A Novel Approach To Improve the Efficiency of Block Freeze

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A Novel Approach to Improve Efficiency of Block Freeze Concentration Using Ice Nucleation Proteins with Altered Ice Morphology Jue Jin, Edward J. Yurkow, Derek Adler, and Tung-Ching Lee J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b03710 • Publication Date (Web): 27 Feb 2017 Downloaded from http://pubs.acs.org on February 28, 2017

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Journal of Agricultural and Food Chemistry

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A Novel Approach to Improve Efficiency of Block Freeze Concentration Using

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Ice Nucleation Proteins with Altered Ice Morphology

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Jue Jin a, Edward J. Yurkow b, Derek Adler b and Tung-Ching Lee a,*

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a

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Road, New Brunswick, New Jersey 08901, USA

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b

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University of New Jersey, 41 Gordon Road, Suite D, Piscataway, New Jersey 08854,

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USA

Department of Food Science, Rutgers, the State University of New Jersey, 65 Dudley

Molecular Imaging Center, Rutgers Translational Sciences, Rutgers, the State

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*

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[email protected] (Tung-Ching Lee).

Correspondent author. Tel: +1 848 9325536, Fax: +1 732 9326776, E–mail:

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ACS Paragon Plus Environment


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ABSTRACT

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Freeze concentration is a separation process with high success in product quality. The

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remaining challenge is to achieve high efficiency with low cost. This study aims to

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evaluate the potential of using ice nucleation proteins (INPs) as an effective method to

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improve the efficiency of block freeze concentration while also exploring the related

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mechanism of ice morphology. Our results show that INPs are able to significantly

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improve the efficiency of block freeze concentration in a desalination model. Using

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this experimental system, we estimate that approximately 50% of the energy cost can

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be saved by the inclusion of INPs in desalination cycles while still meeting the EPA

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standard of drinking water (99.9%), sodium

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chloride,

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magnesium sulfate (MgSO4), and calcium chloride (CaCl2) were obtained from Fisher

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Scientific (Fair Lawn, NJ, USA). L-serine, L-alanine, potassium iodide (KI) and

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magnesium chloride (MgCl2) were purchased from Sigma-Aldrich (St. Louis, MO,

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USA). All reagents were of analytical grade, and deionized water from Milli-Q was

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used throughout the experiments. Seawater was prepared artificially in the lab by

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dissolving 26.73g NaCl, 2.26g MgCl2, 3.25g MgSO4 and 1.15g CaCl2 in 1L of

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deionized water.26

Tris

(Hydroxymethyl)

aminomethane,

potassium

6


sulfate

(K2SO4),

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Preparation and purification of ice nucleation proteins. Erwinia herbicola was

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stored frozen at -60 °C and grown in yeast extract (YE) media (20g/L) containing

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sucrose (10g/L), L-serine (2g/L), L-alanine (2g/L), K2SO4

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(4g/L). Following culture expansion to a density of 108/L, the cells were collected by

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high-speed centrifugation (10,000×g, 20 mins @ 4 oC), and the resulting pellet was

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re-suspended in 20mM Tris buffer containing 20mM MgCl2. The suspension was then

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sonicated on ice, using three brief (10 sec) sonication bursts generated by a Brandson

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sonicator (Danbury, CT) set at 4.5 power output. Following sonication, the suspension

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was centrifuged again as described above and the supernatant was isolated and

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ultra-centrifuged at 4 oC and 160,000×g for 2h. Finally, the resultant pellet was

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re-suspended in 20mM Tris buffer with 20mM MgCl2 and freeze-dried to obtain the

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INP powder. Lyophilized INPs isolated in this manner were stored at -18 oC prior to

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use.27

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Determination of the supercooling point using differential scanning calorimetry.

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The supercooling point of seawater containing different concentrations of INPs was

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measured by differential scanning calorimetry using a DSC 823E thermal analyzer

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(Mettler-Toledo Inc., Columbus, OH) charged with liquid nitrogen and compressed

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nitrogen gas as described in the manufacturer’s instructions. Briefly, 30µL of the

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seawater samples were transferred into 40-µL aluminum crucibles with lids and

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positioned in the unit. The temperature ramp of the DSC unit was set at a freezing rate

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of 1 oC/min, from 4 oC to -25 oC. An empty crucible was used as the reference. The

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temperature point exhibiting the maximum observed heat flow was recorded as the 7


(8.6g/L) and MgSO4


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supercooling point.

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Centrifugal freeze concentration procedures. The artificial seawater (40mL) used

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in this study was frozen in plastic centrifuge tubes (internal diameter of 29mm) by

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radial freezing using a static cooling bath containing a mixture of water and ethylene

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glycol.28 The samples were then removed from the cooling bath and rapidly subjected

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to refrigerated centrifugation to separate the brine from the ice fractions. After

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centrifugation, the frozen ice fractions were thawed and the total dissolved salt (TDS)

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was measured at ambient temperature using a conductivity meter (model 09-326-2,

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Fisher Scientific). The volume of the solutions was also determined. The desalination

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rate was calculated using the following equation: =

− × 100%

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Where Rd is the desalination rate (%); C0 is the TDS of the original seawater; Cice is

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the TDS in the melt ice fraction.

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To evaluate the effect of INPs on the efficiency of freeze concentration, the freeze

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concentration procedures above were performed using different variables to determine

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the desalination rates. The experimental variables tested in our study were INP

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concentration, freezing temperature, and centrifugal time and speed, all of which were

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identified, by other studies, as the main factors that could affect concentration

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efficiency.4,

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freeze concentration experiment are listed in Table 1. For example, the tested variable

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of the experiment group 1 was INP concentration. The controlled variables of group 1

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were freezing temperature at -18 oC and centrifugation condition at 500 rpm for 5

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The levels of tested variables and controlled variables during each

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mins. The salt concentration of seawater in nature (i.e. 33.3 g/L) was used as the

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initial salt concentration during the experiments of these four variables (i.e.

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experiment groups 1 to 4). For desalination cycles (i.e. experiment groups 5&6), both

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control and INP samples started at the concentration of seawater in nature (i.e. 33.3

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g/L). The initial concentration of each subsequent cycle was based on the salt

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concentration in melted ice of its previous cycle. The desalination cycles ended, when

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the salt concentration in the melted ice fraction was less than the Environmental

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Protection Agency (EPA) standard of drinking water (i.e. 0.5 g/L). All experiments

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were performed in triplicates.

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Ice crystal size determination. The evaluation of ice crystal structure was conducted

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using a 10× Olympus lens (0.25 N.A.) (Olympus, Tokyo, Japan) and a Q imaging

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2560 × 1920 pixel CCD camera (Micropublisher, Surrey, Canada) equipped with a

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Linkham temperature-controlled imaging stage (LTS120, Linkham, Surrey, England).

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In a typical experiment, samples of seawater containing INP concentrations ranging

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from 10-7 mg/mL to 10-2 mg/mL were frozen in petri dishes. The frozen preparations

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were placed on the microscope stage setting at -18 oC, and digital images of the ice

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crystal structure were collected by focusing on the surface ice layer. The average size

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of the ice crystals was determined with ImageJ software (version 1.46r, NIH,

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Bethesda, MD), using Feret’s diameter calculation.29-30

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X-ray computed tomography image analysis. Three dimensional imaging analysis

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of frozen and centrifuged seawater samples was obtained using the Albira PET/CT

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Imaging System (Bruker, Billerica, MA) at standard voltage and current settings (i.e., 9



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45kV and 400µA) at the Molecular Imaging Center at Rutgers University. A set of

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400 image projections was then captured throughout a 360o rotation of the sample.

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Reconstruction of X-ray data produced 3D images in which the air, ice and brine

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pockets could be differentiated based on differences in X-ray attenuation properties.

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The morphology of ice structures within different growth heights was studied to

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determine interface evolution in both control and INP samples. The samples were

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frozen on a cold surface at -18 oC until the vertical length of frozen matrix reached

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our targeted growth height. The frozen samples were then removed from the cold

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surface to pour out the remaining liquid and kept in the freezer at -18 oC before

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imaging analysis. A KI contrast agent was included in the solutions prior to freezing

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the samples for better differentiation between ice and brine phases. The morphology

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of ice crystals in both horizontal and vertical directions was characterized by the

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measurement of crystal dimension and brine inclusion width. The hydraulic diameter

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in the cross sections at different growth heights of the entire frozen sample was

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calculated based on flow mechanic theory, in order to compare the brine flow rate in

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control and INP samples.31-32 The hydraulic diameter with different INP

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concentrations was determined at the same growth height. The hydraulic diameter of

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different channel shapes is given by: D =

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4 × Cross Section Area 4A = Wetted Perimeter P

For rectangular cross section: "#$

%#$

D = %(#'$) = #'$

(1)

For triangular cross section: 10


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*

D =

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) ) "× * ×(#* + , ) .*

%#'$

(2)

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Where Dh = hydraulic diameter; a = ice crystal dimension in the cross section; b =

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maximum width of brine inclusion between two ice crystals.

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Frozen samples for the brine distribution study were prepared in centrifugal tubes

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as previously described in the centrifugal freeze concentration procedures and placed

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in a cooling bath at the subzero temperature of -18 oC until completely frozen.

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Segmentation for imaging processing is done using thresholding techniques where the

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volume is partitioned into voxel groups of each region of interest (ROI) inside the

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sample. Volumes of the brine inside frozen samples were determined using VivoQuant

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image analysis software (version 1.23, inviCRO LLC, Boston MA). In a typical study

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volumes of the entrapped brine liquid were resolved and determined using a threshold

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range of 80 to 150 Hounsfield Units.21, 23

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Statistical analysis. One-way ANOVA was performed using Data Processing System

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software v.9.50.

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significant difference (LSD) at 5%.

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Results & Discussions

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Effect of INPs on the supercooling point of seawater. The effect of INPs isolated

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from E. herbicola on the supercooling point of seawater was investigated using

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differential scanning calorimetry (Fig. 2A). The supercooling point was determined as

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the lowest temperature that the supercooled solution could reach before the phase

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transition from water to ice took place, with the release of latent heat (as indicated in

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Figure 2B). With the addition of INPs at final concentrations of 1mg/mL, the

Differences among mean values were established by the least

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supercooling point of the seawater samples was elevated to -6.24 oC, as compared to

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the supercooling temperature of -21.38 oC for the control samples. Even at the lowest

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INP concentration of 10-6 mg/mL, the supercooling point increased to -11.36 oC. As

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suggested by Jung et al., the nonlinear relationship between INP concentrations and

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supercooling point might mainly be due to the dependence of ice nucleation activity

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on the degree of protein aggregation.33 In the theory of heterogeneous ice nucleation,

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a larger nucleating site leads to a higher threshold temperature of ice nucleation

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activity.34 Therefore, the variation of ice nucleation activity at the supercooling point

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(threshold temperature), shown by the DSC measurement, is likely to be the result of

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protein aggregation into different sizes of ice nuclei. Such aggregation process was

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suggested to be limited by stochastic chain-terminating events in the growing ice

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nucleus rather than the availability of INP concentration range.35 The results indicate

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that INPs can function as effective ice nucleators for controlling the supercooling

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level of seawater even at low concentrations. The elevated nucleation temperature

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also suggests significant energy savings for the freezing step, which will be further

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discussed in the energy saving section.

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Effect of INP concentration on desalination rate. The effect of INPs on freeze

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concentration efficiency as a function of desalination rate was investigated at different

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INP concentrations using block freeze concentration assisted by centrifugation (Fig.

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3). In this technique, concentrated solute drains through the porous ice block and is

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separated from the ice under centrifugal force. The addition of INPs at a concentration

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of 10-6 mg/mL increased the desalination rate by 14% as compared to the control 12


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sample. This rate continued to increase from 48% to 53% with increases in INP

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concentrations from 10-6 mg/mL to 10-2 mg/mL. The results demonstrate that INPs

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can increase desalination rates, indicating that INPs can improve concentration

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efficiency and thus reduce related energy cost.

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Desalination rate by INPs at different freezing temperatures and centrifugation

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conditions. To examine the practical application of INPs to the production of drinking

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water from seawater, the effect of these agents on desalination rates at different

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conditions, including freezing temperature and centrifugation speed or time, were

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investigated. INP concentration of 10-2 mg/mL was utilized in the subsequent

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experiments.

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The effect of INPs on desalination rate at different freezing temperatures was

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determined (Fig. 4). The results show that INPs can improve concentration efficiency

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at freezing temperatures of -13 oC and -18 oC compared to the control samples. At the

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temperature of -8 oC, INPs were able to freeze samples while seawater controls

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remained liquid. This elevation in the freezing temperature induced by INPs is

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associated with a 36% increase in the desalination rate, which was two-fold higher

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than the effect of INPs on the desalination rate of samples frozen at -18 oC. This

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increased desalination rate observed at the higher subzero temperature (-8 oC) is

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thought to be due to the higher diffusion rate of solute under warmer temperature that

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then leads to less solute entrapment as compared to samples with INPs at lower

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freezing temperatures.

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The effect of INPs on desalination rate at different centrifugation conditions was 13



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also characterized. Here, desalination rates were determined while varying

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centrifugation times (Fig. 5A). For these studies, frozen samples of the control

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seawater and seawater containing INPs were centrifuged at 500rpm for increasing

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periods of time (i.e., 5, 10, 15, 20 and 30 mins). The results show that INPs exhibit

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marked effect on desalination rates at the lower centrifugation times of 5, 10 and 15

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minutes, with increases ranging from 16% to 20% as compared to control samples

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under the same conditions. Since there was no significant difference (P

A Novel Approach To Improve the Efficiency of Block Freeze

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