Isolation and Purification of Polyhydroxyalkanoates. Scaling in Pilot Production

Исследован процесс выделения и очистки поли‑3-гидроксибутирата в условиях масштабирования. В качестве солюбилизирующих агентов изучены додецилсульфат натрия, гипохлорит натрия и стиральный порошок «Пемос». Наибольший выход 95 %, и чистота полимера 99,5 % достигнут при использовании двухстадийного метода экстракции и порошка «Пемос» в качестве солюбилизирующего агента. В результате чего удалось снизить затраты на стадии солюбилизации до 31 руб/кг поли‑3-гидроксибутирата. По результатам исследований внесены изменения в аппаратурную и технологическую схему процессаThe process of isolating and purifying poly‑3-hydroxybutyrate under scaling conditions has been studied. Sodium dodecyl sulfate, sodium hypochlorite and Pemos washing powder were studied as solubilizing agents. The highest yield of 95 % and polymer purity of 99.5 % was achieved using a two- stage extraction method and Pemos powder as a solubilizing agent. As a result, it was possible to reduce the costs at the solubilization stage to 31 rubles/kg of poly‑3-hydroxybutyrate. Based on the research results, a technology for the isolation and purification of polyhydroxyalkanoates was propose

Selection and Validation of Reference Genes for Quantitative Real-Time PCR in Buckwheat (Fagopyrum esculentum) Based on Transcriptome Sequence Data

Quantitative reverse transcription PCR (qRT-PCR) is one of the most precise and widely used methods of gene expression analysis. A necessary prerequisite of exact and reliable data is the accurate choice of reference genes. We studied the expression stability of potential reference genes in common buckwheat (Fagopyrum esculentum) in order to find the optimal reference for gene expression analysis in this economically important crop. Recently sequenced buckwheat floral transcriptome was used as source of sequence information. Expression stability of eight candidate reference genes was assessed in different plant structures (leaves and inflorescences at two stages of development and fruits). These genes are the orthologs of Arabidopsis genes identified as stable in a genome-wide survey gene of expression stability and a traditionally used housekeeping gene GAPDH. Three software applications – geNorm, NormFinder and BestKeeper - were used to estimate expression stability and provided congruent results. The orthologs of AT4G33380 (expressed protein of unknown function, Expressed1), AT2G28390 (SAND family protein, SAND) and AT5G46630 (clathrin adapter complex subunit family protein, CACS) are revealed as the most stable. We recommend using the combination of Expressed1, SAND and CACS for the normalization of gene expression data in studies on buckwheat using qRT-PCR. These genes are listed among five the most stably expressed in Arabidopsis that emphasizes utility of the studies on model plants as a framework for other species

Comparative Study of Methods of Pha Extraction from Bacterial Biomass

Проведено сравнительное исследование различных методов экстракции полигидроксиалканоатов (ПГА) из биомассы бактерий. Показано, что различные реагенты и технология ведения процесса по-разному влияют на показатели – полноту извлечения полимера и степень его чистоты. Применение хлороформа делает необходимым использование большого количества летучих и токсичных реагентов. При использовании дихлорметана полнота извлечения повышается, но возникает необходимость в реализации процедуры разделения смеси «экстрагент – осадитель». Подобрана пара «растворитель – осадитель» (дихлорметан – гексан), которая не образует между собой азеотропную смесь, что делает возможным вернуть в процесс до 90 % реагентов. В результате снизился расход растворителей с 73,5 кг/кг ПГА (хлороформ – гексан) до 63,7 кг/кг ПГА (дихлорметан – этанол – гексан). При исключении из состава этанола возможно снижение расхода до 7,8 г/г ПГА, но в этом случае необходима дополнительная обработка биомассы с целью разрушения мембранных комплексов или последовательная обработка сначала спиртом, а затем дихлорметаном. Безреагентный метод с применением в качестве детергента додецилсульфата натрия (ДДС-Na) позволяет более экономичным способом получать высокие выходы полимера, не загрязненного примесями жирных кислот, пригодного для технических целей (тара, упаковочная продукция). Разработан комбинированный метод, который значительно снижает расходы реагентов и позволяет получать полимер высокой чистоты при полноте экстракции до 98,5–99,0 %A comparative study of different methods of PHA extraction from the biomass of bacteria was carried out. It was shown that different reagents and process technologies have different effects on the extraction results – the completeness of polymer extraction and degree of its purity. Application of chloroform requires the use of large amounts of volatile and toxic reagents. When dichloromethane is used, the completeness of extraction increases, but it becomes necessary to implement procedures for separating the “extractant-precipitator” mixture. The solvent-precipitator pair (dichloromethanehexane) was selected, which does not lead to formation of an azeotropic mixture, making it possible to return up to 90 % of the reactants to the process. As a result, solvent consumption went down from 73.5 kg/kg of PHA (chloroform – hexane) to 63.7 kg/kg of PHA (chloroform – ethanol – hexane). If ethanol is excluded, it is possible to reduce the consumption to 7.8 g/g of PHA, but in this case additional treatment of biomass to destroy membrane complexes or sequential treatment first with alcohol and then with dichloromethane is needed. A nonchemical method using sodium dodecyl sulfate (NaDS) as a detergent allows for a more economical way to obtain high yields of polymer not contaminated with admixtures of fatty acids and suitable for technical purposes (packaging, packaging products). A combined method was developed, which considerably reduces the cost of reagents and makes it possible to obtain polymers with high purity degree and extraction completeness up to 98.5–99.0

The relative expression level of flower development genes.

<p>(a) <i>ap2-14</i> mutant analysis, (b) <i>ag-1</i> mutant analysis, (c) <i>clv3-2</i> mutant analysis. Error bars represent the standard error of the mean. Asterisks indicates what values are significant to p<0.05. <i>AP2</i> - <i>APETALA2</i>, <i>AP3</i> - <i>APETALA3</i>, <i>PI</i> - <i>PISTILLATA</i>, <i>AG</i> - <i>AGAMOUS</i>, <i>CLV1</i> - <i>CLAVATA1</i>, <i>CLV2</i> - <i>CLAVATA2</i>, <i>WUS</i> – <i>WUCSHEL.</i> Dashed line indicates 1.0 expression level.</p

Flowers of wild type (wt) and of three single mutants of <i>A. thaliana</i>.

<p>Scale bar = 5 mm.</p

Comparative Study of Methods of Pha Extraction from Bacterial Biomass

Проведено сравнительное исследование различных методов экстракции полигидроксиалканоатов (ПГА) из биомассы бактерий. Показано, что различные реагенты и технология ведения процесса по-разному влияют на показатели – полноту извлечения полимера и степень его чистоты. Применение хлороформа делает необходимым использование большого количества летучих и токсичных реагентов. При использовании дихлорметана полнота извлечения повышается, но возникает необходимость в реализации процедуры разделения смеси «экстрагент – осадитель». Подобрана пара «растворитель – осадитель» (дихлорметан – гексан), которая не образует между собой азеотропную смесь, что делает возможным вернуть в процесс до 90 % реагентов. В результате снизился расход растворителей с 73,5 кг/кг ПГА (хлороформ – гексан) до 63,7 кг/кг ПГА (дихлорметан – этанол – гексан). При исключении из состава этанола возможно снижение расхода до 7,8 г/г ПГА, но в этом случае необходима дополнительная обработка биомассы с целью разрушения мембранных комплексов или последовательная обработка сначала спиртом, а затем дихлорметаном. Безреагентный метод с применением в качестве детергента додецилсульфата натрия (ДДС-Na) позволяет более экономичным способом получать высокие выходы полимера, не загрязненного примесями жирных кислот, пригодного для технических целей (тара, упаковочная продукция). Разработан комбинированный метод, который значительно снижает расходы реагентов и позволяет получать полимер высокой чистоты при полноте экстракции до 98,5–99,0 %A comparative study of different methods of PHA extraction from the biomass of bacteria was carried out. It was shown that different reagents and process technologies have different effects on the extraction results – the completeness of polymer extraction and degree of its purity. Application of chloroform requires the use of large amounts of volatile and toxic reagents. When dichloromethane is used, the completeness of extraction increases, but it becomes necessary to implement procedures for separating the “extractant-precipitator” mixture. The solvent-precipitator pair (dichloromethanehexane) was selected, which does not lead to formation of an azeotropic mixture, making it possible to return up to 90 % of the reactants to the process. As a result, solvent consumption went down from 73.5 kg/kg of PHA (chloroform – hexane) to 63.7 kg/kg of PHA (chloroform – ethanol – hexane). If ethanol is excluded, it is possible to reduce the consumption to 7.8 g/g of PHA, but in this case additional treatment of biomass to destroy membrane complexes or sequential treatment first with alcohol and then with dichloromethane is needed. A nonchemical method using sodium dodecyl sulfate (NaDS) as a detergent allows for a more economical way to obtain high yields of polymer not contaminated with admixtures of fatty acids and suitable for technical purposes (packaging, packaging products). A combined method was developed, which considerably reduces the cost of reagents and makes it possible to obtain polymers with high purity degree and extraction completeness up to 98.5–99.0

Three-Dimensional Printing of Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] Biodegradable Scaffolds: Properties, In Vitro and In Vivo Evaluation

The results of constructing 3D scaffolds from degradable poly(3-hydrosbutyrpate-co-3-hydroxyvalerate) using FDM technology and studying the structure, mechanical properties, biocompatibility in vitro, and osteoplastic properties in vivo are presented. In the process of obtaining granules, filaments, and scaffolds from the initial polymer material, a slight change in the crystallization and glass transition temperature and a noticeable decrease in molecular weight (by 40%) were registered. During the compression test, depending on the direction of load application (parallel or perpendicular to the layers of the scaffold), the 3D scaffolds had a Young’s modulus of 207.52 ± 19.12 and 241.34 ± 7.62 MPa and compressive stress tensile strength of 19.45 ± 2.10 and 22.43 ± 1.89 MPa, respectively. SEM, fluorescent staining with DAPI, and calorimetric MTT tests showed the high biological compatibility of scaffolds and active colonization by NIH 3T3 fibroblasts, which retained their metabolic activity for a long time (up to 10 days). The osteoplastic properties of the 3D scaffolds were studied in the segmental osteotomy test on a model defect in the diaphyseal zone of the femur in domestic Landrace pigs. X-ray and histological analysis confirmed the formation of fully mature bone tissue and complete restoration of the defect in 150 days of observation. The results allow us to conclude that the constructed resorbable 3D scaffolds are promising for bone grafting

Scaling of Biodegradable Polyhydroxyalkanoates Synthesis Technology in Pilot Production Conditions

На основе экспериментально достигнутых технико-технологических показателей процессов биосинтеза получены исходные данные и разработан проект создания опытного производства разрушаемых полигидрокисалканоатов (ПГА). Сконструировано, укомплектовано и введено в строй пилотное производство полигидроксиалканоатов, включающее ферментационную линию «Bioengineering» (Швейцария) и блоки получения инокулята и питательных сред, выделения и очистки полимеров. Опытное производство введено в строй. При исходной концентрации инокулята (10±2) г/л и длительности культивирования 65 ч конечные концентрации биомассы клеток и полимера в культуре Cupriavidus eutrophus В10646 составили (110±10) г/л и (85±5) % при продуктивности процесса по биомассе и ПГА 1,7 и 1,4 г/л∙ч соответственно, что вдвое превосходит ранее достигнутые показатели в культуре Ralstonia eutrophus B5786 на фруктозе. Масштабирование технологии обеспечило получение опытных партий полимеров в количествах, необходимых для проведения комплекса регламентированных исследований, разработки и стандартизации специализированной полимерной продукцииOn the basis of experimentally achieved and developed technical and technological parameters of biosynthesis processes the baseline data were obtained and the project was worked out for organization of pilot production of degradable polyhydroxyalkanoates (PHAs). The pilot line for production of polyhydroxyalkanoates was designed, equipped and commissioned, including the “Bioengineering” fermentation line (Switzerland), as well as the blocks for preparation of inoculum and culture media, isolation and purification of polymers. The pilot production was commissioned. When the initial concentration of inoculum was (10 ± 2) g/l and the cultivation duration was 65 h, the final concentrations of cells biomass in the culture of Cupriavidus eutrophus В10646 and polymer were (110±10) g/l and (85±5)%, with the process productivity by biomass and PHA of 1.7 and 1.4 g/l∙h, respectively, which is twice as high as the previously achieved results in the culture of Ralstonia eutrophus B5786 on fructose. Scaling the technology made it possible to obtain the experimental batches of polymers in the amounts needed for conducting a complex of prescribed research, development and standardization of specialized polymer product

Biodegradable Polyhydroxyalkanoates Formed by 3- and 4-Hydroxybutyrate Monomers to Produce Nanomembranes Suitable for Drug Delivery and Cell Culture

Biodegradable polyhydroxyalkanoates, biopolymers of microbiological origin, formed by 3- and 4-hydroxybutyrate monomers P(3HB-co-4HB), were used to obtain nanomembranes loaded with drugs as cell carriers by electrospinning. Resorbable non-woven membranes from P(3HB-co-4HB) loaded with ceftazidime, doripinem, and actovegin have been obtained. The loading of membranes with drugs differently affected the size of fibers and the structure of membranes, and in all cases increased the hydrophilicity of the surface. The release of drugs in vitro was gradual, which corresponded to the Higuchi and Korsmeyer-Peppas models. Antibiotic-loaded membranes showed antibacterial activity against S. aureus and E. coli, in which growth inhibition zones were 41.7 ± 1.1 and 38.6 ± 1.7 mm for ceftazidime and doripinem, respectively. The study of the biological activity of membranes in the NIH 3T3 mouse fibroblast culture based on the results of DAPI and FITC staining of cells, as well as the MTT test, did not reveal a negative effect despite the presence of antibiotics in them. Samples containing actovegin exhibit a stimulating effect on fibroblasts. Biodegradable polyhydroxyalkanoates formed by 3-hydroxybutyrate and 4-hydroxybutyrate monomers provide electrospinning non-woven membranes suitable for long-term delivery of drugs and cultivation of eukaryotic cells, and are promising for the treatment of wound defects complicated by infection

Biodegradation of microbial plastic poly(3-hydroxybutyrate) in soil ecosystems at different latitudes

The features of the degradation of the ''green'' plastic poly(3-hydroxybutyrate) [P(3HB)] in the soil of various geographical regions were studied: in red ferralitic soil under tropical conditions (Kerala, India) and in chernozem soil under conditions of a sharply continental climate (Eastern Siberia, Russia). Significant differences in the chemical composition, temperature, and humidity of the studied soils were revealed. The number of bacteria and mycelial fungi in the Siberian chernozem was higher than in the red soil of India, from 2-3 to 10 or more times. The degradation of P(3HB) films in the chernozem occurred faster than in the red soil, which was drier, with a low content of humus and minerals, and fewer microorganisms than the chernozem. The half-life of polymer samples in Siberia and India was 64.8 and 126.4 days, respectively. During degradation, a decrease in the molecular weight and an increase in the degree of crystallinity of polymer samples were revealed, which indicates a more active biodegradation of the amorphous phase of the polymer by soil microorganisms. The primary degraders of the polymer have been isolated and identified, and it has been shown that the complexes of degrading bacteria and fungi in different types of soils did not have common species. Despite the presence of species with pronounced depolymerase activity, the rate of film degradation in red ferralitic soils was slowed down by unfavorable environmental conditions. The obtained results confirm the importance of studying the process of PHA degradation in natural conditions