal suspension, pigments release, and protein release, were performed on three
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species of microalgae (M. aeruginosa, C. pyrenoidosa, and C. reinhardtii) to develop effective
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techniques for rapid monitoring of cell disruption and to optimize the acoustic energy efficiency. Results
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showed optical density of algal suspensions was not an optimal indicator to monitor cell disruption,
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although it is a common technique for determining cell concentration in microbial cultures. Instead, an
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accurate and reliable technique was to determine the release of intracellular pigments (absorbance peaks
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of supernatant) for indicating cell rupture. The protein released during sonication could also be a useful
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indicator if it is the component of interest. A fitted power functional model showed a strong relationship
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between cell disruption and energy consumption (R2 > 0.87). This model could provide an effective
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approach to directly compare the energy efficiency of ultrasound in different systems or with varying
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microalgae species. This study provides valuable information for microalgae utilization and the
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treatment of algal blooms by ultrasound, so as to achieve energy conservation and environmental safety.
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Keywords: quantitative techniques, microalgae disruption, ultrasonication, acoustic energy efficiency,
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power functional model
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1 Introduction
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Some species of microalgae are of high value by virtue of their use in production of commercial
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products, which have attracted immense scholastic interests.1-3 However, the valuable components in
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microalgae are generally circumscribed by membranes and/or cell walls. It is difficult to extract these
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materials from intact algal cells4-7 but ultrasonic pre-treatment can rupture algal cells for enhancing the 2
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extraction efficiency.8-10
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In natural environments, bloom-forming microalgae, particularly cyanobacteria, have been
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promoted excessively by anthropogenic nutrient enrichment and global warming, leading to serious
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ecological and environmental challenges.
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control blooms due to its high ability to inactivate or rupture algal cells.8,12-14 However, ultrasonic
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treatment for cell degradation in the applications of microalgae utilization and control of cyanobacterial
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blooms is still in the early phase, and this commonly causes the operation to be either insufficient or
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excessive; leading to either incomplete cell disruption15 or destruction of the extracts.16 Therefore, this
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technique needs to be optimized in terms of avoiding an overexposure and for achieving high energy
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efficiency.
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Ultrasonic technique has been frequently employed to
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To monitor the degree of cell rupture accurately and conveniently with reliable techniques is
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primarily vital for optimizing this process. Direct (e.g., cell counting) and indirect techniques (e.g.,
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determination of the release of intracellular components) for measuring cell disruption are widely
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discussed in the available literature.17 Cell counting gives a direct microscopic examination of the
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proportion of fragmented cells, which is commonly employed to monitor cell disruption due to its
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unambiguous nature.5,6,18 Despite various advantages, however, this technique is less effective to such
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extent that it is difficult to be used in large-scale operations. Other direct methods, such as determination
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of biomass loss, are also unsuitable because of either highly conservative estimate or the process being
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time-consuming. Viscosity or electrical conductivity of algal suspension have also been studied to assess
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the disruption levels, but it is difficult to establish relationships between rupture levels and viscosities or
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conductivities.19 Some indirect techniques, for example, determination of particle size distribution or
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measurement of the release of intracellular metabolites are popular in literatures.20-22 In addition, optical 3
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density of algal suspension at special wavelengths is commonly used to measure cell disruption.23-25
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Although most of them are simple, over- or under-estimation for cell disruption frequently occurs.26 In
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summary, the most common methods for monitoring the cell disruption in literature are cell counting
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and the measurement of optical density of algal suspensions.27-30 However, the method of cell counting
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is very time-consuming, and the method of determining optical density of algal suspensions still remains
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to be well established.13 Therefore, more reliable and effective techniques need to be developed for
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monitoring the algal cells disruption.
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Moreover, estimating the correlation between cell reduction and acoustic energy consumption is
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also important for the optimization of ultrasonic treatment. Although a bilinear equation model has been
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proposed to fit the relationship between cell rupture and energy consumption, the cell disruption is not a
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first-order process with ultrasonic energy inputs, which depends on the treated algal species and
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ultrasonic parameters.5,16,31 In order to upgrade ultrasonic energy efficiency, the correlation between cell
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disruption and energy inputs needs to be analyzed quantitatively.
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This study aimed to develop rapid and reliable techniques for monitoring cell disruption induced by
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ultrasound in three representative microalgae (Microcystis aeruginosa, Chlorella pyrenoidosa, and
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Chlamydomonas reinhardtii), and to propose an empirical model, which integrates energy inputs and
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cell lyses. The results from this study can provide valuable information for microalgae utilization and
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algal blooms treatment by ultrasound.
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2 Materials and methods 2.1 Microalgal strains and sample preparation
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M. aeruginosa PCC 7806 (an ovoid or spherical unicellular cyanobacteria; 3-6 µm in diameter) was
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obtained from the State Key Laboratory of Lake Science and Environment, Nanjing Institute of 4
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Geography and Limnology, Chinese Academy of Sciences. C. pyrenoidosa FACHB 5 (a spherical
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unicellular green algae; 3-8 µm in diameter) and C. reinhardtii FACHB 359 (an ovoid unicellular green
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algae with two flagella; 10-12 µm in diameter) were purchased from Freshwater Algae Culture
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Collection at the Institute of Hydrobiology, Chinese Academy of Sciences. M. aeruginosa and C.
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pyrenoidosa were grown in 250-ml Erlenmeyer flasks with BG11 medium, while C. reinhardtii was
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cultured in flasks with SE medium. Cultures were grown in a sterile illumination incubator at 25 ±
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0.5 °C under 30 µ E/(m2 s) light intensity with a light:dark cycle of 12h:12h. M. aeruginosa was selected
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due to its ubiquitous distribution as common and dominant species in bloom-forming cyanobacteria,32
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which could be controlled effectively by ultrasound. The two green algal strains were chosen because
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they have been investigated broadly as promising feedstocks for health products or biofuel
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production.33-35 All algal cells of each species were collected at stationary phase by centrifuging (5000 g)
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for 10 min at 4 °C, and were then re-suspended with fresh media. Standard algal samples of M.
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aeruginosa, C. pyrenoidosa, and C. reinhardtii were prepared with different cell concentrations at
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1.83×107, 1.21×107, and 2.58×106 cells/mL, respectively, or with dry weight concentrations at
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approximately 1.48, 2.26, and 0.26 Kg/m3, respectively. In addition, although cell concentration
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significantly influences the disruption rate of algae by ultrasound,36 this study did not focus on the
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different rupture patterns or responses of the three microalgae to ultrasound, but instead aimed to
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develop rapid and reliable techniques for monitoring the cell disruption. Therefore, the main conclusions
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of this study are not significantly impacted by the use of different cell concentrations.
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2.2 Ultrasonic equipment
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An ultrasonic apparatus (bath-type) equipped with a disk-type transducer (35 kHz; DAS Corp.,
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China) was employed in the subsequent experiments (Figure 1). The transducer was fixed at the central 5
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bottom of the cylindrical stainless steel tank (a maximal capacity of 200 mL). An artificial cooling jacket
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surrounded the tank. The accurate acoustic power entering the system was measured by calorimetry
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method.37 150 ml of distilled water was sonicated in the tank for 15 minutes without operating the
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cooling system, and its temperature was recorded synchronously. The tank was covered with a lid during
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the calibration of the power output. The temperature was measured using a digital thermometer and the
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interval of each measurement was less than ten seconds. The measurement was carried out in triplicate
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and the resulting data were then averaged. There was a strong linear relationship between sonication
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time and temperature (R2 = 0.99, P80%) (Figure 3F).
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Altogether, therefore, OD values highly relied on the feature of algal strains and were not suitable for
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determining cell disruption among all of the microalgae species.
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3.3 Optical densities of the supernatant of algal sample at special wavelengths for monitoring cell
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disruption
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Five steps of the physical disruption of an individual microorganism have been proposed: (i)
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remaining whole, (ii) becoming damaged, (iii) releasing intracellular components while remaining
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nominally whole, (iv) breaking and further releasing cellular inclusions, and (v) approaching complete
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fragmentation.26 According to this proposal, the disruption process indeed can be further simplified into 11
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the fragmentation of intact cell and the release of cellular inclusions. Cell fragments produced from cell
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disruption might be responsible for the complexity of absorbance spectra of algal suspensions mentioned
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above. Therefore, if the cell debris can be separated from the algal suspensions, measuring the release of
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intracellular pigments would be a valid technique for monitoring cell disruption. In order to examine this
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hypothesis, algal samples with various treatments were centrifuged, and the absorbance spectrum of the
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supernatant was then determined. These results are shown in Figure 4.
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Insert [Figure 4]
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As shown in figures 4A, 4C, and 4E, absorbance spectra of all the supernatants of algal samples
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increased with cell disruption and exhibited a typical chlorophyll absorption spectrum. Chlorophyll is a
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lipophilic pigment that is inside the chloroplast for eukaryotic algal cells or the cytosol for cyanobacteria.
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Coupled with the carotenoids, it was possible that they were released into the aqueous medium with
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their membrane substances during sonication, and they were left in the supernatant even after
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centrifugation at 13,000g for 10 min, where green supernatants were observed. All the supernatants had
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the same peak at around 680 nm (S-OD680), but a few differences were observed at the shorter
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wavelengths, which may be due to the different components of pigments in cells between green algae
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and cyanobacteria.44 Despite the slight difference between species, the relationship between S-OD
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values and the extents of cell disruption at peak wavelengths was significantly linear. For instance,
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S-OD440 or S-OD680 of M. aeruginosa achieved the highest reliability (r
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indicators) (Figure 4B). S-OD430 and S-OD680 of the two green algae also appeared to have higher
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reliabilities (r 2Pearson > 0.95, P
