## 5 Potential for Concurrent Production of Biofuels
Microalgae biomass can be effectively used to produce biogas, methane, hydrogen, and bio-hythane (5-25%). The main disadvantage of anaerobic fermentation (AD) leading to biogas production is the difficulty of solution by enzymatic hydrolysis of the microalgae cell wall [35]. AD converts almost all organic components of biomass into biogas through complex microbial action, without drying or extraction processes. By applying hydrothermal pretreatment to biomass, an improvement in the efficiency of biofuel gas production has been found, not only on a laboratory scale but also on an industrial scale [32]. Despite this advantage, the use of microalgae biomass as a feedstock for AD faces a number of technological challenges, which, in addition to the resistance of the cell structure, include the presence of toxic substances in anaerobic bacteria. Pre-treatment of microalgae biomass (residual or total) prior to AD seeks to address these problems. The main methods applied are pre-treatment methods, such as mechanical methods (size reduction, ultrasonic, or microwave), thermal hydrolysis, chemical methods (oxidation, addition of alkalis, acids), biological methods (enzymes), and hybrid methods where a combination of methods is performed. From the comparison of the above pre-treatments, thermal hydrolysis appears to have the highest Energy Return Over Investment (EROI), with the EROI ratio reaching 6.8. In addition to pre-treatment, recycling of AD outputs as inputs to AD has been proposed in order to make the system economically and environmentally sustainable [35]. Bioethanol production is increasing, from 17,250 mL in 2000 to 160,000 L in 2020. Microalgae are an alternative resource for bioethanol production due to their starch and cellulose contents, which can be broken down into fermentable sugars such as glucose. In addition, they have the advantage compared to lignocellulosic matter that no lignin is contained in their cell wall, thus facilitating its breakdown. Through the fermentation of sugars, bioethanol is produced. For the breakdown of carbohydrates into fermentable forms of simple sugars, a saccharification stage is applied before fermentation. The production of bioethanol by microalgae is greatly influenced by the pre-treatment and type of fermentation process. The main methods used for the pre-treatment of microalgae biomass in order to disrupt the cell wall to release more carbohydrates and convert them to monosaccharides are chemical methods, mainly acid hydrolysis and alkaline hydrolysis. For acid hydrolysis, sulfuric and hydrochloric acid have been applied, while for alkaline hydrolysis, caustic soda, soda ash, and ammonia solution have been applied. In the case of acid hydrolysis, inhibitors are formed which have a negative effect on the fermentation process, and therefore neutralization must be carried, out which further increases the production cost of bioethanol. Enzymatic hydrolysis, is more efficient and has lower energy needs compared to chemical hydrolysis, although the cost of selected enzymes is high, thus limiting its commercial application. The choice of pre-treatment technology is closely correlated with the characteristics and efficacy of enzymes. Mechanical methods such as crushing, grinding, or milling, as well as the use of ultrasound, increases the accessibility of the substrate to enzymes, but have high cost and energy requirements compared to chemical methods. In addition, a combination of methods, such as the use of ultrasound with enzymatic hydrolysis, can be used with satisfactory results [36]. The microalgae strains that have been most scientifically investigated for increased carbohydrate production are _Isochrysis galthana, Porphyridium crucuntum, Namochloropsis oculate,_ and species of _Spirogyna_ and _Chlorella_[35]. Biobutanol is considered a more efficient biofuel than biotechnanol or bioethanol due to its higher energy density, and is compatible with gasoline due to its similar molecular structure.