Article of and Eukaryotic Using Exopolysaccharide Extracted from a Glacier Bacterium

Pervaiz Ali 1,2, Daniel Fucich 1 , Aamer Ali Shah 2 , Fariha Hasan 2 and Feng Chen 1,*

1 Institute of Marine and Environmental Technology, University of Maryland Center for Environmental Science, Baltimore, MD 21613, USA; [email protected] (P.A.); [email protected] (D.F.) 2 Applied Environmental and Geomicrobiology Laboratory, Department of , Quaid-i-Azam University, Islamabad 15320, Pakistan; [email protected] (A.A.S.); [email protected] (F.H.) * Correspondence: [email protected]; Tel.: +410-234-8866; Fax: +410-234-8896

Abstract: Exopolysaccharide (EPS) has been known to be a good cryoprotective agent for , but it has not been tested for cyanobacteria and eukaryotic microalgae. In this study, we used EPS extracted from a glacier bacterium as a cryoprotective agent for the cryopreservation of three unicellular cyanobacteria and two eukaryotic microalgae. Different concentrations of EPS (10%, 15%, and 20%) were tested, and the highest concentration (20%) of EPS yielded the best growth recovery for the algal strains we tested. We also compared EPS with 5% (DMSO) and 10% for the cryopreservation recovery. The growth recovery for the microalgal strains after nine months of cryopreservation was better than 5% DMSO, a well-known cryoprotectant for microalgae. A poor recovery was recorded for all the tested strains with 10% glycerol as a cryoprotective agent. The patterns of growth recovery for most of these strains were similar after 5 days, 15 days, and

 9 months of cryopreservation. Unlike common cryopreservants such as DMSO or methanol, which  are hazardous materials, EPS is safe to handle. We demonstrate that the EPS from a psychrotrophic

Citation: Ali, P.; Fucich, D.; Shah, bacterium helped in the long-term cryopreservation of cyanobacteria and microalgae, and it has the A.A.; Hasan, F.; Chen, F. potential to be used as natural cryoprotective agent for other cells. Cryopreservation of Cyanobacteria and Eukaryotic Microalgae Using Keywords: psychrophilic bacteria; exopolysaccharide (EPS); cryopreservation; cyanobacteria; microalgae Exopolysaccharide Extracted from a Glacier Bacterium. Microorganisms 2021, 9, 395. https://doi.org/ 10.3390/microorganisms9020395 1. Introduction Many eukaryotic microalgae and cyanobacteria have been isolated from natural Academic Editor: Maria Luisa Tutino environments. These microalgae contain many specific properties that have research and Received: 27 January 2021 commercial value. They are cultivated and maintained in different laboratories worldwide, Accepted: 11 February 2021 Published: 15 February 2021 and many algal cultures have been deposited in culture collection centers. There is a need to preserve the cultures for longer while ensuring viability, purity, and genetic stability.

Publisher’s Note: MDPI stays neutral Cryopreservation is the maintenance of the biological samples in a state of ‘suspended − ◦ with regard to jurisdictional claims in animation’ at low temperatures [1]. Though cryopreservation (typically at 80 C) has published maps and institutional affil- been successfully used in bacteria and cyanobacteria [2,3], it has not been routinely used iations. to maintain complex eukaryotic microalgae [4]. Some photoautotrophic have been maintained in laboratories through serial sub-culturing [5]. However, this method of culture maintenance has inherent disadvantages, including culture contamination (either bacterial or cross contamination with other strains), time- and labor-intensive processes, and expensive resources when it involves large culture collections [6]. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. Cryopreservation is an important technique for the long-term preservation of cells, This article is an open access article tissues, and organs. However, the ultra-low temperature can cause the formation of distributed under the terms and crystals in the cellular in absence of a suitable cryoprotective agent, resulting conditions of the Creative Commons in lysis the membrane [7]. Cryoprotective agents (CPAs) are added to culture media Attribution (CC BY) license (https:// to protect cells from cryo-injury by lowering the point of water and inhibiting creativecommons.org/licenses/by/ ice crystal formation in the suspension medium and cell interior [8]. Dimethyl sulfoxide 4.0/). (DMSO), methanol, and glycerol are commonly used intracellular cryoprotectants, while

Microorganisms 2021, 9, 395. https://doi.org/10.3390/microorganisms9020395 https://www.mdpi.com/journal/microorganisms Microorganisms 2021, 9, 395 2 of 14

sucrose and large polymers provide extracellular protection because these polymers cannot permeate the [7]. CPAs, at higher concentrations, more effectively prevent ice formation in cells, tissues, and organs during cryopreservation. However, CPAs be- come more toxic at higher concentrations and impede the viabilities of the cryopreserved materials [9]. Successful cryopreservation should therefore involve strategies to eliminate ice crystal formation while minimizing the risk of CPA [10]. The various factors affecting successful cryopreservation depend on the types and concentrations of CPAs, the storage time, and the temperature. The method used for cryopreservation is paramount, as many strains cannot withstand deleterious steps such as pre-cooling and post-thawing. Thus, looking for alternative CPAs with less toxicity and improving protocols to avoid cryo-injury can help in the successful cryopreservation of cells. The use of glycerol and DMSO, which are regarded as universally useful CPAs for most samples, actually marked the beginning of modern cryopreservation technology. Cryopro- tective agents could be penetrating or non-penetrating depending on their permeability when crossing the membrane. Penetrating CPAs are a class of cryoprotectants that cross cell membranes, and they include (EG), , DMSO, glycerol, and methanol. Non-penetrating CPAs are large molecules, usually polymers that inhibit ice growth via the same mechanisms as penetrating CPAs, but they do not enter cells [1]. Examples of non-penetrating cryoprotectants include sucrose, , and polyethylene glycol (PEG). Trehalose is a popular non-penetrating CPA, reported as less toxic and highly efficient in cryopreservation [11,12]. Additionally, a cocktail of non-penetrating (trehalose) and penetrating (glycerol) CPAs results in efficient post-cryopreservation recovery [11]. Non-penetrating CPAs are usually less toxic than their penetrating counterparts at the same concentration [13]. The toxicity of penetrating or permeating cryoprotectants were well-summarized in a recent review article [10]. Extreme environments are a less-explored niche, and these untapped offer novel microbial metabolites with promising industrial applications [14]. Cold temperatures are widely distributed among the extreme environments on . Cold environments have been successfully colonized by the microorganisms, which not only survive but actively metabolize at these freezing conditions. They have developed strategies to success- fully overcome the negative effects of extremely low temperatures, including intracellular freezing [15]. The production of exopolysaccharide (EPS) is one of the many strategies used by cold-adapted microorganisms to cope with extremely low temperatures [16–18]. Exopolysaccharides are glycopolymers secreted by microorganisms in their surrounding environment [19]. These polymers are produced by a diverse group of microorganisms including bacteria, cyanobacteria, , fungi, , and microalgae. ice and in the Antarctic marine environment have abundant microbial EPS, and they could play a role in the survival and adaptation of microbial communities to cope with extreme temperatures and [17,20,21]. Microorganisms are considered to be hidden wealth due to the enormous biotech- nological potential they offer. Despite their huge potential, only few of bacterial EPSs have made their way into the global market, mainly because of their high production cost. Nevertheless, for novel EPSs with unique functional properties and improving their production cost can pave the way for their commercialization. The biodi- versity and functionality of the Karakoram glaciers have not been fully explored and hence provide an opportunity for the discovery of novel microorganisms or their metabolites. Recently, , , and were found to be dominant in the Karakoram glaciers by using both culture and culture-independent methods [22]. A high proportion of bacterial isolates in this region produces antimicrobial compounds. Pseudomonas sp. strain BGI-2 was one of psychrotrophic bacteria isolated from the ice of Batura Glacier, Pakistan [23]. The BGI-2 strain was selected for further studies based on its maximum EPS production among the EPS-producing isolates. BGI-2 produces a high yield of cryoprotective EPSs at low temperatures. EPSs are categorized as stress molecules that assist microorganisms to cope with the extreme temperatures, high salinity, and desic- Microorganisms 2021, 9, 395 3 of 14

cation [16,24]. Previously, the survivability of the EPS producer BGI-2 against a series of freeze–thaw cycles were compared to two other non-EPS-bacteria including Rhodococcus sp., BGI-11 isolated from the same environment, and a mesophilic . The sur- vivability of the EPS-producing BGI-2 strain was significantly higher than BGI-11 and E. coli. The EPS in our study also provided significant cryoprotection to another bacterium (E. coli K12), which was comparable to 20% glycerol [23]. This demonstrated the role of EPS in protecting cells from damage caused by freezing conditions and freeze–thaw events. Several studies have demonstrated the possible cryoprotective role of EPS in these cold and icy environments [16,25,26]. In another study, EPS from an Antarctic Pseudomonas was reported for its cryoprotective role in the producer strain itself, as well as other bacteria [18]. EPSs from cyanobacteria and microalgae have been previously reported for their cryoprotective role in producer strains in extremely cold environments [24,27]. To the best of our knowledge, there has not been a single report of using a bacterial EPS for the cryopreservation of photosynthetic microorganisms including eukaryotic microalgae and prokaryotic cyanobacteria. The preservation of microalgae and cyanobacteria is important in basic research and industry applications. DMSO and methanol, the two most commonly used CPAs, are toxic to cells at room temperature [28]. These chemicals are also hazardous to humans when ingested, inhaled, or contacted through skin. We therefore used the possibility of employing a natural polymer (bacterial EPS) for the cryopreservation of the photosynthetic organisms. In the current study, we tested the possibility of employing a bacterial EPS for the cry- opreservation of photosynthetic microorganisms, including cyanobacteria and microalgae.

2. Material and Methods 2.1. EPS Yielded by Pseudomonas sp. BGI-2 Pseudomonas sp. BGI-2 is a bacterial strain isolated from the ice sample of Batura Glacier, Pakistan. BGI-2 is able to grow in a wide range of environmental conditions [23]. It can grow at temperatures of 4–35 ◦C, pH values of 5–11, and concentrations of 1–5%, and it utilize diverse sources of organic . BGI-2 is known to produce a large quantity of EPSs, which contain rich monomers like , galactose, and glucosamine. A high copy number of EPS-producing was found in the BGI-2 , supporting its capability of high EPS production [29]. In this study, BGI-2 was grown at 15 ◦C and pH 6, in NaCl (10 g L−1), with glucose as the carbon source (100 g L−1), yeast extract as the source (10 g L−1), and a glucose/yeast extract ratio of 10/1 to achieve the maximum EPS yield. Under these growth conditions, BGI-2 can yield 2 g L−1 of EPS.

2.2. Extraction of EPS EPS was extracted following the procedure described by Ali et al. in 2020 [23]. Briefly, EPS produced by the BGI-2 bacterial strain was precipitated with ethanol and dried at room temperature. The crude EPS was further deproteinized with trichloroacetic (TCA), precipitated with ethanol, and dried. The EPS was re-dissolved in deionized water and dialyzed in a dialysis membrane (120 KDa molecular weight cutoff) to remove traces of TCA, , and low-molecular-weight molecules. The purified EPS was freeze-dried in a lyophilizer and used for cryopreservation by dissolving it in deionized water.

2.3. Cyanobacterial and Microalgal Strains Used for Cryopreservation Assay Five strains, including 3 cyanobacteria and 2 microalgae, were used for this study. The cyanobacteria used in this study were sp. CB0101, Synechococcus sp. CBW1003, and aeruginosa PCC7806. The microalgae chosen for this study were Scenedesmus obliquus HTB1 and Chlorella vulgaris UTEX 2714 (Figure S1). Scenedesmus sp. HTB1, Chlorella vulgaris UTEX 2714, and PCC7806 were grown in a BG-11 medium [30], whereas the two Synechococcus strains (CB0101 and CBW1003) were grown in an SN medium [31] with a salinity of 15 ppt. Microcystis aeruginosa PCC7806 Microorganisms 2021, 9, 395 4 of 14

is a cyanobacterium which causes frequent algal blooms around the world. Synechococcus sp. CBW1003 is a picocyanobacteria isolated from the Chesapeake Bay during the winter season [32]. Synechococcus sp. CB0101 is another unicellular cyanobacterium isolated from the water of inner harbor Baltimore, Maryland [33]. This strain is a common inhabitant of the Chesapeake Bay, with a wide growth range for temperature, salinity, and nutrients. Scenedesmus obliquus HTB1 was isolated from the upper Chesapeake Bay (Back River) and has the ability to survive in high CO2 concentrations [34]. Chlorella vulgaris UTEX 2714 was purchased from the UTEX culture collection of at the University of Texas Austin, USA.

2.4. Cryopreservation of Cyanobacteria and Microalgae The cryoprotective effect of EPS for the cryopreservation of cyanobacteria and microal- gae was determined by a method used previously with some modifications [35]. Three different concentrations of EPS (10%, 15%, and 20%) from a stock solution (20 mg/mL) were used for cryopreservation. The cryoprotective effect of EPS was compared to 5% (v/v) dimethyl sulfoxide (DMSO) and 10% (v/v) glycerol, which were used as controls. Glycerol and EPS were sterilized by autoclaving (121 ◦C for 15 min), whereas DMSO was sterilized using 0.2 µm filters. Each culture in their late log phase was transferred from a flask into falcon tubes and centrifuged at 10,000 rpm for 5 min at room temperature. The supernatant was discarded, and the tube with pellet was placed in ice. A fresh medium was added, and the pellet was resuspended. EPS was then added to a final concentration of 10%, 15%, or 20% (v/v), and 5% DMSO and 10% glycerol were added in a similar way. Cells were re-suspended by gentle vortexing and immediately transferred to cryovials (1 mL each). Cryovials were first incubated at 4 ◦C for 30 min and then stored in an ultra-low temperature freezer at −80 ◦C.

2.5. Growth Recovery All 5 strains were checked for growth recovery after 5 days, 15 days, and 9 months of cryopreservation. For growth recovery, cryovials were taken out of the freezer and placed in a water bath at 35 ◦C for 5 min. Cultures were centrifuged at 8000 rpm for 3 min, and the supernatant was discarded to remove any CPAs. Cryopreserved cells were re-suspended in their respective media and transferred to 24-well plates (Figure S2). Cultures were incubated in the dark overnight in order to allow cells to recover under low light and under normal light at room temperature (21 ◦C) afterwards. Optical density at 750 nm was measured in a microplate reader (SpectraMax M5) to monitor growth.

2.6. Effect of Heat Sterilization on the Cryoprotective Activity of EPS Three different concentrations of EPS were tested without autoclaving the stock to observe the impact of heat sterilization on the cryoprotective activity of the EPS.

3. Results 3.1. Growth Recovery of Cyanobacteria 3.1.1. Synechococcus sp. CBW1003 The best growth recovery was obtained from EPS-preserved Synechococcus CBW1003 after five days of freezing at −80 ◦C. The maximum cell density, measured using optical density (OD) by proxy, was recorded at 20% EPS (OD 2.66), followed by 15% EPS (OD 2.13), 5% DMSO (OD 2.1), and 10% EPS (OD 1.98). A low was recorded in 10% glycerol (OD 1.44) and in the control (OD 0.95) after 18 days of incubation under normal shaking and light at room temperature (Figure1a). Microorganisms 2021, 9, 395 5 of 14 Microorganisms 2021, 9, x FOR PEER REVIEW 6 of 15

(a) 10% EPS 3 15% EPS 20% EPS 2.5 5% DMSO 2 10% Glycerol

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Figure 1. Growth recovery forFigureSynechococcus 1. Growthsp. recovery CBW1003 for afterSynechococcus cryopreservation sp. CBW1003 for (a) after 5 days, cryopreservation (b) 15 days, and for (c) ( 9a months.) 5 days, (b) Duplicate samples were measured.15 days, Theand images(c) 9 months. of cultures Duplicate at the samples end points were refer measured. to Figure The S3. images DMSO: of dimethyl cultures sulfoxide; at the end EPS: exopolysaccharide. points refer to Figure S3. DMSO: dimethyl sulfoxide; EPS: exopolysaccharide.

3.1.2. TheSynechococcus maximum sp. CB0101 recovery after 15 days of cryopreservation was also recorded at 20% EPS (OD 3.32), followed by 15% EPS (OD 3.27), 5% DMSO (OD 3.17), and 10% EPS Microorganisms 2021, 9, 395 6 of 14 Microorganisms 2021, 9, x FOR PEER REVIEW 7 of 15

(ODFor 3.01). Synechococcus Again, a low sp. biomass CB0101, recovery the maximum was recorded recovery at was 10% recorded glycerol (ODin 20% 1.24) EPS and (OD in control (OD 1.52), as shown in Figure1b. 2.34), followed by 15% EPS (OD 1.98) and 10% EPS (OD 1.74) after 5 days of cryopreser- The pattern of biomass recovery after 9 months of cryopreservation was similar to 5 vation. A poor recovery was recorded for 10% glycerol and in the control. No biomass and 15 days of cryopreservation. Growth after 9 months of cryopreservation showed the was recovered at 5% DMSO even after 20 days of post-cryopreservation incubation under maximum recovery in 20% EPS (OD 2.32) and 5% DMSO (OD 2.34). A high cell density optimum conditions (Figure 2a). was also recorded at 15% EPS (OD 1.52) and 10% EPS (OD 1.52). The lowest biomass A similar recovery pattern was recorded after 15 days of cryopreservation, with the recovery was recorded at 10% glycerol (OD 0.591) and in the control (OD 0.76), as shown maximum growth observed in cultures with EPS as the cryoprotective agent. The maxi- in Figure1c. mum biomass recovery was observed in the 20% EPS (OD 1.81), followed by 15% EPS (OD 1.45)3.1.2. andSynechococcus 10% EPS (ODsp. CB01011.74). No biomass recovery was recorded at 5% DMSO, 10% glyc- erol, and in the control (Figure 2b). For Synechococcus sp. CB0101, the maximum recovery was recorded in 20% EPS (OD Very similar results were obtained after the prolonged cryopreservation (9 months) 2.34), followed by 15% EPS (OD 1.98) and 10% EPS (OD 1.74) after 5 days of cryopreser- of this strain. The maximum biomass recovery was recorded at 20% EPS (OD 2.26), fol- vation. A poor recovery was recorded for 10% glycerol and in the control. No biomass lowedwas recovered by 15% EPS at 5% (OD DMSO 2.19) evenand 10% after EPS 20days (OD of1.30). post-cryopreservation Likewise, no recovery incubation was observed under inoptimum 5% DMSO, conditions 10% glycerol (Figure, 2anda). the control after 26 days of incubation under optimum conditions (Figure 2c).

10% EPS (a) 15% EPS 3 20% EPS 2.5 5% DMSO 2 10% Glycerol

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Figure 2. Cont. Microorganisms 2021, 9, 395 7 of 14 Microorganisms 2021, 9, x FOR PEER REVIEW 8 of 15

10% EPS Microorganisms 2021, 9, x FOR PEER REVIEW (c) 8 of 15 15% EPS 2.5 20% EPS 2 (c) 10%5% EPS DMSO 1.5 15%10% EPS Glycerol 2.5 20%Control EPS OD750 1 2 5% DMSO 0.5 1.5 10% Glycerol 0 Control OD750 1 1 5 9 15 20 23 26 0.5 Time (Days)

0 1 5 9 15 20 23 26 Figure 2. Growth recovery forFigureSynechococcus 2. Growthsp. recovery CB0101 for after Synechococcus cryopreservation sp. CB0101 for (a )after 5 days, cryopreservation (b) 15 days, and for ( c()a 9) 5 months. days, (b) 15 days, and (c) 9 months.Time Duplicate (Days) samples were measured. The images of cultures at the end Duplicate samples were measured. The images of cultures at the end points refer to Figure S5. points refer to Figure S5. FigureA 2. similar Growth recovery recovery for pattern Synechococcus was recorded sp. CB0101 after after cryopreservation 15 days of cryopreservation, for (a) 5 days, (b) with 3.1.3. Microcystis aeruginosa PCC7806 the15 days, maximum and (c) 9 growth months. observedDuplicate samples in cultures were measured. with EPS The as images the cryoprotective of cultures at the agent. end The maximumpointsMicrocystis refer biomassto Figure aeruginosa recoveryS5. PCC7806 was observed demonstrated in the 20% a EPSgood (OD recovery 1.81), followedin all treatments by 15% EPS in- cluding(OD 1.45) the and control. 10% EPSThe maximum (OD 1.74). recovery No biomass after recovery 5 days of was cryopreservation recorded at 5% was DMSO, recorded 10% atglycerol,3.1.3. 20% MicrocystisEPS and (OD in 3.31), the aeruginosa control followed PCC7806 (Figure by 10%2b). EPS (OD 3.30), 15% EPS (OD 3.26), the control (OD 3.18),Very Microcystisand similar5% DMSO aeruginosa results (OD were 2.89). PCC7806 obtained Recovery demonstrated after was the low prolonged aat good 10% recoveryglycerol cryopreservation (ODin all 2.84) treatments (9compared months) in- to of allthiscluding other strain. treatmentsthe The control. maximum The(Figure maximum biomass 3a). recovery recovery after was 5 recordeddays of cryopreservation at 20% EPS (OD was 2.26), recorded followed byat 20% 15%The EPS EPSrecovery (OD (OD 3.31), 2.19)of this followed and strain 10% by after EPS 10% 15 (ODEPS days (OD 1.30). of 3.30), cryopreservation Likewise, 15% EPS no (OD recovery 3.26),demonstrated the was control observed a (ODsimilar in pattern5%3.18) DMSO,, and, with 5% 10% theDMSO glycerol,maximum (OD 2.89). and recovery R theecovery control recorded was after low at at 26 15% 10% days EPSglycerol of (OD incubation (OD 3.63), 2.84) followed under compared optimum by to 20% EPSconditionsall other(OD treatments3.58) (Figure and2 c). 15%(Figure EPS 3a). (OD 3.53). Biomass recovery in the control (OD 3.45) was betterThe than recovery 5% DMSO of this (OD strain 3.33) after and 1510% days glycerol of cryopreservation (OD 3.21) (Figure demonstrated 3b). a similar 3.1.3.patternBiomassMicrocystis, with recoverythe aeruginosamaximum afterPCC7806 9recovery months recordedof cryopreservation at 15% EPS again(OD 3.63), demonstrated followed by the 20% maxi- mumEPS Microcystis (ODgrowth 3.58) in an aeruginosathed treatment15% EPSPCC7806 (OD with 3.53). 20% demonstrated BEPSiomass (OD recovery a3.75), good 15% recoveryin theEPS control (OD in all 3.72) treatments(OD, and 3.45) 10% was includ- EPS better than 5% DMSO (OD 3.33) and 10% glycerol (OD 3.21) (Figure 3b). (ODing the 3.60). control. The strain The maximum also recovered recovery well after in 5% 5 daysDMSO of (OD cryopreservation 3.54) and even was in recordedthe control at Biomass recovery after 9 months of cryopreservation again demonstrated the maxi- (OD20% 3.26). EPS (OD A poor 3.31), recovery followed was by observed 10% EPS in (OD the 3.30), 10% glycerol 15% EPS (Figure (OD 3.26), 3c). the control (OD 3.18),mum andgrowth 5% DMSOin the treatment (OD 2.89). with Recovery 20% EPS was (OD low 3.75), at 10% 15% glycerol EPS (OD (OD 3.72) 2.84), and compared 10% EPS to (OD 3.60). The strain also recovered well in 5% DMSO (OD 3.54) and even in the control all other treatments (Figure3a). 10% EPS (OD 3.26). A poor recovery was(a) observed in the 10% glycerol (Figure 3c). 15% EPS 4 10%20% EPS EPS 3.5 (a) 15% EPS 3 4 5% DMSO 20% EPS 2.53.5 10% Glycerol 5% DMSO 3 Control

OD750 2 10% Glycerol 1.52.5 Control OD750 1 2 0.51.5 1 0 0.5 1 5 9 11 13 15 18 0 Time (Days) 1 5 9 11 13 15 18

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Figure 3. Cont. Microorganisms 2021, 9, 395 8 of 14 Microorganisms 2021, 9, x FOR PEER REVIEW 9 of 15

(b) 10% EPS 15% EPS 4 3.5 20% EPS 3 5% DMSO 2.5 10% Glycerol 2

OD750 Control 1.5 1 0.5 0 1 5 9 11 13 16 18 Time (Days)

10% EPS (c) 15% EPS 4.5 20% EPS 4 5% DMSO 3.5 3 10% Glycerol 2.5 Control 2 OD750 1.5 1 0.5 0 1 5 9 11 13 15 18 Time (Days)

Figure 3. Growth recovery for Microcystis aeruginosa PCC7806 after cryopreservation for (a) 5 days, Figure 3. Growth recovery for Microcystis aeruginosa PCC7806 after cryopreservation for (a) 5 days, (b) 15 days, and (c) (b) 15 days, and (c) 9 months. Duplicate samples were measured. The images of cultures at the end 9 months. Duplicate samplespoints were measured.refer to Figure The S6. images of cultures at the end points refer to Figure S6.

3.2. TheGrowth recovery Recovery of of this Eukaryotic strain after Microalgae 15 days of cryopreservation demonstrated a similar pattern, with the maximum recovery recorded at 15% EPS (OD 3.63), followed by 20% EPS 3.2.1. Scenedesmus sp. HTB1 (OD 3.58) and 15% EPS (OD 3.53). Biomass recovery in the control (OD 3.45) was better than 5%For DMSO the microalgal (OD 3.33) sp. andScenedesmus 10% glycerol HTB1, (OD the maximum 3.21) (Figure growth3b). recovery after 5 days of cryopreservationBiomass recovery was after observed 9 months at 5% of DMSO cryopreservation (OD 2.15) followed again by demonstrated 10% EPS (OD the 2.04), maxi- mum15% EPS growth (OD in 1.96) the and treatment 20% EPS with (OD 20% 1.78 EPS). Poor (OD biomass 3.75), 15% recov EPSery (OD in 10% 3.72), glycerol and 10% (OD EPS (OD1.05 3.60).) was observed The strain. Viability also recovered and recovery well inwere 5% negligible DMSO (OD in the 3.54) control and with even no in addition the control (ODof any 3.26). cryoprotective A poor recovery agent ( wasFigure observed 4a). The in optical the 10% density glycerol results (Figure presented3c). here are over a period of 18 days of incubation under optimum conditions. 3.2. GrowthThe m Recoveryaximum of biomass Eukaryotic recovery Microalgae after 15 days of cryopreservation was again rec- 3.2.1.ordedScenedesmus at 5% DMSOsp. (OD HTB1 1.87) and 10% EPS (OD 1.86), followed by 15% EPS (OD 1.80) and 20% EPS (OD 1.78). Low growth recovery was recorded at 10% (OD 1.35) and the control For the microalgal sp. Scenedesmus HTB1, the maximum growth recovery after 5 days (OD 1.07) (Figure 4b). The pattern of biomass recovery after 9 months of cryopreservation ofwas cryopreservation similar to 5 and was 15 observed days of cryopreservation. at 5% DMSO (OD Biomass 2.15) followed recovery by for 10% HTB1 EPS (ODafter 2.04),9 15%months EPS of (OD cryopreservation 1.96) and 20% was EPS the (OD maximum 1.78). Poor at 10 biomass% EPS (OD recovery 2.29) followed in 10% by glycerol 15% EPS (OD 1.05)(OD was2.19) observed., 5% DMSO Viability (OD 1.99) and and recovery 20% EPS were (OD negligible 1.92). The inlowest the control growth with recovery no addition was ofagain any cryoprotectiverecorded at 10% agent glycerol (Figure and4 thea). control The optical where density optical results density presented could not here reach are to over 1 aafter period 18 days of 18 of days post of-cry incubationopreservation under incubation optimum (Figure conditions. 4c). Microorganisms 2021, 9, 395 9 of 14 Microorganisms 2021, 9, x FOR PEER REVIEW 10 of 15

(a) 10% EPS 15% EPS 2.5 20% EPS 2 5% DMSO 10% Glycerol 1.5 Control

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Figure 4. Growth recovery for Scenedesmus sp. HTB1 after cryopreservation for (a) 5 days, (b) 15 Figure 4. Growth recovery for Scenedesmus sp. HTB1 after cryopreservation for (a) 5 days, (b) 15 days, and (c) 9 months. days, and (c) 9 months. The images of cultures at the end points refer to Figure S4. The images of cultures at the end points refer to Figure S4. 3.2.2. Chlorella vulgaris Microorganisms 2021, 9, 395 10 of 14

The maximum biomass recovery after 15 days of cryopreservation was again recorded at 5% DMSO (OD 1.87) and 10% EPS (OD 1.86), followed by 15% EPS (OD 1.80) and 20% Microorganisms 2021, 9, x FOR PEER REVIEW 11 of 15 EPS (OD 1.78). Low growth recovery was recorded at 10% (OD 1.35) and the control (OD 1.07) (Figure4b). The pattern of biomass recovery after 9 months of cryopreservation was similar to 5 and 15 days of cryopreservation. Biomass recovery for HTB1 after 9 months Chlorellaof vulgaris cryopreservation clearly demonstrated was the maximumthe maximum at recovery 10% EPS (after (OD 5 2.29) days followedof cryo- by 15% EPS (OD preservation)2.19), in presence 5% DMSO of 5% (OD DMSO 1.99) (OD and 3.32 20%) asEPS a CPA (OD followed 1.92). Theby 10% lowest glycerol growth (OD recovery was again 1.95) and the control (OD 1.67) after 18 days of incubation at optimum conditions. A poor recorded at 10% glycerol and the control where optical density could not reach to 1 after recovery was observed for all concentrations of EPS used (Figure 5a). The optical density 18 days of post-cryopreservation incubation (Figure4c). results presented here were over a period of 18 days of incubation under optimum condi- tions. 3.2.2. Chlorella vulgaris Fifteen days of cryopreservation results again demonstrated 5% DMSO (OD 3.26) as the choice of cryoprotectiveChlorella vulgaris agent forclearly this strain demonstrated. Recovery was the better maximum in the control recovery than (after 5 days of cry- the treatmentsopreservation) containing EPS in presenceas the cryoprotective of 5% DMSO agent (OD (Figure 3.32) 5b) as, a and CPA 5% followed DMSO by 10% glycerol worked well(OD for the 1.95) longer and preservation the control of (OD this 1.67)strain. after 18 days of incubation at optimum conditions. After 9A months poor recoveryof cryopreservation, was observed the maximum for all concentrations biomass was recovered of EPS from used 5% (Figure 5a). The op- DMSO (OD tical2.71), densityfollowed results by 10% presentedEPS (OD 2.13) here and were the control over a(OD period 1.94). ofRecovery 18 days was of incubation under low at all otheroptimum concentration conditions. of EPS and glycerol (Figure 5c). The optical density results presented here were over a period of 18 days of incubation at the optimum conditions.

10% EPS (a) 15% EPS 3.5 20% EPS 3 5% DMSO 2.5 10% Glycerol 2 Control 1.5 OD750 1 0.5 0 1 5 9 11 13 15 18 Time (Days)

10% EPS (b) 15% EPS 3.5 20% EPS 3 5% DMSO 2.5 10% Glycerol 2 Control 1.5 OD750 1 0.5 0 1 5 9 11 13 16 18 Time (Days)

Figure 5. Cont. Microorganisms 2021, 9, 395 11 of 14 Microorganisms 2021, 9, x FOR PEER REVIEW 12 of 15

10% EPS (c) 15% EPS 3.5 20% EPS 3 5% DMSO 2.5 10% Glycerol 2 Control 1.5 OD750 1 0.5 Microorganisms 2021, 9, x FOR PEER REVIEW 12 of 15 0 1 5 9 11 13 15 18 Time (Days) 10% EPS (c) 15% EPS Figure 5. Growth recovery3.5 for Chlorella vulgaris after cryopreservation for (a) 5 days, (b) 15 days, Figure 5. Growth recovery for Chlorella vulgaris after cryopreservation for (a) 5 days, (b) 15 days, and (c20%) 9 EPS months. Duplicate and (c) 9 months. Duplicate samples were measured. The images of cultures at the end points refer 3 5% DMSO samples were measured.to Figure S7. The images of cultures at the end points refer to Figure S7. 2.5 10% Glycerol 3.3. Effect of Heat FifteenSterilization2 days onof the cryopreservation Cryoprotective Activity results of the again EPS demonstratedControl 5% DMSO (OD 3.26) as 1.5 The autoclavingthe choice ofOD750 of EPS cryoprotective had no major agenteffects foron its this cryoprotective strain. Recovery activity was for better majority in the control than the of the strains.treatments However, containing in1 case of Synechococcus EPS as thecryoprotective sp. CB0101, there agent was clear (Figure difference5b), and in 5% DMSO worked biomass recoverywell for inthe the0.5 longertreatments preservation with autoclaved of this andstrain. non-autoclaved EPSs. As dis- cussed above, theAfter CB0101 90 months strain demonstrated of cryopreservation, the maximum the biomass maximum recovery biomass in the was EPS,recovered from 5% while no recoveryDMSO was (OD recorded 2.71),1 followed at 5%5 DMSO by 10%9 and EPS 10%11 (OD glycerol. 2.13)13 The and15 maximum the control18 biomass (OD 1.94). Recovery was recovery waslow recorded at all other at 20% concentration non-autoclaved of EPS EPSTime (OD and (Days) 2.26). glycerol Comparatively (Figure5,c). the The biomass optical density results recovery waspresented low in the here presence were overof autoclaved a period EPS of 18 as daysthe cryoprotective of incubation agent at the (Figure optimum conditions. 6). Figure 5. Growth recovery for Chlorella vulgaris after cryopreservation for (a) 5 days, (b) 15 days, 3.3. Effectand (c of) 9Heat months. Sterilization Duplicate samples on the were Cryoprotective measured. The images Activity of cultures of the at EPSthe end points refer to Figure S7. The autoclaving of EPS had no major effects on its cryoprotective activity for majority of the3.3. strains. Effect of However, Heat Sterilization in case on the of CryoprotectiveSynechococcus Activitysp. of the CB0101, EPS there was clear difference 2.5 in biomassThe recovery autoclaving in of theEPS treatmentshad no major effects with on autoclaved its cryoprotective and activity non-autoclaved for majority EPSs. As discussedof the above,strains. However, the CB0101 in case strain of Synechococcus demonstrated sp. CB0101, the maximumthere was clear biomass difference recovery in in the biomass recovery in the treatments with autoclaved and non-autoclaved EPSs. As dis- 2 EPS, while no recovery was recorded at 5% DMSO and 10% glycerol. The maximum cussed above, the CB0101 strain demonstrated the maximum biomass recovery in the EPS, biomasswhile recovery no recovery was was recorded recorded at at 5% 20% DMSO non-autoclaved and 10% glycerol. EPS The maximum (OD 2.26). biomass Comparatively, 1.5 the biomassrecovery recoverywas recorded was at 20 low% non in-autoclaved the presence EPS (OD of 2.26). autoclaved Comparatively EPS, asthe thebiomass cryoprotective agentrecovery (Figure 6was). low in the presence of autoclaved EPS as the cryoprotective agent (Figure

OD750 1 6).

0.5 2.5 0 1 5 2 9 15 20 23 26 Time (Days) 1.5

Figure 6.OD750 Growth1 recovery for Synechococcus sp. CB0101 after 9 months of cryopreservation in autoclaved and non-autoclaved EPSs as the cryoprotective agents. Duplicate samples were measured. 0.5

0 1 5 9 15 20 23 26 Time (Days)

Figure 6. Growth recovery for Synechococcus sp. CB0101 after 9 months of cryopreservation in Figure 6. Growth recovery for Synechococcusautoclaved and nonsp.-autoclaved CB0101 EPS afters as 9 the months cryoprotective of cryopreservation agents. Duplicate samples in autoclaved were and non- autoclaved EPSs as the cryoprotectivemeasured. agents. Duplicate samples were measured.

Microorganisms 2021, 9, 395 12 of 14

4. Discussion Overall, EPS worked well as the cryoprotective agent for strains including Scenedesmus sp. HTB1, Synechococcus sp. CBW1003, Synechococcus sp. CB0101, and Microcystis sp. 7806. For all these strains, growth recovery was better than 5% DMSO, a common CPA used for the cryopreservation of various cells. A poor recovery was recorded when 10% glycerol was used as the cryoprotective agent. There are conflicting reports regarding the performance and choice of CPAs for the cryopreservation of photosynthetic organisms. Gaget et al. (2017) used different concentrations of methanol, DMSO, and glycerol for the cryopreservation of 196 cyanobacterial isolates and found 5% DMSO to be the preferable choice of CPA for most of the strains [35]. In our study, 5% DMSO (except for the CB0101 strain), along with the EPS, also worked well for most of the strains. In another study, Esteves-Ferreira et al. (2013) used five microalgae and cyanobacterial strains for cryopreservation and found 10% glycerol to be the most efficient CPA [36]. Some studies have demonstrated the effectiveness of CPAs when used in combination rather than alone. Nakanishi et al. (2012) demonstrated the 50% survivability of the four microalgal strains when a combination of CPAs (5% DMSO, 5% ethylene glycol, and 5% proline) was used [37]. According to their findings, little or no cryoprotection was observed when these CPAs were used alone. Aray-Andrade et al. (2018) found DMSO–sucrose and glycerol to be effective cryoprotective agents while working with the cryopreservation of two Chlorella and one Scenedesmus species [38]. In Synechococcus sp. CB0101, EPS was the only cryoprotective agent where growth was recovered after 9 months of cryopreservation, whereas no recovery was observed with DMSO and glycerol. Interestingly, growth recovery was significantly higher in the non-autoclaved EPS than in the autoclaved EPS. This pattern of growth recovery was recorded at all three durations of cryopreservation (5 days, 15 days, and 9 months). For the CB0101 strain, a higher concentration of EPS (20%) worked well, whereas 5% DMSO exhibited a toxic effect. Though 5% DMSO is the preferable CPA, it has previously been reported for its cytotoxic effect to susceptible cyanobacterial strains {36]. Other studies have also reported the toxicity of DMSO [39,40]. Penetrating CPAs that cross cell membranes, namely EG, propylene glycol, DMSO, glycerol, and methanol, have been reported to have cytotoxic activity [10]. For the successful post-thaw survival rate of microalgae and cyanobacteria, the type and concentration of cryoprotectants are crucial. In our study, EPS worked well for all the strains except for Chlorella vulgaris. Likewise, 5% DMSO performed well as a CPA for most of the strains except for Synechococcus sp. CB0101. Similarly, the same cryoprotectant with a different concentration had a different growth recovery. It is therefore critical to test the type and concentration of cryoprotectants for each strain prior to long-term cryopreservation. The choice of CPA, viability, and biomass recovery are all very much strain-dependent [41]. The BGI-2 producer strain is a psychrotrophic bacterium capable of growing at low temperatures. This strain has a high yield of EPS (2 g L−1) at 15 ◦C, which negates the expensive heating steps required for working with mesophilic strains. Working at low temperatures conserves , minimizes contamination with other microorganisms, and minimizes undesirable chemical reactions. Despite the enormous biotechnological poten- tial, only a handful of bacterial EPSs have been successfully commercialized. The major hindrance is the cost of production, which can be overcome by successfully employing a number of measures including the use of an inexpensive substrate, op- timization to improve product yield, the improvement of the producer strain through mutagenesis, genetic and metabolic manipulations to enhance the , and the improvement of downstream processing, which involves extraction and purification.

5. Conclusions Many laboratories worldwide use sub-culturing as the primary method for cyanobac- terial and microalgal culture maintenance despite inherent disadvantages. This study serves as the first step towards successful use of EPS for cryopreservation photosynthetic microorganisms including cyanobacterium and eukaryotic microalgae. The concentration Microorganisms 2021, 9, 395 13 of 14

of EPS used as a CPA is critical factor that is dependent on the sensitivity of a particular strain. Most of the strains demonstrated good recovery and viability at the higher EPS concentrations used. Therefore, more tests with increased EPS concentrations will further improve growth recovery. More research can improve methods and standard operating protocols for this natural polymer to replace the existing toxic chemicals in use today as cryoprotective agents.

Supplementary Materials: The following are available online at https://www.mdpi.com/2076-260 7/9/2/395/s1. Author Contributions: Conceptualization, P.A. and D.F.; methodology, formal analysis, P.A.; re- sources, A.A.S. and F.H.; data curation, P.A.; writing—original draft preparation, P.A. and F.C.; writing—review and editing, D.F., F.C., A.A.S., and F.H.; supervision, F.C.; project administra- tion, F.C.; funding acquisition, F.C. All authors have read and agreed to the published version of the manuscript. Funding: The funding supports (#6503, #6511, and #5903) from the Maryland Industrial Partnership Program, the Maryland Department of Natural Resource, HY-TEK Bio, LLC, and GreatAlga, LLC, and National Science Foundation of the United States of America (Award #1829888). Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: MDPI Research Data Policies. Conflicts of Interest: The authors declare no conflict of interest.

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