Correction: Zhantlessova et al. Advanced "Green" Prebiotic Composite of Bacterial Cellulose/Pullulan Based on Synthetic Biology-Powered Microbial Coculture Strategy. Polymers 2022, 14, 3224.

In the original publication [...].

Caldanaerobacter subterraneus subsp. keratinolyticus subsp. nov., a Novel Feather-Degrading Anaerobic Thermophile.

Caldanaerobacter subterraneus subsp. keratinolyticus subsp. nov. strain KAk was isolated from a geothermal hot spring located in Kazakhstan. Growth occurred at temperatures ranging from 50 to 80 °C, with approximately 70 °C as optimum. It also thrived in pH conditions ranging from 4.0 to 9.0, with the best growth occurring at 6.8. Under optimal conditions in a glucose-containing medium, the cells were predominantly observed singly, in pairs, or less frequently in chains, and did not form endospores. However, under conditions involving growth with merino wool or feathers, or under suboptimal conditions, the cells of strain KAk exhibited a notably elongated and thinner morphology, with lengths ranging from 5 to 8 µm, and spores were observed. The KAk strain exhibited efficient degradation of feather keratin and merino wool at temperatures ranging from 65 to 70 °C. Analysis of the 16S rRNA gene sequence placed KAk within the genus Caldanaerobacter, family Thermoanaerobacteraceae, with the highest similarity to C. subterraneus subsp. tengcongensis MB4T (98.84% sequence identity). Furthermore, our analysis of the draft genome sequence indicated a genome size of 2.4 Mbp, accompanied by a G+C value of 37.6 mol%. This study elucidated the physiological and genomic characteristics of strain KAk, highlighting its keratinolytic capabilities and distinctiveness compared to other members of the genus Caldanaerobacter.

Polycladomyces zharkentensis sp. nov., a novel thermophilic cellulose- and starch-degrading member of the Bacillota from a geothermal aquifer in Kazakhstan.

A thermophilic, aerobic and heterotrophic filamentous bacterium, designated strain ZKZ2T, was isolated from a pipeline producing hydrothermal water originating from a >2.3 km deep subsurface geothermal source in Zharkent, Almaty region, Kazakhstan. The isolate was Gram-stain-positive, non-motile, heat-resistant and capable of producing a variety of extracellular hydrolases. Growth occurred at temperatures between 55 and 75 °C, with an optimum around 70 °C, and at pH values between 5.5 and 9.0, with an optimum at pH 7.0-7.5 with the formation of aerial mycelia; endospores were produced along the aerial mycelium. The isolate was able to utilize the following substrates for growth: glycerol, l-arabinose, ribose, d-xylose, d-glucose, d-fructose, d-mannose, rhamnose, d-mannitol, methyl-d-glucopyranoside, aesculin, salicin, cellobiose, maltose, melibiose, sucrose, trehalose, melezitose, raffinose, starch, turanose and 5-keto-gluconate. Furthermore, it was able to hydrolyse carboxymethylcellulose, starch, skimmed milk, Tween 60 and Tween 80. The major cellular fatty acids were iso-C15 : 0, iso-C17 : 0, iso-C16 : 0 and C16 : 0. Our 16S rRNA gene sequence analysis placed ZKZ2T within the genus Polycladomyces, family Thermoactinomycetaceae, with the highest similarity to the type species Polycladomyces abyssicola JIR-001T (99.18 % sequence identity). Our draft genome sequence analysis revealed a genome size of 3.3 Mbp with a G+C value of 52.5 mol%. The orthologous average nucleotide identity value as compared to that of its closest relative, P. abyssicola JIR-001T, was 90.23 %, with an in silico DNA-DNA hybridization value of 40.7 %, indicating that ZKZ2T represents a separate genome species. Based on the phenotypic and genome sequence differences from the other two Polycladomyces species, we propose that strain ZKZ2T represents a novel species, for which we propose the name Polycladomyces zharkentensis sp. nov. The type strain is ZKZ2T (=CECT 30708T=KCTC 43421T).

Biopolymers as Seed-Coating Agent to Enhance Microbially Induced Tolerance of Barley to Phytopathogens.

Infections of agricultural crops caused by pathogen ic fungi are among the most widespread and harmful, as they not only reduce the quantity of the harvest but also significantly deteriorate its quality. This study aims to develop unique seed-coating formulations incorporating biopolymers (polyhydroxyalkanoate and pullulan) and beneficial microorganisms for plant protection against phytopathogens. A microbial association of biocompatible endophytic bacteria has been created, including Pseudomonas flavescens D5, Bacillus aerophilus A2, Serratia proteamaculans B5, and Pseudomonas putida D7. These strains exhibited agronomically valuable properties: synthesis of the phytohormone IAA (from 45.2 to 69.2 µg mL-1), antagonistic activity against Fusarium oxysporum and Fusarium solani (growth inhibition zones from 1.8 to 3.0 cm), halotolerance (5-15% NaCl), and PHA production (2.77-4.54 g L-1). A pullulan synthesized by Aureobasidium pullulans C7 showed a low viscosity rate (from 395 Pa·s to 598 Pa·s) depending on the concentration of polysaccharide solutions. Therefore, at 8.0%, w/v concentration, viscosity virtually remained unchanged with increasing shear rate, indicating that it exhibits Newtonian flow behavior. The effectiveness of various antifungal seed coating formulations has been demonstrated to enhance the tolerance of barley plants to phytopathogens.

The Effect of Encapsulating a Prebiotic-Based Biopolymer Delivery System for Enhanced Probiotic Survival.

Orally delivered probiotics must survive transit through harsh environments during gastrointestinal (GI) digestion and be delivered and released into the target site. The aim of this work was to evaluate the survivability and delivery of gel-encapsulated Lactobacillus rhamnosus GG (LGG) to the colon. New hybrid symbiotic beads alginate/prebiotic pullulan/probiotic LGG were obtained by the extrusion method. The average size of the developed beads was 3401 µm (wet), 921 µm (dry) and the bacterial titer was 109 CFU/g. The morphology of the beads was studied by a scanning electron microscope, demonstrating the structure of the bacterial cellulose shell and loading with probiotics. For the first time, we propose adding an enzymatic extract of feces to an artificial colon fluid, which mimics the total hydrolytic activity of the intestinal microbiota. The beads can be digested by fecalase with cellulase activity, indicating intestinal release. The encapsulation of LGG significantly enhanced their viability under simulated GI conditions. However, the beads, in combination with the prebiotic, provided greater protection of bacteria, enhancing their survival and even increasing cell numbers in the capsules. These data suggest the promising prospects of coencapsulation as an innovative delivery method based on the inclusion of probiotic bacteria in a symbiotic matrix.

Prebiotic Cellulose-Pullulan Matrix as a "Vehicle" for Probiotic Biofilm Delivery to the Host Large Intestine.

This study describes the development of a new combined polysaccharide-matrix-based technology for the immobilization of Lactobacillus rhamnosus GG (LGG) bacteria in biofilm form. The new composition allows for delivering the bacteria to the digestive tract in a manner that improves their robustness compared with planktonic cells and released biofilm cells. Granules consisting of a polysaccharide matrix with probiotic biofilms (PMPB) with high cell density (>9 log CFU/g) were obtained by immobilization in the optimized nutrient medium. Successful probiotic loading was confirmed by fluorescence microscopy and scanning electron microscopy. The developed prebiotic polysaccharide matrix significantly enhanced LGG viability under acidic (pH 2.0) and bile salt (0.3%) stress conditions. Enzymatic extract of feces, mimicking colon fluid in terms of cellulase activity, was used to evaluate the intestinal release of probiotics. PMPB granules showed the ability to gradually release a large number of viable LGG cells in the model colon fluid. In vivo, the oral administration of PMPB granules in rats resulted in the successful release of probiotics in the colon environment. The biofilm-forming incubation method of immobilization on a complex polysaccharide matrix tested in this study has shown high efficacy and promising potential for the development of innovative biotechnologies.

Advanced "Green" Prebiotic Composite of Bacterial Cellulose/Pullulan Based on Synthetic Biology-Powered Microbial Coculture Strategy.

Bacterial cellulose (BC) is a biopolymer produced by different microorganisms, but in biotechnological practice, Komagataeibacter xylinus is used. The micro- and nanofibrillar structure of BC, which forms many different-sized pores, creates prerequisites for the introduction of other polymers into it, including those synthesized by other microorganisms. The study aims to develop a cocultivation system of BC and prebiotic producers to obtain BC-based composite material with prebiotic activity. In this study, pullulan (PUL) was found to stimulate the growth of the probiotic strain Lactobacillus rhamnosus GG better than the other microbial polysaccharides gellan and xanthan. BC/PUL biocomposite with prebiotic properties was obtained by cocultivation of Komagataeibacter xylinus and Aureobasidium pullulans, BC and PUL producers respectively, on molasses medium. The inclusion of PUL in BC is proved gravimetrically by scanning electron microscopy and by Fourier transformed infrared spectroscopy. Cocultivation demonstrated a composite effect on the aggregation and binding of BC fibers, which led to a significant improvement in mechanical properties. The developed approach for "grafting" of prebiotic activity on BC allows preparation of environmentally friendly composites of better quality.

Characterization of cadmium-tolerant endophytic fungi isolated from soybean (Glycine max) and barley (Hordeum vulgare).

Cadmium stress disrupts plant-microbial interactions and reduces plant growth and development. In plants, the tolerance to stress can be increased by inoculation with endophytic microorganisms. The aim of this study was to investigate the distribution of endophytic fungi in various plant organs of barley and soybean and evaluate their Cd removal ability. Two hundred fifty-three fungal strains were isolated from various organs of barley (Hordeum vulgare cv Arna) and soybean (Glycine max cv Almaty). The colonization rate ranged from 13.6% to 57.3% and was significantly higher in the roots. Ten genera were identified: Fusarium, Penicillium, Aspergillus, Metarhizium, Beauveria, Trichoderma, Rhodotorula, Cryptococcus, Aureobasidium and Metschnikowia. Twenty-three fungal strains have a Cd tolerance index from 0.24 to 1.12. Five strains (Beauveria bassiana T7, Beauveria bassiana T15, Rhodotorula mucilaginosa MK1, Rhodotorula mucilaginosa RH2, Metschnikowia pulcherrima MP2) with the highest level of Cd tolerance have minimum inhibitory concentrations from 290 to 2400 µg/ml. These fungi were able to remove Cd up to 59%. The bioaccumulation capacity ranged from 2.3 to 11.9 mg/g. Selected fungal strains could be considered as biological agents for their potential application in the bioremediation of contaminated sites.

Biochar Amendments Improve Licorice (Glycyrrhiza uralensis Fisch.) Growth and Nutrient Uptake under Salt Stress.

Licorice (Glycyrrhiza uralensis Fisch.) is a salt and drought tolerant legume suitable for rehabilitating abandoned saline lands, especially in dry arid regions. We hypothesized that soil amended with maize-derived biochar might alleviate salt stress in licorice by improving its growth, nutrient acquisition, and root system adaptation. Experiments were designed to determine the effect of different biochar concentrations on licorice growth parameters, acquisition of C (carbon), nitrogen (N), and phosphorus (P) and on soil enzyme activities under saline and non-saline soil conditions. Pyrolysis char from maize (600 °C) was used at concentrations of 2% (B2), 4% (B4), and 6% (B6) for pot experiments. After 40 days, biochar improved the shoot and root biomass of licorice by 80 and 41% under saline soil conditions. However, B4 and B6 did not have a significant effect on shoot growth. Furthermore, increased nodule numbers of licorice grown at B4 amendment were observed under both non-saline and saline conditions. The root architectural traits, such as root length, surface area, project area, root volume, and nodulation traits, also significantly increased by biochar application at both B2 and B4. The concentrations of N and K in plant tissue increased under B2 and B4 amendments compared to the plants grown without biochar application. Moreover, the soil under saline conditions amended with biochar showed a positive effect on the activities of soil fluorescein diacetate hydrolase, proteases, and acid phosphomonoesterases. Overall, this study demonstrated the beneficial effects of maize-derived biochar on growth and nutrient uptake of licorice under saline soil conditions by improving nodule formation and root architecture, as well as soil enzyme activity.

Metagenomics and Culture-Based Diversity Analysis of the Bacterial Community in the Zharkent Geothermal Spring in Kazakhstan.

Diversity of the microbial community in the Zharkent geothermal hot spring, located in the southeastern region of Kazakhstan, was assessed using both culture-dependent and -independent approaches. Shotgun metagenomic sequencing of DNA extracted from the spring water yielded 11,061,725 high-quality sequence reads, totaling >1,67 Gb of nucleotide sequences. Furthermore, water samples were enriched in nutrient broth at varying high temperatures, and colonies isolated by being streaked onto nutrient agar. Finally, DNA extraction and amplification, as well as sequencing and phylogenetic analysis, were conducted. Bacteria constituted more than 99.97% of the total prokaryotic abundance, with Archaea contributing only an extremely small component; Firmicutes, Proteobacteria, and Actinobacteria dominated the community. At genus level, Firmicutes reads affiliated with Desmospora, Parageobacillus, Paenibacillus, and Brevibacillus, accounting for more than 60% of total prokaryotic abundance. Eight morphologically distinct, aerobic, endospore-forming thermophilic bacteria were recovered; isolates differed significantly in substrate utilization patterns, as well as their production of thermophilic, extracellular, hydrolytic enzymes for degradation of starch, lipids, cellulose, and protein. Five strains could degrade all four macromolecular types at temperatures ranging from 55 to 75 °C. Phylogenetic analyses based on 16S rRNA gene sequences placed all isolates into the genus Geobacillus with some of them possibly representing novel species. The results indicate that this hot spring represents a rich source of novel thermophilic bacteria and potentially useful thermostable enzymes.