1. Introduction
Humanity uses natural gas, coal, peat and oil as the main sources of energy
[1,2][1][2]. All these resources are nonrenewable, and their reserves are being depleted. In this regard, the issue of finding alternative renewable energy sources is acute, and plant raw materials are of particular interest. Such renewable plant raw materials as trees are a slowly renewable energy resource. Although 23% of the territory of the Russian Federation is covered with forest, wood accounts for more than 71.4 million hectares of the forested territory—81.5 billion m
3 [3].
Miscanthus is used in landscape design, for the manufacture of paper and roofing. This is a promising biomass resource that can replace fossil hydrocarbon fuels
[4]. It belongs to the
Andropogoneae herb family, which includes corn, sorghum and sugar cane—these crops are high yielding and important all over the world, and they can be grown as sources of food, feed and biofuels. Miscanthus is unpretentious and easy to grow. It can thrive in marginal areas, requires only limited fertilizer, tolerates drought and low temperatures well and uses a more efficient form of C4 photosynthesis
[5].
In addition to its historical role as an ornamental plant and in paper production, wild miscanthus can also produce highly productive biomass, thereby protecting and potentially increasing the carbon content in the soil
[6]. The increase in the carbon content in the soil is associated with the absorption of light, the absorption of nutrients and the efficient use of water, which makes miscanthus a promising bioenergy crop. Bioenergy raw materials (miscanthus) are constantly being genetically improved to increase yields
[6]. Before continuous biotic and abiotic stress, it is necessary to guarantee the yield and stability of these crops. This is particularly true for perennial grasses, as the economic and environmental sustainability of perennial grasses, compared to annual crops, depends on the life expectancy of the plants
[7]. Kosolapov et al. demonstrated that perennial crops have a huge potential to increase yields and minimize environmental impacts, which makes them a promising, affordable and environmentally friendly resource for biofuels
[7]. Dubouzet et al. established that genetic engineering can accelerate the development and reproduction of varieties, but it is not essential to increase biomass yield
[8].
It is reported that the global availability of hydrocarbons is increasing due to the modern hydraulic fracturing of rock formations
[9]. However, studies of Pärt et al. showed that these isolation methods, which ensure the production of fuel and other biological products, use toxic chemical additives, allergens, mutagens and carcinogens and cause the decomposition of radioactive materials
[10]. Muscanthus can provide a more environmentally friendly solution for the extraction of bioenergy from the second-generation crops muscanthus provides as it is a potential supplier of sustainable biomass
[11]. Miscanthus is the most environmentally friendly fuel because miscanthus plants can grow in one location for more than 20 years, reducing the need for circulating capital inputs significantly. It is resistant to pests and diseases and can be grown without the use of chemicals. Furthermore, miscanthus successfully performs ecological and environmental improvement functions: it protects landscapes from erosion, promotes organic matter accumulation in the soil and significantly reduces CO
2 emissions
[12].
Kukk and Sõber demonstrated in their study that due to miscanthus’s high-biomass yield, long-term growth, soil carbon sequestration potential, reduced soil erosion and lower fertilizer requirements, the most related varieties of miscanthus are used, including
M. sinensis,
M. sacchariflorus and
M. × giganteus [10]. In the UK, miscanthus is typically planted in the spring and harvested after rooting for 15 years. New shoots usually appear in mid-spring and grow quickly in the next few months, depending on the genotype, they can reach a height of several meters in mid-summer
[13]. Arnoult and Brancourt-Hulmel established that autumn frost causes aging, and as the miscanthus ages, nutrients are transferred from the aboveground organs to the rhizomes
[14]. Therefore, usually, only fully aging plants are harvested, which guarantees the resumption of crop growth in the next season
[14].
Rosen and Kishawy found that the efficient and sustainable development of the world economy requires mandatory and comprehensive consideration of environmental aspects
[15]. The constant increase in global energy consumption, as well as the limited availability of fossil raw materials and energy, necessitate the replacement of existing industrial technologies with energy-, nature-, and resource-saving biotechnologies
[16]. Environmental pollution caused by plastic packaging materials and products made from synthetic polymers is a serious global environmental problem of our time
[17]. This necessitates the urgent development and increase in the production of biodegradable and biocompatible packaging materials, including those derived from renewable raw materials, in order to contribute to the environmental stabilization of the natural environment
[18]. Bhatia and Goli demonstrated that there are countries in the world that are actively developing the direction of biotechnology based on the production of valuable substances and products with high added value from plant materials (biomass of energy crops with intensive rates of photosynthetic activity)
[19].
According to Baibakova et al., one of the most promising plants in this regard is miscanthus (
Miscanthus spp.), which has a relatively high adaptive potential (it can be grown on unproductive degraded lands, on fields with a slope of up to 7°, with soil acidity pH 5.5–7.5). The largest energy yield per unit area was 13.55 GJ/ha
[20].
Its aboveground cellulose-containing biomass belongs to non-traditional renewable sources of raw materials and energy, the production of which does not require significant capital investments
[21].
The study of miscanthus bioengineering and molecular biology is a vital task in this regard
[10]. For the first time, this re
vise
w arch gathered information on miscanthus species, their properties, and their application in bioengineering to obtain a substantial amount of plant biomass for biofuel production. This
work research aimed to summarize the research of the world’s leading scientists on the methods of bioengineering and molecular biology of miscanthus to obtain promising sustainable biofuels.
2. The Importance of the Cell Wall in Miscanthus Bioengineering
Although there are many potential applications in bioprocessing, lignocellulose biomass is still largely untapped due to the stability of the cell wall, i.e., resistance to deconstruction, relative abundance and interaction between cell wall components
[22]. Therefore, as demonstrated by Ochoa-Villarreal et al., in order to effectively use the cell wall as a renewable source of useful molecules, it is important to increase knowledge on how to assemble the wall in terms of composition and structure
[23]. The cell walls of commelinoid monocots, including grasses, differ from other plant groups. In addition to lignin, these cell walls also contain a high percentage of cellulose, a low percentage of xyloglucan, mixed bonds and a high content of 4-β-xylan
[24]. Xylanes usually contain acetyl substituents, arabinose and/or glucuronic acid attached to certain xylose residues in the main chain, hence, the name arabinoxylan (AX) and glucuronic acid arabinoxylan (GAX)
[25]. The walls of miscanthus cells also contain a small amount of pectin. The studies of da Costa demonstrated that these pectins are polysaccharides rich in α-galacturonic acid. Polysaccharides are thought to be composed of three domains (homogalacturonan, rhamnogalacturonan-I and rhamnogalacturonan-II), linked by glycosidic bonds
[26]. Cellulose was determined by the Kürschner method. The fiber content was determined by the Kürschner method, which is based on the oxidative destruction of all substances, except fiber, in a mixture of acetic and nitric acids. Cellulose fibers were separated and dried. The percentage to the weight of a raw or anhydrous sample was determined as a result of weighing
[27]. In
Table 1, the chemical composition of samples
[28,29][28][29] of various miscanthus types are presented.
Miscanthus plants (
Table 1) of the species
M. × giganteus,
M. sinensis and
M. Sacchariflonis were grown in open ground in the conditions of the Siberian Forest steppe, in a temperate climate with a sufficient amount of heat and solar energy, at a moisture level of up to 500–600 mm of precipitation per year. Miscanthus was planted in open ground in sunny locations in the spring, when the temperature is high and the earth heats up to +20–25 °C. No more than 1–2 plants were planted per 1 m
2. The soil for growing miscanthus was well drained, but not waterlogged. Miscanthus plants grew just as well on acidic and heavy clay soils as it did on black or fertilized soils. A well-developed root system makes the plant suitable for growing on various types of soils, from sands (with a low level of groundwater) to soils with a high organic matter content
[30]. Miscanthus grew well in a wide range of soil pH, but the ideal pH was between 5.5 and 7.5. The fallen leaves of the plant itself were an excellent fertilizer. Nitrogen fertilizers were applied in autumn and organic fertilizers were applied several times during the growing season. It is critical not to overuse nitrogen fertilizers, as this will affect the color of the leaves as well as the quality of the biomass. Fertilizers, pesticides and inter-row tillage were applied in the same manner as for traditional crops, but mostly in the first year of growth. Such measures are very limited in subsequent years (if appropriate, they are carried out only in early spring, at the beginning of the growing season). Weed management was essential for miscanthus plantings, particularly in the first and second years of cultivation. After that, it was not necessary, since the miscanthus plants were already well rooted and competed with weeds. Miscanthus is cut-harvested as biomass grows (2–3 times per season). Miscanthus samples were chemically analyzed after four years of cultivation
[30].
Galacturonans (HG) makes up most of the cell wall pectin and includes unbranched chains of α-galacturonic acid residues connected by methylated bonds. In addition, rare ingredients, such as hydroxycinnamate (HCA) and structural proteins, can be found
[31]. The complexity of cell wall composition is complicated by the fact that various components of the cell wall are linked together by a process, much like the molecular structure that maintains the integrity of plant tissue and resists external attacks. Therefore, according to Marowa et al., it is not easy to understand these complex connections, especially considering that about 10% of plant genomes are related to the assembly/disassembly of cell walls
[32].
Research into improving lignocellulose biomass for biological purification purposes has stimulated the structure study of plant cell walls
[32]. Determining the required characteristics of the cell wall and cultivating cultures containing such characteristics are key steps to optimize the bioremediation of lignocellulose
[32]. However, the study of
[30] showed that since the composition of all cell walls is different, often, there are difficulties in the complex assessment of the quality of different raw materials, especially throughout different studies that consider the profile of the reference cell wall to increase the biomass of leaves and stems of Miscanthus
[30]. In this material, particular attention is paid to the composition of cell wall glycans
[31].
Research on the structural composition and distribution of glycans in miscanthus organs and/or tissues is important not only for optimizing the use of lignocellulose biomass as a raw material for renewable biological products and bioenergetic solutions, but also for understanding the significance of the cell wall
[31]. Studies based on mid-infrared Fourier transform spectroscopy in the mid-infrared region show that structural polysaccharides are a major factor in composition variability during trunk development and between organs
[32].
The study of
[33] identified factors causing this variation in components (temperature, humidity, pH, organic and mineral composition of the soil, type and age of the plant and its productivity) and discussed some important observations about possible interactions between components and cell wall structures. It has been shown that various monoclonal antibodies targeting plant glycans can be markers of changes in characteristics of glycans in the matrix (distribution, structure and extractability)
[33]. This set of molecular markers of Miscanthus has sufficient variability to identify most types of non-cellulose polysaccharides in the plant cell wall
[19]. This was the first time that researchers used a complex of glycan-targeted antibodies to comprehensively characterize cell wall glycome variation across cultures to create a representative sample of Miscanthus wall biomass data (reference map)
[32]. Several global breeding programs are known to genetically improve the biomass and yield characteristics of Miscanthus, based on the use of genotypic and phenotypic differences between and within the species diversity of Miscanthus
[34].
The detailed miscanthus cell wall portrait in these studies contributes to genetic transformation, providing important information for the formation of the necessary characteristics in different types of miscanthus, which are planned to be processed into biofuels and other biological materials
[34].
Thus, this section discussed some important observations about possible interactions between the components and structures of cell walls and presented the factors that contribute to the transformation of miscanthus into a renewable source of biofuel. The complexity of the miscanthus cell wall composition, which affects miscanthus bioconversion into biofuel, was revealed.