**5. Fermentation**

Fermentation is a process that based on disciplines of chemistry, biochemistry and microbiol‐ ogy and which fermentable sugars are converted to ethanol by microorganisms [84]. Process consists of conversion of glucose to alcohol and carbon dioxide:

$$\rm{C}\_6\rm{H}\_{12}\rm{O}\_6 \rightarrow 2\rm{C}\_2\rm{H}\_5\rm{OH} + 2\rm{CO}\_2 \tag{1}$$

In this process 0.51 kg bioethanol and 0.49 kg carbon dioxide are obtained from per kg of glucose in theory maximum yield. However practically, microorganisms also use glucose for their growth, the actual yield is less than 100% [85]. Microorganisms used in fermentation are utilized from 6-carbon sugars in ethanol production. Therefore, cellulosic biomass which have high amount of glucose are the materials that have easiest conversion capability. One of the most effective yeast which produces bioethanol is *Saccharomyces cerevisiae.* Besides having high bioethanol production yields, it has a resistance to high bioethanol concentration and inhibitor components which can be occurred after acid hydrolization of lignocellulosic biomass. Because reaction occurs under anaerobic conditions, oxygen molecules must be removed with nitrogen gas as a swept gas. Yeast and fungi can tolerate 3.5-5.0 pH ranges [86]. *S.cerevisiae* has high osmotic resistance and can tolerate low pH levels like 4.0. *Zymomonas* stands out with rapid bioethanol production and high productivity compared to other traditional yeasts. However *Z.mobilis* cannot tolerate the toxic effects of asetic acid and various phenolic compounds in the lignocellulosic hydrolysate [87]. Bioethanol yields of microorganisms are depend on temper‐ ature, pH level, alcohol tolerance, osmotic tolerance, resistance for inhibitors, growth rate and genetic stability [54]. Fermentation processes generally are carried out with two basic processes as *simultaneous saccharification and fermentation* and *separate hydrolysis and fermentation*, yet new production processes have been developed [1].

### **5.1. Separate Hydrolysis and Fermentation (SHF)**

dase hydrolyzes the cellulose units and enables the formation of glucose [64]. Enzymatic hydrolysis can be affected by certain factors which are enzyme-related and substrate-related factors. Substrate-related factors have a directly influence on enzymatic hydrolysis. These factors are connected to each other and effect the enzymatic conversion. These factors can be defined as *degree of polymerization and crystallinity of cellulose, accessibility of the substrate, lignin*

Hydrolysis rates of biomass depend on the degree of polymerization and crystallinity of cellulose. Degree of polymerization is related to crystallinity. Cellulase enzymes can hydrolyze the crystalline structure of cellulose. Endoglucanase enzymes decrease polymerization degree of cellulosic component by cutting the internal sites of cellulose chains in the enzymatic hydrolysis [80]. Accessibility of the substrate is another main factor effect hydrolysis rate. The rate of hydrolysis increases with increasing substrate accessibility because of being surface area more available for enzymatic attack [80]. Lignin and hemicellulose are complex structures to hydrolyze in lignocellulosic materials. Due to have a role like cement, lignin acts as physical barrier and prevents the digestible parts of cellulose to hydrolyze and it becomes very difficult for enzymes to access cellulose. For this reason, they reduce the efficiency of hydrolysis. Removal of hemicellulose enhances the pore size and provides accessibility to cellulose for enzymes in order to perform hydrolysis efficiently [81,82]. Pore size of the substrate is one of the limiting factors in enzymatic hydrolysis process. In many lignocellulosic material, external area of the biomass is smaller than internal area and this situation causes cellulase enzymes to entrap in the pores of the material. In order to increase hydrolysis rate, porosity of the biomass

Fermentation is a process that based on disciplines of chemistry, biochemistry and microbiol‐ ogy and which fermentable sugars are converted to ethanol by microorganisms [84]. Process

In this process 0.51 kg bioethanol and 0.49 kg carbon dioxide are obtained from per kg of glucose in theory maximum yield. However practically, microorganisms also use glucose for their growth, the actual yield is less than 100% [85]. Microorganisms used in fermentation are utilized from 6-carbon sugars in ethanol production. Therefore, cellulosic biomass which have high amount of glucose are the materials that have easiest conversion capability. One of the most effective yeast which produces bioethanol is *Saccharomyces cerevisiae.* Besides having high bioethanol production yields, it has a resistance to high bioethanol concentration and inhibitor components which can be occurred after acid hydrolization of lignocellulosic biomass. Because reaction occurs under anaerobic conditions, oxygen molecules must be removed with nitrogen gas as a swept gas. Yeast and fungi can tolerate 3.5-5.0 pH ranges [86]. *S.cerevisiae* has high

C H O 2C H OH+2CO 6 12 6 2 5 ® <sup>2</sup> (1)

consists of conversion of glucose to alcohol and carbon dioxide:

*and hemicelluloses content and pore size*.

150 Biofuels - Status and Perspective

should be increased [83].

**5. Fermentation**

Enzymatic hydrolysis is performed separately from fermentation in this process. Liquid which comes from hydrolysis reactor first converted to ethanol in a reactor that glucose fermented in, and then ethanol is distilled and remained unconverted ksilose is converted to ethanol in a second reactor. Advantage of the process is performing reactions in optimum conditions. On the otherhand,usage ofdifferentreactors is increasing the cost.Also glucose andcelluloseunits that obtainedafterhydrolysis,inhibit activity ofthe enzyme anddecreasehydrolysis rate [3,54].

### **5.2. Simultaneous Saccharification and Fermentation (SSF)**

In this process, pre-treatment and enzymatic hydrolysis steps are carried out with fermentation step in the same reactor. It is very efficient when dilute acid or hot water at high temperature is applied in the process. High bioethanol yields can be achieved with SSF process. Also inhibiton of enzyme activity is very low due to fermenting glucose and cellulose units in the same media by yeast. Therefore, this process needs low amount of enzyme. In addition to that, process cost is reduced because of the reactions are carried out in one reactor. As a disadvant‐ age, temperatures differences between saccharification and fermentation cause various effects in growth of microorganisms. *Saccharomyces* cultures are used in pH of 4.5 and temperature of 37 °C this process [3,54,88].

### **5.3. Simultaneous Saccharification and Co-Fermentation (SSCF) & Separate Hydrolysis and Co-Fermentation (SHCF)**

*Saccharomyces cerevisiae* which used in fermentation cannot convert carbohydrates like pentos under moderate conditions and this causes impurity for biomass and decreases bioethanol yield. In order to overcome this, recombinant yeasts can be used to convert residues such as pentose to ethanol. In SSCF, recombinant yeasts and cellulase enzyme complex are fed to the same vessel to convert biomass to ethanol. This system is generally the same as SSF process. SCHF process is a combination of SSCF and SHF. In this process, fermentation and hydrolysis are carried out in different vessel. This process can produce ethanol with high productivity in comparison with SHF process [88].

Due to their simple structure and being a new raw material for bioethanol production, most of these pre-treatment techniques have not applied to algal biomass yet, and just few studies have been found in literature which is presented in Table 4.


**Table 4.** Studies of ethanol production from micro and macroalgae
