What Can Contribute to Weakening of Poly(Vinyl Alcohol) Cryogels Used for Cell (Self)Immobilization?

Abstract

This work was undertaken in order to identify the presence of changes in the characteristics of poly(vinyl alcohol) (PVA) cryogels that can contribute to the degradation of such polymer matrices under the influence of their contact with various microorganisms used in immobilized form in different biotechnological processes using various complex media and conditions. Immobilized cells of bacteria, yeasts, microalgae, fungi, and microbial consortia were involved in the investigations. It was established that the presence of microorganisms can indirectly (through media transformed by them, in particular, containing lipids) or directly (through high rates of metabolite production, in particular, the fast accumulation of gases in the pores of polymer matrices, or due to the colonization of cryogels (self)immobilization by fungi with the growing mycelium) decrease rheological characteristics of PVA cryogel. Such weakening of PVA cryogels can be expected as a result of the first stage of further degradation of polymer matrices. The values of both the modulus of elasticity and the shear modulus of PVA cryogels confirmed this. The effect of high pressure accumulated in the reactors with PVA cryogel-immobilized cells, as well as their use in flow systems, was not revealed. These factors can be taken into account for the sustainable use of matrices based on PVA cryogels as biocatalysts with microorganisms or soil-structuring elements in artificial or natural environments.

1. Introduction

Polyvinyl alcohol (PVA) is a unique material that has found application in a wide variety of fields, as it is a water-soluble synthetic polymer, which is generally recognized as safe (GRAS) [1]. Gels of PVA formed due to multiple noncovalent interactions (hydrogen bonds) that appear between polymer chains during freezing of PVA solutions at subzero temperatures, with their further thawing, are named “cryogels”. They are a very popular polymer matrix in various areas of application. They are offered for medical use as drug delivery systems or cryogel-based implants for bone regeneration and replacement [2,3]. Functionalized PVA cryogels are considered relevant candidates among polymers for use as packaging or dressing materials with antioxidant and antimicrobial properties [4,5]. PVA cryogels obtained by freezing/thawing PVA solutions are actively used as matrices for the production of biocatalysts (BCs) based on enzymes and cells of various microorganisms [5,6,7]. Such BCs have the potential for widespread use in obtaining valuable microbial metabolites [7], wastewater treatment systems, detection and degradation of xenobiotics in environmental objects (aquatic environments and soil) [8,9]. Often, processes using PVA cryogels can be a combination of the above-mentioned technical approaches to their application [5,7].

The wide potential of PVA cryogels raises questions about the possible degradation of these polymer matrices in their application processes and environmental conditions. These issues are particularly relevant in the case of the use of PVA cryogels as components of building materials, the use of which is expected in the Arctic regions or structure-forming agents for soils containing various microorganisms and susceptible to erosion under cold-weather conditions [10,11,12]. Currently, the question of the possible appearance of microparticles of polymer matrices obtained on the basis of PVA in environmental objects has already been formulated in the scientific literature, and it has been noted that such particles can reside under environmental conditions for up to 50 years [13].

A search in Google Scholar for the combination of words “degradation”, “PVA cryogel”, and “microbial cultures” brings up about a thousand publications, which generally indicates the relevance of this topic. However, the degradation of cryogels, as a rule, is investigated under physiological conditions typical of human organisms (pH 7.4, PBS buffer, 14–37 °C, 3 weeks) [14,15] or the properties of cryogel are being studied in a single biotechnological process involving only specific cells [16]. Previously published research results with a wide range of microorganisms that can be (self)immobilized and potentially affect the properties of PVA cryogels, weakening them, have not been identified. Therefore, this topic has become the subject of this study. The use of rheological research methods allows us to obtain an important understanding of the stability of polymers and the kinetics of their decomposition [17]. It should be noted that under environmental conditions, polymer particles can swell over time and freeze and thaw due to natural changes in weather conditions, which can hypothetically lead to the formation of new PVA cryogels in natural ecosystems. Already such matrices can act as carriers for the sorption of aboriginal microorganisms and various substances, including microbial metabolites and environmental pollutants. Such polymer particles can further serve as a “Trojan horse” [18], capable of both the positive formation of stable matrices with the formation of natural consortia on their basis, performing an important ecological function for the mineralization of various organic wastes, and leading to a negative effect in the form of the appearance of foci of resistant pathogenic and toxic loci. In this case, the interest in the stability of polymer matrices is increased.

It is known that there are some factors affecting PVA cryogels that can significantly enhance them rather than weaken them. Among them are

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The presence of components of various wastes (for example, salt) used as media for exposing the polymer matrices under discussion [19];

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The inclusion of additional components in PVA cryogels (for example, various cellulose derivatives) that turn them into samples of composite materials [5,20];

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Reduction in ambient humidity and drying of PVA cryogels [21].

At the same time, differences in the pH values of media, characteristic of various biocatalytic and microbial processes, slightly change the characteristics of pure PVA cryogels [8,12]. The presence of microorganisms usually brings some factors that cannot be taken into account prognostically when discussing the stability of PVA cryogels.

High efficiency of degradation of different polymers, including PVA cryogels, has been found for many cells of microorganisms (bacteria, fungi, microalgae) and their consortia [22]. However, our own significant research experience in the work with PVA cryogels as cell carriers led us to the idea that it is often not possible to isolate and identify individual metabolites of microbial cells that can contribute to the weakening or degradation of such polymer carriers. These can often be complex effects from microbial cells and media in which PVA cryogels are exposed, reflecting a combination of factors leading to the discussed changes in the rheological characteristics of polymer carriers. The predisposition of PVA cryogels to degradation in biotechnological processes may become a limiting factor for their practical application as carriers for immobilized BCs. Or, on the contrary, to open up new horizons for their use under environmental conditions. In this regard, it is important and necessary to study the responsiveness of the rheological properties of PVA cryogels in the presence of various microorganisms and the media they use. Until now, attention has not been paid to the systematization of knowledge in this practically significant area of research [22].

Gaining knowledge about the properties of PVA cryogels in a variety of application conditions and establishing certain patterns between several interrelated factors (microorganisms/media, conditions/characteristics of PVA cryogels) can serve as a scientific base for predicting the strength characteristics of such matrices and possible environmental risks of their use in the development and application of new BCs and processes with them. The purpose of this work was to identify the presence of changes in the rheological characteristics of PVA cryogels, which can contribute to the degradation of such polymer matrices under the influence of their contact with various microorganisms used in immobilized form in different biotechnological processes using various complex media and conditions (Figure 1).

For a wide range of microorganisms the PVA cryogel was estimated as a carrier, allowing immobilization of cells by their entrapment or colonization, when the main attention was drawn to the changes in the rheological characteristics of the PVA cryogel granules as a summarizing function of influence of various factors connected with viability of cells (aerobic and anaerobic conditions of their existence, different chemical content of nutritional media, flow and static regimes of cultivation, etc.).

2. Materials and Methods

2.1. Materials

The components of nutrient media for biomass accumulation were purchased from Chimmed (Moscow, Russia). Dimethyl sulfoxide was purchased from Chimmed (Moscow, Russia) and used to extract adenosine triphosphate (ATP) from cells. The PVA (Type 16/1, 84 kDa) was purchased from Sinopec Corp. (Beijing, China). Paraoxone was purchased from Sigma (St. Louis, MO, USA).

2.2. Microorganisms and Conditions of Their Cultivation

Filamentous fungi (Aspergillus foetidus F-359, Aspergillus niger F-326, Rhizopus oryzae F-814), yeast-like fungi (Aureobasidium pullulanus Y-4137), yeast (Saccharomyces cerevisiae Y-185, Pachysolen tannophilus Y-475), bacteria (Clostridium acetobutylicum B-1787, Desulfovibrio desulfuricans B-1799, Leuconostoc mesenteroides B-5481, Komagataeibacter xylinus B-12429, Photobacterium phosphoreum B-1717) were obtained from the All-Russian Collection of Industrial Microorganisms, Moscow, Russia (http://www.genetika.ru/vkpm/ has been accessed on 30 July 2025). Bacteria Rhodococcus erythropolis AC-1514D, Pseudomonas esterophilus V-1436D were obtained from the All-Russian Collection of Microorganisms, Moscow region, Russia (https://vkm.ru/ has been accessed on 30 July 2025).

Microalgae Chlorella vulgaris C-1 was obtained from the Collection of Microalgae IPPAS Moscow, Russia (http://collection.cellreg.org/ has been accessed on 30 July 2025). Thalassiosira weissflogii CCMP 1336 diatoms were obtained from Department of Biophysics, Biological Faculty, Moscow State University.

Natural methanogenic consortium was taken from the wastewater treatment division of Bryntsalov plant (Moscow region, Russia).

The anaerobic consortium was maintained on 0.1 M potassium phosphate buffer (pH 7.2) with the addition of 1 g/L of glucose. For the accumulation of spores and germination of fungal mycelium, a nutrient medium of the following composition (g/L) was used: glucose—50; yeast extract—5; K₂HPO₄—3; KH₂PO₄—2; MgSO₄—0.5; pH—6.8 with or without agar addition (20). The cells were cultured at 30 ± 2 °C for 48 ± 1 h with constant agitation (180 ± 10 rpm) at a shaker Lab-Therm (Adolf Kühner, Switzerland). A nutrient medium of the following composition (g/L) was used to accumulate biomass of yeast cells and yeast-like fungi: glucose—20; yeast extract and peptone-5; K₂HPO₄—3; KH₂PO₄—2; citric acid—1; ascorbic acid—1; MgSO₄—0.5; pH—5.6. Cells were cultured at 26 ± 2 °C for 20 ± 1 h with constant agitation (150 ± 10 rpm). A nutrient medium of the following composition (g/L) was used to accumulate the biomass of R. erythropolis and P. esterophilus bacteria: peptone—10; yeast extract -5, NaCl—10; pH—7.0.

The C. acetobutylicum was cultivated in a medium with the following composition (g/L): glucose—50; tryptone—10; yeast extract—5 (pH 6.8). Cultivation was carried out under anaerobic conditions in an argon atmosphere at 37 °C for 24 h. Same medium was also used for the accumulation of organic solvents. The concentration of the immobilized BC was 20 g dry weight/L.

For D. desulfuricans the medium of the following composition was used (g/L): K₂HPO₄—0.5, NH₄Cl—1.0, Na₂SO₄—1.0, CaCl₂ × 2 H₂O − 0.1, MgSO₄ × 7 H₂O − 2.0, sodium lactate − 2.0, yeast extract-1.0, FeSO₄ × 7 H₂O − 0.5, Na-thioglycolate − 0.1, ascorbic acid − 0.1, FeCl₂ × 4 H₂O −0.001 g, ZnCl₂—0.068, MnCl₂ × 4 H₂O − 0.1, H₃BO₃—0.062, CoCl₂ × 6 H₂O − 0.12, Na₂MoO₄ × 2 H₂O − 0.024; pH—7.4.

The nutrient medium used to accumulate L. mesenteroides biomass at 28 ± 1° C had the following composition (g/L): sucrose-50; yeast extract-5; tryptone-5; K₂HPO₄—1.5; MgSO₄—0.01; MnCl₂—0.01; CaCl₂—0.05; pH—7.0. Cultivation lasted 18 h at 200 rpm.

To accumulate K. xylinus biomass at a temperature of 28 ± 1 °C, a Hestrin–Schramm medium was used (g/L): glucose-20; yeast extract-5; peptone-5; K₂HPO₄—2.5; MgSO₄—0.3; citric acid—1.15; pH—5.6.

Farghaly growth medium (g/L) was used to accumulate P. phosphoreum cell biomass: NaCl—30.0; Na₂HPO₄—5.3; KH₂PO₄ × 2H₂O − 2.1; (NH₄)₂HPO₄—0.5; MgSO₄ × 7H₂O − 0.1; yeast extract-1.0; peptone-5.0; glycerol-3.0; pH—7.5. Cultivation was carried out at 16 °C, 60 rpm (IRC-1-U temperature-controlled shaker, Adolf Kuhner AG Apparatebau, Birsfelden, Switzerland). The use of immobilized photobacterium cells in a flow reactor (5 mL) was carried out at 15 °C and a flow rate of 360 mL/h.

Tamiya medium (g/L) was used for microalgae biomass accumulation: KNO₃—5.00, MgSO₄ × 7H₂O − 2.50, KH₂PO₄—1.20, FeSO₄ × 7H₂O − 0.03, with addition of solution A (1 mL) containing (mg/L) H₃BO₃—2.80, MnCl₂ × 4H₂O − 1.80, ZnSO₄—0.20, and solution B (1 mL) containing (mg/L) MoO₃—17.6, NH₄VO₃—22.9, pH 7.0. Cultivation of the microalgal cells was performed at 25 °C and under round-the-clock lighting with Osram Fluora 77 luminescent lamps (30 W, Munich, Germany).

The same medium was used to study the PVA cryogel samples with immobilized cells under flowing conditions. The working volume of the photobioreactor was 5 mL, and the flow rate of the medium was 360 mL/h.

The pH was monitored potentiometrically using a PBL pH meter (Mettler Toledo, Greifensee, Switzerland).

Ash content was determined in crude glycerol using a Nabertherm LT3/11 muffle furnace (Lilienthal, Germany) at 590 ± 10 °C for 24 h. Ash is expressed as a percentage of the difference in weight before and after ashing.

2.3. Immobilization and Application of Microorganisms

To obtain microbial cells immobilized in PVA cryogel, the inclusion method was used, which was described earlier [23]. Yeast, bacteria, microalgae, and natural methanogenic consortium were immobilized as cell biomass, while fungi were immobilized as spore material.

Biomasses of yeast and bacteria were separated from the culture broth by centrifugation (Avanti J25, Beckman, Germany) for 15 min at 8000 rpm. Naturally sedimenting biomass was used to immobilize methanogenic consortia and microalgae.

To immobilize mycelial fungi, spores were previously accumulated by solid-phase cultivation, which were incorporated into the PVA cryogel and then grown as part of a BC in a nutrient medium.

Artificial immobilized consortia (methanogenic and bacterial) were prepared as described previously [23]. The composition of the resulting mixture of biomass of different cells was immobilized by inclusion into PVA cryogel and used in experiments (biomass % by wet substances, humidity 85 ± 3%): I sample (80% natural methanogenic consortium + 10% R. erythropolis + 10% D. desulfuricans) and II sample (33% R. erythropolis + 67% P. esterophilus).

The immobilization procedure consisted of the following: an aqueous PVA solution of the appropriate concentration was autoclaved at 121 °C and 0.5 atm, cooled and mixed with the corresponding microbial biomass. The suspension was poured into special forms and frozen at −20 °C or −70 °C (for microalgae). After 24 h, the frozen suspension was placed at +4 °C until it was completely defrosted. Similarly, but without the addition of microbial biomass, “empty” PVA cryogel matrices were prepared. The geometric dimensions of the granules were as follows: 0.7 ± 0.1 × 0.4 ± 0.05 cm. The mass of one pellet was 200 ± 10 mg. The resulting immobilized BCs were used in various processes with different media, which are shown in

Table 1. Substrates and processes using PVA cryogel-immobilized cells of microorganisms studied in this work.

Medium and Its Content *	Process Conditions
Starch processing wastewater, g/L: Lip-5; Ch-50; Pr-20; Sl-3	Process: Wastewater treatment with multi-enzyme complexes of filamentous fungi. Conditions: The use of each type of model wastewater for cell cultivation was carried out on a shaker Lab-Therm (Adolf Kühner, Switzerland) under aerobic conditions, 180 rpm, 28 °C. Concentration of each BC was 20 g DW/L. The media were refreshed after each working cycle (30 h).
Meat processing wastewater, g/L: Lip-10; Ch-35; Pr-1; Sl-12.3
Wastewater from abattoirs, g/L: Lip-8; Ch-30; Pr-20; Sl-4.3
Mixed dairy processing, g/L: Lip-21; Ch-0.1; Pr-0.5; Sl-10.3
Soybean processing factory, g/L: Lip-0.5; Ch-0.1; Pr-0.9; Sl-3
Apple cake, g/g: Ch-45.9; Pr-6.2
Domestic wastewater, g/L: Lip-0.08; Ch-0.04; Pr-0.06	Process: Wastewater treatment and accumulation of microalgae biomass. Conditions: BC concentration—3.5 g DW/L; 28 °C. The media were refreshed after each working cycle (3 days).
Dairy wastewater, g/L: Lip-0.35; Ch-0.56; Pr-0.28
Farming wastewater, g/L: Lip-0.17; Ch-0.27; Pr-0.13
Treated potato pulp: for its treatment (enzymatic hydrolysis), the α-amylase of Aspergillus oryzae (Sigma-Aldrich Inc., Missouri, United States) was added to raw material (5 mg per 1 g of dry biomass). The hydrolysate contained 25 g/L glucose.	Process: Biotransformation of waste and production of pullulan with A. pullulanus. Conditions: BC concentration–12 g DW/L; 200 rpm, 26 °C. The media were refreshed after each working cycle (50 h).
The treated beet pulp was obtained by enzymatic hydrolysis of 50 g/L of dried beet pulp in 0.1 M citrate buffer (pH 5.0) with a complex enzyme preparation consisting of cellulases and β-xylosidase isolated from Penicillium canescens cells (5 mg protein/g substrate) for 48 h at 50 °C and constant stirring (250 rpm) (IRC-1-U Adolf Kuhnner, Apparaebau, Switzerland).	Process: Biotransformation of waste and production of dextran and bacterial cellulose by L. mesenteroides and K. xylinus cells, correspondently. Conditions for L. mesenteroides cells: BC concentration—30 g DW/L; 28 °C, 200 rpm, working cycle—24 h; Conditions for K. xylinus cells: BC concentration-12 g DW/L; 28 °C, working cycle-140 h.
Crude glycerol, obtained as a by-product of biodiesel production from the lipids of C. vulgaris biomass, was used for P. tannophilus cell cultivation. KOH in CH3OH was used for lipid transesterification of microalgal lipids, and the reaction was carried out for 10 min at 60 °C. The product was cooled and separated by centrifugation at 5000 rpm for 5 min. Crude glycerol feedstock contained 81.9% glycerol, 6.5% ash, 0.6% CH3OH, and 11% H2O.	Process: Ethanol production. Conditions for P. tannophilus cells: BC concentration—12 g DW/L; 28 °C, 200 rpm, working cycle-24 h.
Soil with pesticide (paraoxon), humidity 90 ± 5%, pesticide concentration—100 mg paraoxon/kg of soil	Process: Soil decontamination from pesticides Conditions: The immobilized cells were buried in the soil to a depth of 15 cm. BC concentration was 20 g DW/kg soil. A new dose of pesticide was added every 24 h. The soil moisture was constantly controlled and maintained.
Medium with benzothiophene sulfone (0.15 mM) was prepared using 0.1 M phosphate buffer (pH 7.2) with addition of 5 g/L ethanol	Process: Biotransformation of sulfur-containing waste from chemical and biocatalytic desulfurization of hydrocarbon raw materials. Conditions: BC concentration—20 g DW/L; 35 °C, anaerobic conditions

* Lip—lipids; Ch—carbohydrates; Pr—proteins; Sl—salts; DW—dry weight.

.

Immobilized yeast cells were used in anaerobic treatment of starch processing wastewater and ethanol production from treated beet pulp (media compositions are shown in Table 1). The concentration of the BC in such processes was 20 g dry cell weight/L. The process was carried out at 28 °C. The period of BC use was 3 days.

The natural methanogenic consortium was used to treat wastewater from the Soybean processing factory, the composition of which is shown in Table 1. The concentration of the BC in methanogenic process was 20 g dry cell weight/L. The process was carried out at 35 °C. The duration of process was 7 days.

The possibility of colonization of PVA cryogel was determined in the same nutrient media, which were applied to maintain the used microorganisms. The concentration of bacterial inoculum and filamentous fungal spores was 1%, and cultivation was carried out for 4 days. In experiments with C. vulgaris microalgae and a methanogenic anaerobic consortium, granules of PVA cryogel at a concentration of 10 g dry cell weight/L were placed in a suspension of microorganisms (with a cell concentration of 2.25 g dry cell weight/L) and exposed for 14 days.

2.4. Rheological Characteristics of PVS Cryogel

The modulus of elasticity of the PVA cryogel with immobilized cells was determined using standard method [24]. The modulus of elasticity of cryogels samples (E) was determined by the method of uniaxial compression using a Kargin–Sogolova dynamometric balance at a constant load of 4.9 mN. The values of E were calculated from the equation: E = (F/h^3/2) • [3(1 − σ²)/(2R)^1/2], where F is the load (4.9 mN), h is the deformation (decreasing of granule diameter in meters), σ is the Poisson’s ratio (assumed to be equal to 0.49), and R is the initial granule radius (in meters). The measurements of the mechanical strength of cryogel samples were carried out for five granules at each investigated point; the obtained E values were averaged. The shear modulus of PVA cryogel was determined using a Lamy RM 200 rheometer (Lamy Rheology, Champagne-au-Mont-d’Or, France) according to a well-known technique [25].

For such media options as waste from a soybean processing plant and soil with pesticides (paraoxon), the modulus of elasticity was controlled for samples of PVA cryogels without microbial cells. This was performed to compare the changes in the rheological characteristics of the polymer matrix under the influence of viable cells and the media used.

2.5. Analytical Methods

Concentrations of carbohydrates, lipids, and protein were determined using standard procedures [26].

When studying cell colonization of gel matrices, the concentration of PVA solution used for cryogel formation was varied in the range of 7–13%. The samples of PVA cryogel were colonized by cells, and the level of colonization was estimated by detection of ATP concentration in the investigated samples. This parameter is in direct correlation with the number of viable cells on a carrier. Intracellular ATP concentration of immobilized cells was estimated by a bioluminescent ATP-metry on a 3560 microluminometer (New Horizons Diagnostics Co., Columbia, MD, USA) using a standard ATP reagent (Biochimmak, Moscow, Russia) based on a recombinant firefly luciferase. To extract ATP, the granules (~100 mg) were transferred to dimethyl sulfoxide (1 mL) and incubated at 25 °C for 1 h.

To study the changes in the rheological characteristics of microalgae cells immobilized in PVA cryogel before and after their exposure in a flow system, the polymer granules with immobilized cells (C. vulgaris, T. weissflogii, or P. phosphoreum) were placed in a 5 mL cuvette of flow bioreactor through which a nutrient medium was pumped for 240 h under following conditions: medium with flow rate 360 mL/h at 16 ± 1 °C for both C. vulgaris and T. weissflogii, medium with flow rate 90 mL/h at 15 ± 1 °C for P. phosphoreum. The cells C. vulgaris and T. weissflogii were kept under round-the-clock lighting (Osram Fluora 77 luminescent lamps, 30 W, Osram, Munich, Germany).

Determination of relative fluorescence variable (Fv/Fm) of microalgae cells was performed when fluorescence excitation was undertaken at 455 nm with an electronic photomultiplier through a KS-18 filter. The intensity of chlorophyll fluorescence under the conditions of open reaction centers of Photosystem II (F₀) and the maximum chlorophyll fluorescence intensity under the conditions of completely closed reaction centers of Photosystem II (Fm) were measured at the intensity of an excitation light with a density of 0.8 and 6000 μmol quanta/(m²/s), respectively. The potential efficiency of the primary photosynthesis processes (maximum photochemical quantum yield of Photosystem II), Fv/Fm = (Fm − F₀)/Fm, Fv = Fm − F₀, is the variable fluorescence. The fluorescence values were presented in relative units.

The dry weight (DW) was determined via a standard gravimetrical method. Samples were dried at 80 °C to a constant weight.

Bioluminescence of immobilized bacteria Photobacterium phosphoreum was analyzed using a 1250 LKB-Wallac luminometer (LKB Wallac, Turku, Finland). The maximum level of luminescence (I₀) was determined for 10 s at 15 °C after thermal equilibration of the flow-through system. The tests were performed in triplicate. For practical purposes, the residual intensity of bioluminescence was used (I/I₀), which was expressed as a percentage of the baseline signal (I₀).

The gas pressure under anaerobic conditions was controlled by pressure gauge (Fiz-tech, Moscow, Russia). The amount and composition of gases produced under anaerobic conditions were analyzed using a Crystallux-4000M gas chromatograph (RPC ‘Meta-chrom’, Yoshkar-Ola, Russia) equipped with a katharometer. Argon was used as the carrier gas, the flow rate was 20 mL/min, and the column temperature was 51 ± 1 °C. The gas phase sample volume was 0.2 mL. A gas mixture of H₂, CH₄, CO₂, and H₂S (5%) in argon was used as a standard (PGS-Service, Moscow, Russia). The obtained data were analyzed using NetChrom software (Meta-chrom ver. 2.1, Yoshkar-Ola, Russia). The accumulation rate of gases (mL/g BC/day) was calculated as the ratio of the total volume of gases produced to the duration of the process. Statistical analysis was performed using SigmaPlot (ver. 12.5, Systat Software Inc., San Jose, CA, USA) with paired t-test and one-way analysis of variance (ANOVA). The data are presented as means of at least three independent experiments ± standard deviation (±SD) unless otherwise stated. A value of p < 0.05 was considered statistically significant.

3. Results

3.1. Changes in the Properties of PVA Cryogels in Presence of Microorganisms Operating Under Aerobic and Microaerophilic Conditions

Samples of model and real wastewater (Table 1), as well as a number of treated waste products, were used as media to study their effect in the presence of microorganisms on the strength properties of PVA cryogels. The selected media were applied for their biotechnological transformations under the action of immobilized microbial cells acting as biocatalysts (BCs) for various processes. Despite the fact that the selected media had different compositions and properties (the pH in the media varied from 4.2 to 9.5), all the biocatalytic processes listed in

Table 2. Dynamics of the modulus of elasticity (kPa) change in PVA cryogel used in obtaining immobilized microbial biocatalysts and applied in the process of wastewater treatment or biotransformation of wet wastes of different origins.

Medium	pHstart/ pHfinal	Immobilized BCs		Modulus of Elasticity (kPa) *
		Concentration of PVA Solution, %	Cell Biomass	Time of Exposition of BCs in the Medium, h
				30	90	300	480
Starch processing	4.3/6.5	11	Filamentous fungi Rhizopus oryzae	60.3 ± 2.3	69.2 ± 6.4	77.1 ± 6.1	79.6 ± 3.9
Meat processing	7.5/5.7			59.8 ± 3.3	56.2 ± 3.4	44.4 ± 5.5	37.7 ± 7.4
Wastewater from abattoirs	6.0/5.1			58.7 ± 6.3	54.3 ± 5.5	50.1 ± 6.6	46.3 ± 4.5
Wastewater from mixed dairy processing	5.9/4.5			56.4 ± 6.5	30.1 ± 5.0	29.9 ± 3.9	26.0 ± 4.2
Wastewater from soybean processing factory	6.3/4.2			58.5 ± 9.5	62.8 ± 9.2	77.1 ± 6.1	75.2 ± 5.1
	6.3	11	Without cells	56.3 ± 3.5	56.1 ± 3.1	56.8 ± 3.3	58.3 ± 2.9
Apple cake	5.5/4.2	10	Filamentous fungi Aspergillus foetidus	61.7 ± 9.2	65.6 ± 9.7	67.8 ± 7.2	70.1 ± 8.2
Domestic wastewater	6.9/9.5	7	Microalgae Chlorella vulgaris	20.1 ± 2.4	26.3 ± 2.7	35.2 ± 2.3	42.7 ± 2.2
Dairy wastewater	6.8/9.2			17.4 ± 1.7	16.2 ± 1.6	14.1 ± 1.7	10.4 ± 1.1
Farming wastewater	6.5/9.0			18.3 ± 1.8	17.5 ± 1.5	16.9 ± 1.5	16.4 ± 1.2
Treated potato pulp	5.5/5.4	11	Yeast-like fungi Aureobasidium pullulanus	68.6 ± 3.1	90.6 ± 4.8	87.4 ± 4.8	89.2 ± 5.2
Treated beet pulp	7.0/5.4	12	Bacteria Leuconostoc mesenteroides	78.8 ± 3.1	79.3 ± 2.3	79.1 ± 2.6	78.7 ± 3.4
	5.5/3.4	12	Bacteria Komagataeibacter xylinus	79.2 ± 3.4	80.2 ± 3.2	79.7 ± 3.3	81.7 ± 4.1
Crude glycerol	5.6/3.2 5.6/4.7			79.2 ± 3.4	76.2 ± 4.1	77.4 ± 3.7	78.2 ± 2.9
		12	Yeast Pachysolen tannophilus	63.4 ± 3.5	65.5 ± 3.3	67.2 ± 3.0	66.4 ± 2.9
Soil with pesticide (paraoxon)	6.4/6.4	12	Bacteria Pseudomonas sp.	74.2 ± 3.1	72.7 ± 3.0	68.9 ± 2.8	68.8 ± 2.3
		12	Artificial bacterial consortium of Pseudomonas sp. and Rhodococcus erythropolis	72.5 ± 2.7	70.6 ± 2.7	65.9 ± 3.3	66.7 ± 2.9
			Without cells	71.7 ± 2.4	71.2 ± 1.7	68.9 ± 1.4	68.1 ± 1.2

* Table cells highlighted in light pink show cases where there was a statistically significant change (p ≤ 0.05) between values at different exposition times; blue shows data for granules of PVA without cell biomass.

proceeded aerobically, which served as the basis for their possible comparative analysis.

In a number of cases, the same media were used for different BCs, which made it possible to assess the influence of the cells themselves on the properties of cryogels. BCs were formed as a result of the inclusion of cells of different microorganisms (filamentous and yeast-like fungi, microalgae, and bacteria, as well as artificial microbial consortium) in PVA cryogels prepared using polymer solutions with different concentrations (Table 2).

An analysis of the changes occurring with the PVA cryogel in the composition of the same BC, which is composed of the same matrix and microorganism, but used in the transformation of different media (in particular, it concerns bacterial cells Leuconostoc mesenteroides and Komagataeibacter xylinus, filamentous fungi Rhizopus oryzae, and microalgae Chlorella vulgaris) (Table 2), showed that the strength of the cryogel carrier in such systems is less affected by the pH values of the media used, and more by the biochemical compositions of the media themselves. Thus, the presence of lipids in the composition of the media in quite significant concentrations (for example, 21 g/L and 10 g/L of lipids in mixed dairy processing and meat processing, respectively) led to a significant decrease in the modulus of elasticity of the PVA cryogel (by 37–54%) for 300 h of using the BCs.

An interesting result should be noted, which was established by the example of BC obtained on the basis of C. vulgaris microalgae cells included in the PVA cryogel. To ensure the survival of C. vulgaris cells during the formation of PVA cryogel granules and the simultaneous incorporation of microalgae cells into the resulting BC, a PVA solution with a relatively low polymer concentration (7%) was used (Table 2). The obtained matrix was characterized by a rather low modulus of elasticity in relation to other BC samples. However, during the use of immobilized phototrophic cells as an inoculum for the accumulation of suspended biomass of phototrophs and their use for the treatment of Domestic wastewater applied as a medium for cell cultivation and BC exposure, the pH value of the medium shifted to the alkaline range (from 6.9 to 9.5). Such conditions for the cultivation of immobilized cells gradually led to the hardening of the PVA cryogel matrix. However, a similar shift towards alkaline pH values (the pH shift was from 6.8 to 9.2) in the Dairy wastewater used as a medium for the same BC led to a 40% decrease in the studied strength index of the PVA cryogel. Thus, a comparison of the data on the initial composition of the media allowed us to say that it is the lipids present in the media into which BCs obtained on the basis of PVA cryogels are introduced that contribute to a decrease in their strength characteristics and the possible subsequent degradation of stable matrices.

3.2. Changes in the Properties of PVA Cryogels in the Presence of Microorganisms Operating Under Anaerobic Conditions

PVA cryogels are also actively used to produce biocatalysts that operate under anaerobic conditions in the processes of producing ethanol, organic solvents, and biogas [27,28]. It is interesting to note that microorganisms immobilized in PVA cryogels, used to produce active BCs for anaerobic processes, are members of natural consortia formed in landfills. In such an ecosystem, not only anaerobic but also aerobic cells can be involved in situ, where synthetic polymers can also be presented [29], including those artificially introduced as soil structure-forming agents. Our experiments have shown that in such systems, excessive pressure resulting from the metabolic activity of microorganisms immobilized in PVA cryogel and the release of gaseous metabolites can lead to a violation of the integrity of the carrier matrices (

Table 3. Changes in granules of BCs, obtained by inclusion of different microorganisms in PVA cryogel, during their use in anaerobic processes.

Microorganisms in BC	Process	Period of BC Use, Days	Pmax, Atm *	Accumulated gases	Velocity of Gas Accumulation, mL/g BC/day	Damaged Granules, % of Total Amount	Type of Damage in Granules
Clostridium acetobutylicum	Production of organic solvents	4	2.6	H2	1.12 ÷ 1.20	15 ± 1	Granule rupture
Saccharomyces cerevisiae	Ethanol production	1095	4.0	CO2	0.92 ÷ 1.01	95 ± 5	Deformation of granules
Saccharomyces cerevisiae	Treatment of wastewater	3	1.1	CO2	0.18 ÷ 0.20	2 ± 0.2	No significant changes
Artificial methanogenic consortium	Conversion of sulfones to hydrogen sulfide within methanogenesis	1095	1.6	Biogas (H2, CH4, CO2, H2S)	0.10 ÷ 0.21	1 ± 0.1	No significant changes
Natural methanogenic consortium	Treatment of wastewater	1095	1.7	Biogas (H2, CH4, CO2)	0.17 ÷ 0.25	1 ± 0.1	No significant changes
Desulfovibrio desulfuricans	Conversion of sulfones to hydrogen sulfide within methanogenesis	3	0.3	H2S	0.01 ÷ 0.03	1 ± 0.1	No significant changes

* Pmax is the maximal pressure registered in the reactors with BCs during investigated processes.

, Figure 2). According to the data obtained, the maximum overpressure in reactors used for the cultivation of anaerobic cells, which can reach 4 atm, is not the reason for the destructive effect on the PVA cryogels. This pressure is due to the accumulation of various gas metabolites produced by cells under anaerobic conditions in the closed reactors. The main damaging effect on the granules of PVA cryogel with the cells of microorganisms included in it was exerted by the gases rapidly accumulated as gaseous metabolites and exited through the pores of the matrix with its rupture (Table 3).

It was found that in the case of prolonged exposition of granules of BC, based on the PVA cryogel and yeast Saccharomyces cerevisiae, at a pressure of 2.1 atm developed in a closed system, the polymer matrix underwent deformation and acquired a changed shape, but retained its integrity (there was no rupture of granules).

As for natural and artificial consortia based on methanogenic sludge, their immobilization in a PVA cryogel and operation in an immobilized state did not lead to a change in the shape and integrity of the biocatalyst granules during a similar duration of use in anaerobic processes. This fact, indeed, confirmed the importance of the intensity of the processes occurring in BC granules using PVA cryogel as a carrier for its mechanical damage or stability. Anaerobic processes, prolonged but with a slow rate of releasing of gas products, especially with the participation of cell consortia, did not lead to damage to polymer cryogels. In this regard, PVA cryogels in the composition of biocatalysts used in biotechnological processes, despite changes in rheological characteristics, maintain the stability of their functioning for a long time.

3.3. The Effect of Microbial Colonization of PVA Cryogels on Their Characteristics

The immobilized BCs developed for the biocatalytic processes described above were obtained by incorporating cell biomass into the carrier matrix during its cryoformation. When PVA cryogels are purposefully introduced into the environment as a soil-structuring agent [30], they can serve as a matrix for colonization by aboriginal microorganisms. Such properties of PVA cryogels are well known and discussed for using in medicine for the adsorption and cultivation of tissue cultures [31]. Presumably, the effectiveness of PVA cryogel colonization by microorganisms can be regulated by the pore size in the carrier matrix, which is primarily determined by the concentration of the polymer in the solution used to form the cryogel. This was estimated in this work, when the colonization of PVA cryogel samples, obtained using PVA solutions with a concentration of 7–13% polymer, by various microorganisms was studied (

Table 4. Characteristics of PVA cryogel samples formed using polymer solutions with different concentrations after their colonization by microorganisms.

Concentration of PVA Solution Used for Cryogel Formation, %	Average Pore Size, µm	Microorganisms
		Microalgae Chlorella vulgaris	Bacteria Pseudomonas putida	Methanogenic Anaerobic Consortium	Filamentous Fungi Aspergillus niger
[ATP], ×10−11 mole/g PVA cryogel colonized by cells
7	70 ± 4	7.3 ± 0.2	14.2 ± 0.1	0.07 ± 0.01	57.3 ± 2.4
9	40 ± 3	12.7 ± 0.6	17.3 ± 0.1	0.04 ± 0	56.8 ± 2.1
11	25 ± 2	19.2 ± 1.2	19.6 ± 0.1	0.01 ± 0	46.9 ± 1.9
13	15 ± 1	14.1 ± 0.7	21.2 ± 0.1	0.01 ± 0	40.5 ± 1.3
* Shear modulus, kPa
7	70 ± 4	3.0 ± 0.07	2.7 ± 0.06	2.8 ± 0.02	2.4 ± 0.02
9	40 ± 3	3.3 ± 0.07	3.2 ± 0.06	3.3 ± 0.08	3.0 ± 0.04
11	25 ± 2	3.8 ± 0.1	3.8 ± 0.1	3.7 ± 0.1	3.5 ± 0.1
13	15 ± 1	4.1 ± 0.1	4.2 ± 0.1	4.2 ± 0.1	4.0 ± 0.1

* Initial values of the shear modulus determined for PVA cryogels formed without cells were 3.2 ± 0.1, 3.7 ± 0.1, 4.1 ± 0.2, and 4.5 ± 0.2 for the 7, 9, 11, and 13% polymer solutions, respectively. Cells highlighted in light pink show cases where there was a statistically significant change (p ≤ 0.05) between values at different PVA concentrations.

).

The choice of applied PVA concentrations for conducting experiments was due to the fact that at polymer concentrations higher than 13%, the PVA solution became very viscous and technologically difficult to work with, while the polymer concentration in solution below 7% led to the formation of a slimy cryogel. In addition, the matrix with a lower polymer concentration was characterized by a large pore size (Table 4) and poorly retained microbial cells. On the other hand, high polymer concentrations ensured the production of matrices with small pore sizes, which could lead to diffusion limitations for mass transfer processes provided by cryogel samples. In the case of PVA cryogel colonization by cells, the main part of them developed on the surface of the polymer matrices (Figure 3).

In studies on microbial colonization of the formed “empty” matrices of PVA cryogels, cells of various microorganisms, including a methanogenic consortium, were used.

It is known that in the aquatic environment, various polymer matrices can become overgrown with cells of different microalgae [32,33]. To study such colonization in vitro, a suspension of Chlorella vulgaris microalgae cells was used, into which PVA cryogel samples formed from solutions with different initial polymer concentrations and exposed for 6 months were placed (Figure 3). Next, cryogel samples were washed with physiological solution to remove weakly adhered cells, and the concentration of intracellular ATP was determined by bioluminescent ATP-metry, which makes it possible to estimate the presence of living cells that (self)immobilized on the carrier [34] (Table 4).

It was found that the highest concentration of (self)immobilized C. vulgaris cells is achieved at 9% concentration of the PVA solution used in polymer cryo-formation. That is confirmed by the maximal total concentration of intracellular ATP among all samples of PVA cryogels. It is this concentration of polymer solution that makes it possible to create pores in the cryogel, the size of which (25 µm) (Table 4) allows retaining the largest concentration of cells (10 µm) in the pores of the polymer matrix.

Bacterial Pseudomonas putida cells are widely distributed in different environmental objects, in particular in the soil, and therefore they are also of interest as an object of research, as they can potentially act as colonizers of PVA cryogels introduced into soils as structure-forming agents. The maximum concentration of intracellular ATP in P. putida, the size of which is in the range of 0.5–4 µm, was detected in samples of PVA cryogel formed from solutions with 13% polymer. However, it is possible to note the effective colonization of all types of studied samples of PVA cryogels by these bacteria (Table 4).

Anaerobic sludge is formed in various environmental objects under conditions of reduced atmospheric oxygen concentrations, most often in landfills, where used packaging materials made of different synthetic polymers end up. The recorded concentrations of intracellular ATP for PVA cryogel samples placed in a suspension of the methanogenic consortium were significantly lower than for microalgae and bacterial cells. It should be noted that most anaerobic cells are typically characterized by lower concentrations of intracellular ATP in comparison with aerobes [34]. In general, the data obtained indicate that the anaerobic methanogenic consortium is also capable of colonizing the PVA cryogel matrix (Figure 3).

Today, various filamentous fungi present in soil ecosystems are considered as potential destructors of PVA [13]; however, among them, preference is given to representatives of the genus Aspergillus [13,35,36]. Invasive growth on various porous matrices is characteristic of such fungi. Aspergillus fungal spores can penetrate the pores with further mycelium development. From this point of view, Aspergillus niger cells are considered to be the most active colonizers, which were used in our study (Table 4, Figure 3).

The fungi successfully colonized PVA cryogel samples, with the most active being the more macroporous variants obtained using 7% and 9% polymer solutions. At the same time, studies of deformation changes in PVA cryogels, namely the shear modulus, have shown that it is the accumulation of fungal biomass in the polymer matrix, as evidenced by the detected concentrations of ATP, which leads to a maximum decrease in this indicator of carrier strength compared with other microorganisms.

3.4. Changes in the Strength Characteristics of PVA Cryogels as Part of BCs Used Under Flow Conditions

It seemed relevant to evaluate the possibility of degradation changes in PVA cryogels with cells included in them as a result of their use in flow systems. For these purposes, microalgae Chlorella vulgaris and Thalassiosira weissflogii cells, as well as Photobacterium phosphoreum cells with their own bioluminescence, were selected as research objects. The idea of using phototrophs in an immobilized form to evaluate changes in the properties of PVA cryogel samples used in flow systems was as follows. Microalgae cells are microorganisms whose photosynthetic apparatus is sensitive to various deformation effects on cells. It was decided to use changes in the functioning characteristics of the fluorescence systems of these cells located in the matrices of PVA cryogels as a response to assess the presence of deformation effects of the medium flow on the polymer matrices themselves.

In turn, the use of P. phosphoreum photobacteria in the work was due to the fact that these bacteria are characterized by the presence of membrane-bound luciferase, which is responsible for the bioluminescent glow of cells and is sensitive to external deformation effects on the cell membrane. In this case, it was also decided to use the change in the bioluminescence level of photobacteria as a response to assess the effect on the polymer matrix with the cells of the medium flow used for exposing cells immobilized in the PVA cryogel. When conducting experiments with microalgae cells immobilized in PVA cryogel (Figure 4,

Table 5. Changes in the characteristics of microalgae cells immobilized in PVA cryogel before and after their exposure for 10 days in a flow system at a medium flow rate of 360 mL/h.

Microalgae	Initial Value			After Exposition in the Flow
	[ATP], mole/g BC	F0	Fv/Fm	[ATP], mole/g BC	F0	* Fv/Fm
C. vulgaris	(2.0 ± 0.1) × 10−11	1.79	0.71	(1.9 ± 0.2) × 10−11	1.71	0.70
T. weissflogii	(1.4 ± 0.3) × 10−9	1.75	0.63	(1.2 ± 0.3) × 10−9	1.69	0.63

* Average value from Figure 4.

) and having known specific characteristics (concentration of intracellular ATP, F₀, Fv/Fm), levels of ATP concentration, bioluminescence, and chlorophyll fluorescence before and immediately after immobilization were comparable (variation in parameters did not exceed 5%).

Thus, 17,280 volumes of photobioreactor with granules of PVA cryogel (5 mL), which included microalgae cells, were passed through it. During this test, as well as after its completion, the fluorescence parameters characterizing the state of the photosynthetic apparatus of immobilized microalgae cells (Figure 4), as well as the residual concentration of ATP in polymer granules, were determined (Table 5).

It follows from the data obtained that the PVA cryogel provided effective cell retention in the pores of the carrier, regardless of the type of microalgae used. It was confirmed by the ATP values. In addition, an analysis of the Fv/Fm fluorescence values also suggests that the PVA cryogel provides almost complete preservation of the functional characteristics of immobilized microalgae cells under the applied conditions. Fluctuations in the relative variable fluorescence values of the immobilized microalgae cells did not exceed 5% of the initial value. In fact, there were no serious deviations from the functioning of the photosynthetic apparatus of cells due to the possible deformation of the PVA cryogel granules.

The results also suggest that photobacteria immobilized into PVA cryogel can stay in a flowing medium for a long time (at least 10 days) while maintaining 95% of their primary luminescence level at a flow rate of 90 mL/h, ensuring that 4320 volumes of medium pass through the reactor with polymer granules (5 mL).

4. Discussion

Today, PVA is one of the largest synthetic resins produced in the world [37]. It is widely used as an emulsifier and stabilizer for various colloidal suspensions, as a sizing, adhesive, and coating agent in the biomedical, food, textile, and paper industries. There are many methods implemented to produce various films and composite materials based on PVA [38,39]. One of them is the production of PVA cryogel. The simple implementation by freezing PVA solutions, holding them at subzero temperatures, and thawing the formed cryogels attracts the attention of specialists from various fields of science and technology to PVA cryogels. Their use as a basis for structuring the soil or as carriers of various (self)immobilizing microorganisms capable of catalyzing different biotechnological processes is of particular interest to industrial, environmental, and biological chemists. Possible approaches to chemical methods of decomposition of PVA are well described in the modern scientific literature, and most of the methods are based on oxidation processes [40,41]. However, the initial stage of decomposition of polymer matrices is very important. Since then, an assessment of the influence of factors that can lead to a weakening of the PVA cryogels when their use is associated with the presence/action of microorganisms has been undertaken in this work.

The modulus of elasticity was chosen as a criterion for evaluating changes in the properties of PVA cryogels. The periodic observation of these characteristics of PVA cryogel for all samples used in the studied processes involving different immobilized microbial cells and media (Table 1) was similarly undertaken and reached a maximum of 20 days in the experiments (Table 2). It is known that the modulus of elasticity of PVA cryogels can be adjusted initially during the formation of a matrix with cell, depending on the following: the concentration of the polymer solution used to prepare a suspension of microbial cells before freezing, the rate of freezing (depending on the temperature of the process) and thawing of the cryogel, its exposure time at subzero temperatures, when multiple hydrogen intermolecular bonds are formed [42]. In this study, special attention was paid to assessing the effect of the initial concentration of the PVA solution used to produce BCs (ranging from 7 to 12% for different samples) intended for certain processes, all other things being the same, under the same conditions for the formation of polymer cryogels.

It follows from literature data [43,44] that the concentration of PVA determines its porosity and the strength of the gel matrix, which, in turn, affects the mass-exchange cellular processes and the intensity of metabolic processes carried out by microorganisms in different media. In this regard, in a number of experiments, the same concentration of the initial polymer solution was used to prepare different BCs intended for different processes and media in order to evaluate the role of the latter ones in a possible change in the modulus of elasticity of PVA cryogels acting as cell carriers.

Significant changes in the rheological characteristics of the PVA cryogels, as expected, occurred in the presence of those microorganisms that actively altered the properties of the medium, possessing the velocities of metabolites’ production. When analyzing the results obtained (Table 2), it was found that the microorganisms that were used to produce BCs also contributed to the change in the strength characteristics of cryogel matrices. The filamentous and yeast-like fungi, penetrating the pores of carrier granules with further growth of mycelium, obviously influenced them, which led to an increase in the modulus of elasticity of polymer matrices. Whereas, it should be noted that if such BCs get into environmental conditions, the detected increase in the strength of the cryogel matrix due to the presence of mycelium in it will not serve as an obstacle to the biodegradation of such mycelium, since the fungal biomass itself should become an organic object of biotransformation by other cells of microorganisms.

For microbial participants in anaerobic processes, the dependence of the presence of degradation effects on the polymer size of granules on the rate of formation of gas substrates by cells included in PVA cryogels was shown. This was clearly revealed by the example of cells of the genus Clostridium (Table 3). Ruptures of polymer granules can theoretically contribute to the subsequent degradation of the polymer [22], but this is possible in the presence of strong oxidizing agents, which are absent under anaerobic conditions. In this case, their introduction from the outside, for example, potassium persulfate or sodium percarbonate, which are actively used in practice to solve various environmental problems [45,46,47], can probably contribute to the degradation of PVA.

It should be noted that in this work, for the first time, the possibility of active colonization by different microorganisms of porous matrices such as PVA cryogels, formed from polymer solutions with different concentrations, was shown. Microscopic fungi, for which growth on the surfaces of various polymer matrices is typical, most clearly influenced the magnitude of the determined shear modulus due to the formation of mycelium on the granules of PVA cryogel. Moreover, this growth was more active on samples of PVA cryogel (Table 4), which were obtained from polymer solutions at the lowest polymer concentrations studied in the work.

Intensive studies on the possible degradation of PVA cryogels under flow conditions, conducted in this work using immobilized cells with their own fluorescence (microalgae) and bioluminescence (photobacteria), sensitive to deformation effects on cells that appeared from the outside, did not reveal obvious signs of the estimated negative effects. At the same time, flow-through displacement reactors were used in the work without mixing the granules inside the flow media. It is possible that in the presence of other conditions in which abrasion effects on granules with cells will occur, the degradation effect for PVA cryogels will manifest itself.

This result must be taken into account when using BCs in biotechnological processes and kept in mind when using PVA cryogels as soil-structuring agents to strengthen roadside slopes and field roads [10,48,49], where water flows may occur, respectively, with active movement of granules in a closed volume or appearance of natural precipitation flows with abrasive particles acting on polymer matrices.

5. Conclusions

It is necessary to note that in undertaken long-term experiments with permanent residence of PVA cryogels in aqueous media of different chemical composition in the presence of various cells of microorganisms, including flow conditions with very high flow rates, information was confirmed that in most cases this does not lead to significant changes in the strength characteristics of PVA cryogels, which could lead to degradation estimated polymer matrices. However, in a number of cases that were identified in this work, the presence of microorganisms can indirectly (through media transformed by them, in particular, containing lipids) or directly (through high rates of metabolite production, in particular, the fast accumulation of gases in the pores of polymer matrices, or due to the colonization of cryogels (self)immobilization by fungi with the development of mycelium) decrease rheological characteristics of PVA cryogel. These factors should be taken into account when using matrices based on PVA cryogels in artificial or natural environments where microorganisms are present.

In slow anaerobic microbial processes, PVA cryogels retain their strength best; only gas metabolites that quickly escape through the pores of the matrix can damage the matrix. In aerobic processes, the strength of the PVA cryogel is less affected by the pH values of the media used for microorganisms, and more by the biochemical compositions of the media. Microalgae cultivation is accompanied by a shift in pH to the alkaline range and hardening of the PVA cryogel matrix. The presence of lipids in the medium avoids this effect. During the colonization of PVA cryogels by microorganisms, the microalgae, bacteria, and fungi prefer to develop on the surface of polymer matrices. The accumulation of fungal biomass in the polymer matrix leads to a maximum decrease in carrier strength compared with other microorganisms. Under flow conditions, the PVA cryogel provided effective cell retention in the pores of the carrier, regardless of the type of cells with bioluminescence (microalgae or photobacteria).