Self-standardization of quality of bacterial cellulose produced by Medusomyces gisevii in nutrient media derived from Miscanthus biomass
Graphical abstract
Introduction
Bacterial cellulose (BC) represents a linear homopolymer composed of d-glucopyranose linked with β-1,4 bonds and can be synthesized with natural or genetically modified microorganisms. Compared to plant cellulose, BC is a chemically pure cellulose. Besides, BC is distinguished by a high degree of polymerization and a high index of crystallinity. A peculiar feature of BC is its architecture. BC has a 3D structure consisting of an ultrathin network of nanofibers. Hence, BC possesses such properties as mechanical strength, elasticity and formability, permeability for gases and liquids, hydrophilicity and high water-retaining capacity, as well as biocompatibility, biodegradation, and cell adhesion and proliferation abilities (Campano, Balea, Blanco, & Negro, 2015; Gama, Dourado, & Bielecki, 2016; Keshk, 2014; Wang, Tavakoli, & Tang, 2019).
Due to the aforementioned properties, BC offers a plenty of different applications in food industry, pulp and paper industry, biotechnological industry, biomedicine, electronics and many more (Albuquerque et al., 2020; Barud et al., 2016; Campano et al., 2015; Gama et al., 2016; Keshk, 2014; Wang et al., 2019; Zahan et al., 2020; Zharikov et al., 2018), given that various ester derivatives and diverse composite materials have been created starting from BC (Volova et al., 2018; Yin, Du, Zhao, Han, & Zhou, 2020; Malmir et al., 2020).
Even though BC is highly demanded in different fields of application, the manufacture of BC is a high-cost process: the cost of synthetic nutrient media may constitute up to 65 % of total value of the process (Velásquez-Riaño & Bojacá, 2017). Therefore, research focused on finding solutions that minimize the prime cost of nutrient broths is highly relevant. The use of cheap cellulosic raw materials to obtain highly valuable BC has become one of such solutions in the world practice. In this context, the synthesis of this high-value-added product from renewable resources conforms to the concept of circular economy (Geissdoerfer, Savaget, Bocken, & Hultink, 2017; Kim, Lee, & Kim, 2016; Taherzadeh, 2019). A successful use of plant biomasses, woody species, agricultural residues, textile and pulp and paper industrial wastes has been described (Abol-Fotouh et al., 2020; Campano et al., 2015; Chen et al., 2019; Hussain, Sajjad, Khan, & Wahid, 2019; Luo et al., 2017; Velásquez-Riaño & Bojacá, 2017).
Here we suggest using a fast-growing plant, Miscanthus sacchariflorus (Maxim.) Hack, as a source of cellulosic feedstock. This is a perennial grass referring to the family Poaceae, which exhibits high growth indices of the aboveground vegetative mass: after a single planting of Miscanthus, the annual gain of straw accounts for 10−15 ton/ha throughout 15–25 years (Gismatulina & Budaeva, 2017; Xue, Lewandowski, Wang, & Yi, 2016). In this study, we used biomass of Miscanthus that had been introduced to and grown in Western Siberia. Compared to other Miscanthus varieties, M. sacchariflorus stands out with active sprout formation, which endows it with an advantage of vegetative biomass accumulation, with the BC production being 10 ton/ha even in the Siberian conditions (Dorogina et al., 2019; Gismatulina et al., 2019).
A cellulosic biomass represents a matrix in which hemicelluloses, lignin and cellulose are bound together with physical and chemical bonds; therefore, the cellulosic feedstock cannot directly be converted by cellulose-producing microorganisms into BC and must be subjected to pretreatment (Hussain et al., 2019). At present, the chemical pretreatment stage of a feedstock is considered by most researchers to be decisive for the whole technology of cellulosic biomass conversion, and it is this stage that determines how successful enzymatic hydrolysis, biosynthesis of target metabolite and its purification will be (Gaurav, Sivasankari, Kiran, Ninawe, & Selvin, 2017; Kim et al., 2016; Zabed, Sahu, Boyce, & Faruq, 2016). That is, pretreatment determines the feasibility of scaling up a technology under development in industry (Zabed et al., 2016). For the bacterial cellulose technology, scale-up is a crucial factor (Velásquez-Riaño & Bojacá, 2017). Despite the broad discussion about pretreatment of cellulosic biomass in the bioethanol production, we have not found studies on the effect of the feedstock pretreatment method on the BC yield and quality. Meanwhile, this is an extremely important factor. Most of the studies describe the variation in structural characteristics of BC when replacing synthetic nutrient media by alternative broths because BC-producing strains are very sensitive to the compositional variation of nutrient media (Hussain et al., 2019; Keshk, 2014; Khan, Kadam, & Dutt, 2020; Luo et al., 2017; Velásquez-Riaño & Bojacá, 2017).
For the pretreatment of Miscanthus biomass, we used our proprietary methods based on dilute HNO3 solutions. The key feature of the HNO3 action on a cellulosic feedstock is that, in addition to partial hydrolysis of hemicelluloses and cellulose, there also occurs oxidative nitration of all the feedstock constituents, thereby augmenting the pretreatment performance (Skiba, Budaeva, Baibakova, Zolotukhin, & Sakovich, 2017). The proprietary methods we used herein have been applied many times under pilot-production conditions in order to obtain semiproducts for biotechnological processes or for technical chemistry (Gismatulina & Budaeva, 2017; Kashcheyeva, Gismatulina, & Budaeva, 2019), and are distinguished by reproducibility, high processability, cheapness and eco-friendliness. However, this is the first study in which the prolonged biosynthesis of BC in nutrient media prepared from Miscanthus biomass pretreated by four different methods has been examined in detail and in-depth physicochemical characterization of the resultant BC samples has been performed.
We chose symbiotic Medusomyces gisevii Sa-12 as the BC producer. This SCOBY (symbiotic culture of bacteria and yeast composed of up to 50 microbial species) is unpretentious, capable of utilizing different sugars, and tolerant to phagous infections due to its multispecies composition (Yurkevich & Kutyshenko, 2002a). These features allow the biosynthesis of BC under non-sterile conditions. A number of authors have already reported the necessity of such pilot studies (Kiziltas, Kiziltas, & Gardner, 2015; Velásquez-Riaño & Bojacá, 2017), and we are glad to present such a study. Due to the instability of highly productive, genetically modified BC-producing strains and the spontaneous uncontrollable decrease in their cellulose-producing capability (Campano et al., 2015; Keshk, 2014), there arises an acute need to find and develop cellulose-producing strains, which is of especial importance in scaling up biotechnological processes. Among such approaches can be the use of microbial consortia (Gama et al., 2016: Marsh, O’Sullivan, Hill, Ross, & Cotter, 2014). The Medusomyces gisevii Sa-12 symbiotic culture represents a natural, very stable consortium which can be employed as a microbial producer for scaling up the biosynthesis of BC.
The objective of the present study was to explore if the production and quality of bacterial cellulose synthesized by Medusomyces gisevii Sa-12 were dependent on the pretreatment method of biomass of Miscanthus that had been grown under plant introduction conditions in West Siberia.
Section snippets
Materials
Miscanthus sacchariflorus (Maxim.) Hack had been grown for one vegetative period on a pilot plot of land in Biysk city (South-Western Siberia); the plantation age was 6 years. The Miscanthus biomass was harvested in October 2018. Prior to the pretreatment, the Miscanthus biomass was ground with a Gardena straw-cutter to a particle size of no more than 10 mm.
Chemical pretreatment of plant biomass
Pretreatment was performed under pilot production conditions at atmospheric pressure and 90−96 °C. A standard 250-L vessel was used; all
Chemical composition of substrates
The chemical compositions of the substrates obtained by different chemical pretreatments of Miscanthus biomass are summarized in Table 1.
All the four chemical pretreatments increased the cellulose content by almost two times compared to the feedstock (from 50.3 % in Miscanthus biomass to 79.2–95.8 % in the pulps) and diminished the content of non-cellulosics, that is, the methods were efficient.
In terms of the contents of cellulose and hydrolyzables, the pulps can be arranged in the row: 3 > 4
Conclusion
Here we examined the bioconversion process of Miscanthus biomass into bacterial cellulose. Four types of enzymatic hydrolyzates were derived by single- and two-stage chemical pretreatments of Miscanthus biomass with dilute HNO3 and NaOH solutions and by hydrolysis of the resultant pulps with commercially available CelloLux-A and BrewZyme BGX enzymes. Nutrient media were prepared from the enzymatic hydrolyzates with added black tea. Symbiotic Medusomyces gisevii Sa-12 was inoculated into the
CRediT authorship contribution statement
Ekaterina А. Skiba: Conceptualization, Methodology, Investigation, Writing - original draft. Evgenia K. Gladysheva: Investigation, Formal analysis. Dmitri S. Golubev: Investigation. Vera V. Budaeva: Conceptualization, Writing - review & editing. Lyudmila А. Aleshina: Validation, Formal analysis, Visualization, Software. Gennady V. Sakovich: Supervision.
Acknowledgement
The research was supported by the Russian Science Foundation (grant No. 17-19-01054).
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