# Natural Materials as Carriers of Microbial Consortium for Bioaugmentation of Anaerobic Digesters
## 1 Introduction  
According to the latest statistical report by the European Biogas Association (EBA), gas import dependency in Europe rose from 83% in 2021 to 97% in 2022. Therefore, it is necessary to enhance the production of alternatives to natural gas, and biogas is one of them [1]. One of the ways to produce biogas is through anaerobic digestion using organic matter as a substrate [2]. During the process, lipids, carbohydrates, and proteins are digested into smaller compounds in the absence of oxygen. AD consists of four biochemical steps, hydrolysis, acidogenesis, acetogenesis, and methanogenesis, which are conducted by bacteria and archaea [2]. The most commonly used organic matter in AD is maize silage, an edible component also used as livestock feed [3]. Another option is lignocellulosic biomass such as dedicated energy crops with biogas yields comparable to maize silage [4]. In Mediterranean countries, an example of a promising substrate for energy production is _Miscanthus x gigentes_ (MxG) or _Arundo donax_[5]. MxG was investigated as a potential biomass due to its abundant carbohydrate content, non-edible nature, rapid growth, andability to be grown on marginal land. Moreover, cultivation is simple due to low nutrient and water requirements [4]. In order to convert MxG crops into biogas, the crucial step is the presence of physiological groups of bacteria that will degrade the biomass to substrates (acetate, H2, and CO2) for further methanogenesis.  
Immobilization of the selected bacterial species or microbial consortium onto solid materials as carriers has frequently been employed in biotechnological processes, especially in the bioremediation of soil or wastewater and the biodegradation of food waste and different types of silage [6]. Lignocellulose biomass is to a large scale quite resistant to microbial enzymatic biodegradation. Therefore, the immobilization of desired microbes accompanied by bioaugmentation is promising for the enhancement of lignocellulosic degradation [3]. The usage of immobilized microbes provides many advantages, such as higher cell density and consequent metabolic activity, great stability, and protection from extreme environmental conditions, subsequently resulting in higher efficiency in bioremediation and biodegradation [7]. However, not all materials are appropriate for the immobilization of microbes and selecting a suitable carrier is crucial for successful immobilization. The properties of an ideal carrier of microbes should be a porous structure with a rough and irregular surface for bacterial colonization, inexpensiveness, and non-toxicity [7]. Numerous types of microbial carriers can be divided into organic and inorganic materials and synthetic and natural materials [8]. The usage of natural materials has many ecological advantages against man-made plastic carriers. Natural organic materials such as corncob, straw, plant fibers, and rice are promising, but their utilization in AD is limited due to low resistance to biodegradation and stability in different pHs [8]. Natural inorganic materials such as zeolite, perlite, and porous glass have high chemical and physical resistance [8].  
In this research, four different natural materials (natural zeolitized tuff, ZeoSand(r), perlite, and crushed corncob) were tested as carriers of a microbial consortium which is intended for bioaugmentation of anaerobic digesters using MxG as a substrate.