Assessing storage of stability and mercury reduction of freeze-dried Pseudomonas putida within different types of lyoprotectant

Pseudomonas putida is a potential strain in biological treatment to remove mercury contained in the effluent of petrochemical industry due to its mercury reductase enzyme that able to reduce ionic mercury to elementary mercury. Freeze-dried P. putida allows easy, inexpensive shipping, handling and high stability of the product. This study was aimed to freeze dry P. putida cells with addition of lyoprotectant. Lyoprotectant was added into the cells suspension prior to freezing. Dried P. putida obtained was then mixed with synthetic mercury. Viability of recovery P. putida after freeze dry was significantly influenced by the type of lyoprotectant. Among the lyoprotectants, tween 80/ sucrose was found to be the best lyoprotectant. Sucrose was able to recover more than 78% (6.2E+09 CFU/ml) of the original cells (7.90E+09CFU/ml) after freeze dry and able to retain 5.40E+05 viable cells after 4 weeks storage at 4 °C without vacuum. Polyethylene glycol (PEG) pre-treated freeze dried cells and broth pre-treated f...


I. INTRODUCTION
REEZE drying of bacteria has been widely used in pharmaceutical, food industry and other application that related to bio-preservation process. Attention has been given to the method of freeze-drying of certain bacteria due to the beneficial effect on the stability after long storage period, appreciable number of rehydrate cells and the transportable product [1]. High concern has been emerged from the effluent of wastewater in petrochemical industry. High mercury content is often detected from wastewater in industry related to the chemical manufacture such as chloralkali plant [2], [3] and petrochemical industry [4]. High mobility of the mercury in environment and its toxic effect has been a major concern. Hence, both reduction and elimination of mercury concentration in effluent is highly appreciable and demanding. In Malaysia, several wastewater plants contain mercury in the amount that excess the permission level by Environmental Quality Act 2011 for standard A and B [4].
Bacteria become one of green solution to overcome many industrial problems and enhance the performance of the process in industry. Biological treatment is known to have a low cost on the process than any available methods, also often no environmental threat as there is no secondary pollutant produced. Importantly, it can be performed in situ at the site interest [5]. Bioremediation using bacteria is considered as one of potential method that can worldly applicable and acceptable in the near future because of its excellent ability to remove heavy metal such as mercury efficiently without causing significant harm to environment [6].
Pseudomonas putida is a gram-negative bacterium that has been extensively study for its ability to reduce the mercury ion from Hg 2+ to Hg 0 [2]- [4]. P. putida draws many interests because of its broad application in bioremediation, in biotechnology and in supporting of plant growth. P. putida can handle environmental stress better and can grow even at low temperature. P. putida can grow at 30 o C which is claimed as the optimum growth condition of this strain [7]. The use of P. putida is considerably safer than other bacteria such as Escherichia coli and Pseudomonas Aeruginosa. The reason of that is non-pathogenic property of P. putida, hence the use of the bacteria pronounces less negative effect for both living creature and environment.
Dried bacteria offer convenient way to distributing the bacteria. Unlike other liquid preservation bacteria such as glycerol that must be stored at very low temperature for the cells to keep viable, dried cells reduce the damage of the cell and offer more stable product by maintaining higher viable cells [8]. The use of P. putida in industry requires suitable form in which the bacteria can be more easily to transporting and must easy to handle. Furthermore, when there is a need to store a large number of cells, availability and cost for storage the frozen suspension become problematic. Conveniently, pure P. putida can be obtained in dried form. Dried cells allow storage for long period due to the minimal moisture content around the cells inside the container [9]. Gram-negative bacteria often show lower survival rates than gram-positive bacteria [10], [11]. Hence, gram-negative P. putida is one of species bacteria that are very likely to suffer of huge loss in number of rehydrating cells after drying and storage.
Freeze drying take a lead as most effective method compared to other due to the process of drying that using sublimation process to remove water. Sublimation process reduces the damaging effect of the cell especially for sensitive bacteria by avoid treatment using high temperature and high pressure. Hence, viability and stability of most bacteria is high. Freeze drying is preservation technique that is used by American Type Culture Collection (ATCC) and the National Collection of Type Cultures (NCTC). Freeze drying is suitable for drying susceptible bacteria [12].
There are fewer studies about the parameter that may influence P. putida tolerance in freeze drying. The purpose of this study is to understand the parameters that effecting freeze drying tolerance of P. putida. This work aims to maximize the survival rate of dried P. putida using freeze drying technique and obtain transportable dried P. putida in laboratory scale. The stability of P. putida towards freeze drying is studied for future used. This works also will provides the data for second consumers of freeze dried bacteria that want to preserve the bacteria.

II. MATERIALS AND METHODS
The aseptic technique was used during handling the experiment to reduce the risk of contamination of unwanted microbes. The experiment was carried out in laminar flow for optimal controllable work. The surface work and gloved was disinfectant with 70% ethanol and wiping clean [13].

A. Bacteria
Bacteria P. putida was obtained from BIOREV SDN BHD and has been growth by previous student in agar medium. The type of bacteria used in this study is P. putida. Before undergo the experiment, it is necessary to ensure that the bacteria are pure P. putida. Microscope was used to identify the type of bacteria in agar. Only pure P. putida was used in this experiment.

B. Chemicals
Nutrient Agar and Nutrient Broth were purchased from Merck (Germany). Sucrose, Tween 80, and Polyethylene glycol (PEG) 1000 were obtained from FKKSA laboratory. Mercury (II) chloride was obtained from Merck.

C. Solution Preparation
Distilled water (DI) was used to prepare the agar and broth solution at room temperature and followed by autoclave at 121 o C for 15 minutes. DI water was used to dissolve sucrose, tween 80 and PEG. Sterilized DI water is used to dilute mercury stock solution to concentration of 1 ppm at room temperature.

D. Microbial Culture Preparation
The cell culture was prepared by inoculating a single colony from agar plate into 20 ml broth in universal bottle. The cells were incubated for 24 h, 180 rpm in 30 o C. The cells were removed into 180 ml of fresh broth for another 16 h at the same condition.
Cell were harvested by centrifuge (EPPENDORF 5810 R) at 5000xg for 5 minute at room temperature and then washed three times with phosphate buffer. The cells concentrations were adjusted to absorbance near 2.3 (A600nm) [14].

E. Freeze-Dried Cells Preparation by Using PEG
One hundred milliliters of 2.5 g/l PEG 1000 solution was prepared using sterile DI water ad boiled for 5 min. The PEG solution was cooled down to room temperature prior to mixing it with 50 ml of cells (A600 nm, 2.5). After mixed, the solution was left to stand to ensure that the lyoprotectant media had sufficient time (Approximately 10 min) to toughly permeate the cells of the suspension [8]. After standing, 2 ml of the mixture was added into each vial (15 ml). The mouth of the vials was filled with sterile cotton to avoid the dry cells to escape from the vial. The cells were freeze inside the freeze dryer (BIOTRON/CLEANVAC) to let the slow freezing occur. The frozen cells were then freeze-dried for 24 h in freeze dryer. All the dry samples were stored in 4 o C.

F. Freeze-Dried Cells Preparation using Tween 80/sucrose
A 500 ml of 50% sucrose stock solution was prepared using DI water and was sterilized by passing the solution through a 0.22-μm filter. The sucrose stock solution was exposed to UV light for 5 min. Twenty five milliliters of 50% sucrose and 0.2 ml of tween 80 were mixed with 80 ml of cells (A600 nm, 2.5). This solution was left to stand to ensure that the lyoprotectant media had sufficient time (Approximately 10 min) to toughly permeate the cells of the suspension [8]. After standing, 2 ml of the mixture was added into each vials vial (15 ml). The mouth of the vials was filled with sterile cotton to avoid the dry cells to escape from the vial. The cells were freeze inside the freeze dryer (BIOTRON/CLEANVAC) to let the slow freezing occur. The frozen cells were then freezedried for 24 h in freeze dryer. All the dry samples were stored in 4 o C.

G. Cell Viability Test
Freeze dried were rehydrate by shaking dried cells with broth in incubator at 180 rpm, 30 o C for 24 h. After 24 h, the cells were shaking with 180 ml of broth at 180 rpm, 30 o C for 24 h. Cell viability was evaluated based on the petri-dish plate count standard method. A drop of cells and a drop of sterile DI water were spread into an agar plate. Serial dilution was performed to ensure that cells were growth in colony, therefore the number of colony in the plate were able to read.

H. Data Analysis
Each test with new batch of trial sample P. putida was repeated in triplicate. The viability of P. putida was determined as CFU (Colony Forming Unit). The number of colony forming unit (CFU) was calculated according to [15]: Mercury analysis was performed using mercury analyzer. The efficiency of mercury removal was calculated according to [4]: where, the initial concentration of mercury is 1000 ppb. The amount of moisture content was determined using moisture analyzer.

A. Cell Viability Study
The effects of freeze drying and storage condition in P. putida were evaluated using plate counts during 0, 1, 2 and 3 weeks of storage. Three of rehydrated freeze-dried P. putida for each lyoprotectant were streaked into agar plate and incubated for 24 h.
The result in Table I reveals that the highest viability immediately after freeze drying (time 0) was obtained from PEG pre-treated freeze-dried P. putida cells. The result indicates that lyoprotectant PEG could give an effective protection for frozen cells when undergoing freeze-dried process [16], [17]. Although there was no significant different CFU number between PEG pre-treated freeze-dried cells and Tween80/sucrose pre-treated freeze-dried cells at time 0, but a rapid lost was exhibited from rehydrate cells prepared in PEG after storage (time 1, 2 and 3). PEG pre-treated cells seemed to be more susceptible to storage after freeze-drying due to the higher toxicity level [8]. In contrast, freeze-dried P. putida cells prepared in Tween 80/Sucrose were able to maintain low decrement along the time during storage in 4 o C. Tween80/Sucrose pre-treated freeze-dried cells were able to give an effective protection for the freeze-dried cells when undergoing freeze-dry and during storage at atmospheric pressure and 4 o C [8], [18]. Sucrose allows the cells to survive during the storage time mainly because sucrose is not toxic compared to PEG [19]. Sucrose was proved as a good cryoprotectant and even suitable for human oocyte cryopreservation [20]. Sucrose is able to enhance the protection of the cell during desiccation by replacing the water around polar residue within macromolecular structure. When sucrose forming hydrogen bonding, it maintain the lipid in the liquid crystalline phase at room temperature and lowering the temperature of the membrane phase transition [21]. It protects the structure and avoids denaturation of protein in the cells during drying. In addition, sucrose able to forming a glass state in dry tissue, avoids the cells to burst and deterioration [22]. The viability of the cells was improved by addition of tween 80 in the solution. is Tween 80 solution yielded a gel-like residue and insoluble in water It has very little anti-bacteria activity and prevents damage to cytoplasmic [8].

B. Moisture Content
In this experiment, all the samples were run at the same time in one freeze drying machine. The difference of moisture content shows that moisture content is depending on the type of lyoprotectant used [21]. The results in Table II prove that the water activity has significant effect on the storage stability of freeze-dried P. putida. Higher CFU indicates high viable cells after rehydration. The lower range of moisture content was found in Tween80/Sucrose pre-suspended cells. Based on the result, lowering the moisture content had increased the stability of P. putida survival during storage. Freeze-dried pre-suspended in PEG has higher range of moisture content and greatly decrease the CFU along the time. Maintaining the minimum water contain in the cells of P. putida is essential to obtain tenacious preserved cells for storage [23].
Under the microscope, it seemed that there is no visual difference between PEG pre-treated freeze-dried P. putida cells and Tween80/Sucrose pre-treated freeze-dried P. putida cells as shown in Fig. 2. Without any rehydration process the cells movement were monitored. For both samples, P. putida was not shown any physical movement, but a few of the cells were less active movement after thawing to room temperature.

C. Mercury Reduction
Mercury reduction testing was performed using freeze-dried P. putida cells taken from the sample that has been stored for 3 weeks to relate the effect of lyoprotectant with the performance of P. putida in removing mercury as shown in Table III. Both PEG and Tween80/Sucrose pre-treated freezedried P. putida cells were able to reduce mercury. The overall total mercury reduction for both lyoprotectant was not higher may due to the less viable P. putida cells after rehydration. Mercury reduction of PEG pre-treated freeze-dried P. putida cells was lower compared to tween80/sucrose pre-treated freeze-dried P. putida cells. It seems reasonable; as the cell viability tween80/sucrose pre-treated freeze-dried P. putida cells were higher during 3 weeks storage. The lower percentage reduction of mercury for both freeze-dried cells compared to the fresh culture may indicates that the cells suffer an internal damage due to the extreme freezing and drying process that affecting the ability of P. putida to remove mercury. The ability of Pseudomonas strain in remove mercury is related to enzyme of mercury reductase. This enzyme catalyses the reduction process from toxic mercury ion (Hg 2+ ) to elemental mercury (Hg 0 ). The enzyme of merT, merP and merA are involved in mercury reduction for gram-negative bacteria [24]. The cells were stress by the physical damage of the process and resulting in sublethally injured the cells. A sufficient amount of recovery time and appropriate environmental conditions may require repairing the metabolic system of the cells. The types of stress that contribute in the cells damage were freeze injury cause by physical damage, exposure to concentrated solutes and osmotic stress that is cause by the move of water in or out from the cells. These factors lead to the membrane damage, protein, and enzyme change [25].

IV. CONCLUSION
A good protectant attribute to the high CFU. It also depends on the drying purpose which is high CFU immediately after freeze drying or high CFU after storage time. The result shows that P. putida freeze dry in tween80/sucrose solution was quite resistant to freeze drying which is useful from commercial viewpoint. The moisture content in tween 80/sucrose solution was the lowest. The maximum viability is essential for mercury reduction. The highest mercury reduction (25.03%) after 3 weeks storage was from tween80/sucrose freeze-dried P. putida cells. In contrast, 56.78% mercury reduction was obtained from PEG pre-suspended cells after immediate rehydration. PEG freeze-dried P. putida cells have higher moisture content. Hence, has a low cell viability and low mercury reduction after storage time. This study proved that Tween80/sucrose is a suitable freeze dry lyoprotectant for P. putida.