In this work, the yeast cells of Ogataea polymorpha BKM Y-2559 were immobilized in a organosilicon sol-gel membrane. It was shown that yeasts immobilized in a matrix containing 85 vol.% diethoxydimethylsilane (DMDES) and 15 vol.% tetraethoxysilane (TEOS) have the highest activity. Long-term storage of the resulting biohybrid material at a temperature of − 18 ºС demonstrated that the low temperature does not lead to a drop in the catalytic activity of the immobilized yeast. Maintaining the activity of the yeast encapsulated in the sol-gel matrix is important in the production and storage of commercial biocomposite samples. The study of the structure of the obtained material showed that the yeast cells of Ogataea polymorpha are located in the sol-gel matrix and are not able to be washed out, while the substrates and metabolites can diffuse to the cells without restrictions through the pores in the material.
Research Article
Immobilization of Microbial Cells in a Sol-Gel Matrix Using a Precursor with Two Non-Hydrolyzed Bonds
https://doi.org/10.21203/rs.3.rs-3324911/v1
This work is licensed under a CC BY 4.0 License
Journal Publication
published 05 Dec, 2023
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sol-gel
organosilicon membrane
Ogataea polymorpha
biocomposites
diethoxydimethylsilane
tetraethoxysilane
Currently, research in the field of biocomposite materials is of great interest for modern scientific research in green chemistry, biotechnology and ecology [1–3]. In this regard, the development of biocomposites continues and accelerates significantly in the last decade, as demonstrated by a large number of published works in this field [4, 5]. Cost-effectiveness, environmental friendliness, no need for energy-intensive and expensive equipment, as well as mild flow conditions make sol-gel synthesis indispensable in the development of biohybrid materials based on living cells of microorganisms [6, 7]. Immobilized cells are able to maintain their viability for a long time, which provides additional advantages when they are used in industrial and environmental biotechnology [6]. One of the modern approaches to the immobilization of microorganisms is encapsulation[8]. Synthetic shells are able to protect cells from physical and chemical stress factors like a natural "spore"; therefore, the similarity of artificial materials with natural ones is often emphasized [9].
One of the main components in the creation of biohybrids in the sol-gel synthesis are alkylalkoxysilanes (a hydrophobic additive containing non-hydrolysable bonds). The sol-gel method is often used to form hybrid materials containing organic functional groups that are attached to an inorganic structure [10, 11]. For this, various precursors are used, which contain not only efficiently hydrolysable Si-OR groups, but also hydrolysis-resistant Si-C bonds [12]. As a result, the final material contains non-hydrolyzed organic groups that do not change. The use of this synthesis scheme makes it easy to incorporate organic functional groups into the resulting inorganic lattice. These groups can impart certain optical or electronic properties to the material, while branching and an increase in the chain length of the precursor substituent reduces the rate of hydrolysis [11, 13].
Over the past ten years, our research team has been conducting research on the creation of porous hybrid biocatalysts using the sol-gel method. Simple and mild synthesis conditions have been proposed under which spontaneous formation of an organosilicate capsule around methylotrophic yeast cells occurs [14] using tetraethoxysilane and methyltriethoxysilane as silane precursors [15]. Previously, we used the following substances as a structure-control agent: polyethylene glycol (PEG) or polyvinyl alcohol (PVA). We have shown that when using PVA, the cells retained their activity longer, and the metrological characteristics of the biosensor were better [16].
However, it is necessary to use alkylalkoxysilanes with nonhydrolyzable Si–C bonds as precursors to create more favorable conditions for the functioning of the biomaterial [15]. Diethoxydimethysilane contains two nonhydrolyzable bonds and is the starting compound in the synthesis of silicones [17–19]. The surface of the silicone polymer is coated with hydrophobic methyl groups. This molecular structure gives silicones their unique properties [10]. It can be expected that the use of DMDES will lead to the formation of a less rigid but strong shell around the biomaterial, which will allow the cells to retain their catalytic activity longer (Fig. 1).
Cultivation of microorganisms
The yeast strain Ogataea polymorpha VKM Y-2559 was obtained from the All-Russian Collection of Microorganisms of the Institute of Biochemistry and Physiology of Microorganisms, Pushchino.
Obtaining a biosensor receptor element by encapsulation of cells by sol-gel method and measurement of the catalytic activity
The formation of the bioreceptor element and the measurement of the catalytic activity of the cells were carried out according to an earlier article [20].
Scanning electron microscopy and EDX
Before measurements, samples of the organosilicon sol–gel matrix with cells were applied to a conductive carbon adhesive tape fixed on the surface of an aluminum object stage 25 mm in diameter and covered with a thin film (15 nm) of carbon. The observations were made using a field emission scanning electron microscope Hitachi SU8000 (FE-SEM).
The images were obtained in the secondary electron mode at an accelerating voltage of 10 kV. EDX-SEM studies were carried out using an Oxford Instruments X-max 80 EDX system at an accelerating voltage of 10 kV.
Sample pore structure study by low-temperature nitrogen adsorption method (BET method)
The resulting material was studied by the method of low-temperature oxygen adsorption (BET method) according to the description given in the article [20].
Analysis of the catalytic activity of cells in biocomposites using the biosensor approach
Yeast cells of Ogataea polymorpha were used in the work. This is a species of mesophilic methylotrophic yeast [14]. They are able to utilize lower alcohols due to the presence of methanol metabolism enzymes, which will be an advantage in the development of a sol-gel matrix, since ethanol is released in hydrolysis reactions during self-assembly of the organosilicate matrix.
The biosensor approach was used to characterize the biocomposites. The installation for determining the respiratory activity of immobilized cells is shown in Fig. 2.
As a hydrophobic additive, we used diethoxydimethylsilane (DEDMS) in the amount of 100, 90, 85, 80 vol.% with the addition of tetraethoxysilane (TEOS), studies were also carried out in the absence of DEDMS. Polyvinyl alcohol was chosen as a structure-controlling agent.
The principle of functioning of the biosensor is based on the fact that the catalytic activity of microorganisms increases in the presence of methanol, which leads to a decrease in the oxygen concentration in the near-electrode space. This change fixes the oxygen electrode. The biosensor response was taken as the rate of change in the oxygen content upon addition of the analyte (mg (O2) min− 1 l− 1). According to the recommendations of the division of the International Union of Pure and Applied Chemistry (IUPAC) on physical and analytical chemistry, the following characteristics are important characteristics of biosensors: sensitivity, working or linear concentration range, and detection limit [21].
Whole-cell-based bioreceptors are of the catalytic type, i.e. the biological response in such systems is provided by enzymatic reactions, so the relationship shown in Fig. 3A is well described by the Michaelis-Menten-type enzymatic kinetics Eq. (1):
where rmax is the maximum reaction rate, achieved at [S]→∞; K M is the effective Michaelis constant numerically equal to the substrate concentration (mmol/dm3) at which the rate of the enzymatic reaction reaches half of the maximum value (r = rmax/2); [S] is the substrate concentration (mmol/dm3).
According to the Michaelis-Menten equation, the biosensor response will be proportional to the substrate concentration only at substrate concentrations below KM.
To reduce analysis errors, a linear section of the calibration curve is often used, which represents the working range of the determined concentrations. The upper limit of the linear dependence was determined from the equation for a hyperbola when approximating the experimental data in the Sigma Plot program. The numerical value of the upper limit of the determined concentrations is equal to the Michaelis constant, KM.
The sensitivity of the biosensor is determined using a quantitative characteristic of the sensitivity coefficient, which is defined as the maximum value of the derivative value of the response from the concentration.
The sensitivity coefficient was 4.2 ± 0.1 mg (O2)·min− 1·mmol− 1 for a biosensor based on a receptor element containing a hydrophobic additive DEDMS of 85% vol. For other receptor elements the results are presented in Table 1.
To characterize the capabilities of the technique in terms of quantitative analysis, the lower limit of the determined contents is used. This is the minimum component content that can be determined with a given degree of accuracy, which is characterized by the maximum permissible value of the relative standard deviation Sr. The results are presented in Table 1.
Long-term stability characterizes the stability of the sensor over a long period of time. Long-term stability was determined by measuring the sensor response daily. The criterion for determining long-term stability was the time in days until the response dropped by 50%. A typical time dependence of the response of a biosensor based on yeast cells encapsulated in organosilicate matrices is shown in Fig. 2B.
The operating time of the biosensor without replacement of the receptor element (DEDMS content 85 vol. %) is 36 days. This indicates a high viability of methylotrophic yeasts encapsulated in an organosilicon matrix of this composition. The results of determining the long-term stability of all studied bioreceptor elements are presented in Table 1.
| The content of DEDMS, about. % | Sensitivity coefficient, mg (О2)·min− 1·mmol− 1 | Determined concentration range, mol/dm3 | Long-term stability, day |
|---|---|---|---|
| 100 | 0,86 ± 0,04 | 5–290 | 9 |
| 90 | 3,1 ± 0,1 | 3–270 | 22 |
| 85 | 4,2 ± 0,1 | 2–440 | 36 |
| 80 | 2,6 ± 0,1 | 2–400 | 17 |
| 0 | 1,6 ± 0,1 | 5–500 | 15 |
Therefore, it has been shown that the best characteristics in terms of catalytic activity are characterized by a biosensor obtained on the basis of a biocomposite containing a hydrophobic additive of diethoxydimethysilane 85 vol. %. It is likely that this ratio of silane components forms a comfortable environment for yeast cells and ensures effective access of the analyte and removal of metabolites, while microorganisms are not washed out of the matrix. This assumption was investigated by us using analysis by the method of low-temperature nitrogen adsorption, as well as by SEM and EDX methods.
The stability of the catalytic activity of immobilized cells during storage at low temperatures is characterized by a change in the activity of microorganisms under certain storage conditions. In this work, yeast cells encapsulated in a sol-gel matrix based on silane precursors 85/15 DEDMS/TEOS were placed under different conditions for a long time: at − 18°C and + 2°C. After 90 and 150 days, the effectiveness of the functioning of samples of hybrid biomaterials was checked by their respiratory activity (Fig. 3C, Table 2).
| Days | Storage temperature Characteristic | + 2 ºС | – 18 ºС |
|---|---|---|---|
| 7 | Sensitivity coefficient, mg (О2)·min− 1·mmol− 1 | 4,1 ± 0,1 | 4,1 ± 0,1 |
| 90 | 0,31 ± 0,02 | 3,9 ± 0,4 | |
| 150 | 0,28 ± 0,04 | 3,8 ± 0,4 |
Analyzing the results, we can conclude that storage at + 2 ºС affects the performance of the biomaterial. So, in the first 90 days there is a decrease in the catalytic activity of cells, which subsequently stabilizes at a low level. This can be explained by the fact that this sample at this temperature becomes infected with foreign microflora or yeast cells die. However, long-term storage of the obtained samples at a temperature of − 18°C has practically no effect on the sensitivity of the receptor element. Samples stored at different temperatures retain their activity during the experiment, which is important in the production and storage of commercial biocomposite samples.
Surface study of obtained biocomposites by SEM and EDX methods
The biocomposite synthesized using 85 vol.% DEDMS was studied by scanning electron microscopy to get an idea of the structure and morphology. The image is shown in Fig. 4. The optimization of analytical measurements was carried out within the framework of the approach described earlier [22]. Before recording, the samples were placed on the surface of an aluminum table 25 mm in diameter, fixed with a carbon adhesive tape, and a conductive metal layer (Au/Pd, 60/40) 10 nm thick was deposited on them using the magnetron sputtering method described earlier [23]. The microstructure of the samples was studied by field emission scanning electron microscopy (FE-SEM) on a Hitachi SU8000 electron microscope. The images were taken in the secondary electron recording mode at an accelerating voltage of 5 kV and a working distance of 8–10 mm. The morphology of the samples was studied taking into account the correction for the surface effects of deposition of the conductive layer [23].
Therefore, when using the hydrophobic additive DEDMS, a capsule is formed around each cell. In this case, the encapsulated cells form a single architecture of the hybrid biomaterial (Fig. 4A). The organosilicon matrix has a looser structure and contains separate spheres from 1 to 5 µm. We assumed that the centers of initiation of sol-gel reactions are concentrated at the surface of microorganisms. As a result, small sol particles are formed around the cells. In the case of using the hydrophobic additive DEDMS, a structure is observed where the cells are covered with a smoother shell, which is similar to a film and on which spheres are present in a much smaller size (Fig. 4B), which is possibly associated with the formation of linear structures. It should be noted that the presence of two non-hydrolyzable Si-C bonds in DEDMS leads to the formation of predominantly linear molecules of alkyl-modified polysilicates, which ensures the formation of a silicone-like shell around yeast cells. In addition, DEDMS contributes to the formation of a special structure of the hybrid biomaterial in the form of chains of encapsulated cells, which can be clearly seen in Fig. 4B.
Therefore, when using the hydrophobic additive DEDMS, as well as when using MTES [15], the structure of encapsulated microorganisms is formed. Also, at the same ratios of silane precursors (85 vol. % MTES and 15 vol. % TEOS), the formation of an organosilicon capsule is observed, which has a different surface morphology when using various hydrophobic additives.
For the obtained biocomposites, the analysis of the local elemental composition was carried out by the method of energy dispersive X-ray spectroscopy. An example of local elemental analysis for a matrix containing 85 vol. % DEDMS with immobilized yeast cells is shown in Fig. 4F. The EDX study of the samples was carried out using an Oxford Instruments X-max energy-dispersive spectrometer.
The carbon content exceeds the silicon content, which is natural, since DEDMS contains two methyl groups and the structure-controlling agent polyvinyl alcohol also contains carbon. The absence of nitrogen, phosphorus, and sulfur indicates the absence of microorganisms on the surface of the matrix and their complete immobilization in the sol-gel matrix.
Study of the chemical structure of biocomposites using the BET method
The low-temperature nitrogen adsorption method was used to determine the porosity and specific surface area of the resulting biocomposites, the results are shown in Fig. 4D.
The resulting isotherm (Fig. 4D) belongs to type V. This type of isotherm is typical for mesoporous and microporous materials in a system where the solid-gas interaction is weaker than between gas atoms. Based on the BJH model (Barrett-Joyner-Halenda), the distribution of pores by volume was obtained for a biocomposite with yeast and the composition DEDMS/TEOS 85/15 (Fig. 4E).
As can be seen from the figure, the size of the majority of pores in the sample is in the range from 20 to 80 nm, i.e., mesopores predominate. Therefore, yeast (about 2 µm in size) is securely fixed in the sol-gel matrix and is not able to be washed out, while metabolites and substrates can diffuse through pores of such sizes without restriction. The sample also contains pores smaller than 6 nm in diameter, which is consistent with the shape of the isotherm, which indicates the presence of meso- and micropores.
In this work, yeast cells were immobilized in a sol-gel matrix using different ratios of the silane components of tetraethoxysilane and diethoxydimethysilane. It has been shown that yeasts have the highest activity when immobilized in a matrix containing 85 vol.% DEDMS; long-term storage of the obtained sample at a temperature of – 18°C practically does not affect the functioning of biosensitive elements, which is important in the production and storage of commercial samples of hybrid biomaterial. When studying the structure of the obtained material, it was demonstrated that yeast cells are not able to be washed out, while metabolites and substrates can diffuse without restrictions through pores of such sizes and the transport of substances to and from the cell is not limited.
When using 85 vol. % DEDMS during the formation of the sol-gel material, the most favorable conditions for the functioning of the biomaterial were created. DEDMS will form a less rigid but strong shell around the biomaterial. Using the SEM and EDX method, it was shown that each cell of microorganisms is immobilized with a sol-gel material; long-term storage of the obtained sample at a temperature of -18ºС demonstrated that the samples retain their activity during the experiment, which is important in the production and storage of commercial samples of the hybrid biomaterial. When studying the structure of the obtained material, it was demonstrated that yeast cells are not able to be washed out, while metabolites and substrates can diffuse without restrictions through pores of such sizes and the transport of substances to and from the cell is not limited.
Electron microscopy characterization and determination of the local composition were performed in the Department of Structural Studies of Zelinsky Institute of Organic Chemistry, Moscow.
Author Contributions
All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Elizaveta A. Lantsova, Pavel V. Rybochkin, Vyacheslav A. Arlyapov, Evgenia A. Saverina and Anatoly N. Vereshchagin. The first draft of the manuscript was written by Olga A. Kamanina and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.
Acknowledgements
This research was funded by the Ministry of Science and Higher Education of the Russian Federation within the framework of a state assignment on the topic "Synthesis of targeted biologically active ionic compounds and new biocomposite materials" (FEWG-2021-0011).
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No competing interests reported.