Versatile-peroxides (VP) are found in various Bjerkandera species and Pleurotus species [114]. VA can oxidize both phenolic and non-phenolic compounds of lignin as well as Mn2+ [32]. The catalytic mechanism is similar to LiP [101]. For example, VP oxidize nonphenolic model compounds such as veratrylglycerol в-guaiacyl ether and results to the formation of veratraldehyde. VP also oxidize Mn2+ to Mn3+, VA to veratraldehyde and p-dimethoxybenzene to p-benzoquinone [32].

3.3.1 Batch Steam Pre-Treatment Processes

Large scale steam pre-treatment of lignocellulosic biomass has mainly been car­ried out using batch processes, where quantities of the biomass samples are radically modified from the high pressure steam conditions resulting in an alteration of the cell wall structures. This is usually visualised by a change of the original biomass colour to a near dark brown material after the treatment step, from which the partially hy­drolysed hemicellulose can be obtained by water-washing, leaving a water-insoluble hydrolysate fraction made up of cellulose, residual hemicellulose and modified lignin components which can be further extracted using mild alkali, ethanol or oxidative methods [13, 23].

Regarding industrial batch pre-treatment processes, the Cambi and Inbicon pro­cesses have enjoyed successful applications for improving the digestibility of a wide variety of biomass especially aimed for subsequently biological conversion processes, that is, for methane and ethanol production. These processes will be highlighted below as useful industrial batch process case studies.

Pellet mill has two types of pellet presses: ring die and flat die. Ring die is a more common type of pellet mill used in commercial unit. For the ring die pellet mill, the die remains stationary and the rollers rotate. Some models have the dies to rotate and the rollers remain stationary during pellet production. The die of a pelletizer is made of hardened steel that is perforated allowing the ground wood particles to be forced through by the rotating die or rollers. As the die revolves, the friction driven rollers force the feed through holes in the die (steps A and B, Fig. 5.4). A layer of biomass (step C, Fig. 5.4) is needed to develop between the roller and the flat die in order to start flowing the particles into the die channels (step D, Fig. 5.4) to push the materials to extrude through the flat die. Cut-off knives mounted on the swing cover cut the pellets when they extruded from the die (step E, Fig. 5.4). The advantage of the ring die type pellet mill allows a higher production throughput compared to other types of presses (e. g., piston and screw presses), while maintaining the power consumption in the range of 15-40 kW h/t [53].

Flat die pellet mill is widely used for producing animal feed. Similar to the ring die pellet mill, the ground particles are loaded into the densiflcation area. The rollers keep rotating in a clockwise direction in a vertical plane while the flat die is rotating anticlockwise in a horizontal plane. A layer of biomass powder is needed to develop between the roller and the flat die to start flowing into the die channels. Pressure is built up due to friction in the press channel and exponential correlation between press channel length and pressure is found [55,57]. Finally, the materials are extruded through the flat die. A cut-off knife is installed at the exit of the flat die to cut the pellets into a certain specific length.

According to Tumuluru’s review [54], die geometry refers to the size and shape of the die. The die geometry determines the pellet dimensions and the resulted density and durability. The aspect ratio (length to diameter of the pellet) can be a metric for the degree of compression during pelletization. An increase in pelletizing pressure increases the length of the pellet, whereas an increase in pellets diameter decreases the pelletizing pressure. Hence, the geometry of the die has a significant determination on the pressure applied onto the resulted pellet. A mathematical model for hardwood and softwood was developed to show how the variation in the model parameters (sliding friction coefficient, the ratio of compression, and the material — specific parameters, such as elastic modulus and Poisson ratio) significantly changes the necessary pelletizing pressure along the press channels of the matrix [55, 56]. Later, the co-relations of processing conditions between single die and commercial pellet mill was attempted [57].

Fig. 5.4 Mechanisms of pelletization by a ring die

The inner diameter (D) and the effective length (L) of the die determine the pellet density. The effective length is the die thickness that actually performs work on the feed. L/D ratio is the effective length divided by the inner diameter of the die. High L/D ratios provide high pellet die resistance as feed moves through the die. Low L/D ratios provide less resistance. Each material has an L/D ratio requirement to form the material into a pellet. The durability of the pellets improves when a smaller die with higher L/D ratios is used [58]. However, if a longer die length is used, which will introduce a higher friction with no more improvement in the pellet durability. This will consume excess energy for production. Therefore, there should have optimum L/D ratios of the die for different types of biomass to produce durable pellets.

Wet oxidation is an oxidative pretreatment method that employs oxygen or air as catalyst. It allows reactor operation at relatively low temperatures and short reac­tor times [103]. It has been proven to be an efficient method for solubilization of hemicelluloses and lignin and to increase digestibility of cellulose, specially.

8.3.3.1 Process Description

Typically, the procedure for wet oxidation consists of drying and milling lignocel — lulosic biomass to obtain particles that are 2 mm in length, to which water is added at a ratio of 1 L to 6 g biomass. A compound, usually Na2CO3, is introduced to the mixture to reduce the formation of by-products. Na2CO3 addition has been shown to decrease formation of inhibitory compounds by maintaining pH in the neutral to alkaline range. Air is pumped into the vessel until a pressure of 10-12 bar is reached. This method of pretreatment is performed at 170-200 °C for a range of 10-20 min [104, 105]. The addition of air/oxygen at temperatures above 170 °C makes the process exothermic reducing the total energy demand. In general, low formation of inhibitors and efficient removal of lignin are achieved with wet oxidation pretreat­ment. On the other hand, cost of oxygen and catalyst are considered one of the main disadvantages for wet oxidation development technologies [2].

Ebru Toksoy Oner

Abstract The interest in polysaccharides has increased considerably in recent years, as they are candidates for many commercial applications in different industrial sec­tors like food, petroleum, and pharmaceuticals. Because of their costly production processes, industrial microbial polysaccharides like xanthan, dextran, curdlan, gel — lan, and pullulan constitute only a minor fraction of the current polymer market. Therefore, much effort has been devoted to the development of cost-effective and environmentally friendly production processes by switching to cheaper fermentation substrates. In this chapter, various microbial polysaccharide production processes utilizing cheap biomass resources like syrups and molasses, olive mill wastewater, cheese whey, various vegetable and fruit pomace, pulp and kernels as well as carbon dioxide and lignocellulosic biomass like rice hull and bran, sawdust, and fibers are discussed with a special focus on the employed pretreatment methods.

Keywords EPS ■ Microbial exopolysaccharides ■ Polysaccharides ■ Biomass ■ Fermentation

2.1 Introduction

Since the beginning of twentieth century, technologies related to microbial pro­duction of biomolecules like enzymes, antibiotics, metabolites, and polymers have matured to a great extend. Currently, microbes are used for commercial production of a wide variety of products such as pesticides, fertilizers, and feed additives in agrochemical sector, biopharmaceuticals and therapeutics in the healthcare sector, biopolymers and biofuels in the energy and environment sectors. According to re­cent market reports, growing environmental concerns and increasing demands from end-use sectors are expected to increase the global market for microbial products to about 250 billion US dollars by 2016 [1].

E. T. Oner (H)

IBSB—Industrial Biotechnology and Systems Biology Research Group, Bioengineering Department, Marmara University,

Goztepe Campus, 34722 Istanbul, Turkey e-mail: ebru. toksoy@marmara. edu. tr

Z. Fang (ed.), Pretreatment Techniques for Biofuels and Biorefineries,

Green Energy and Technology,

DOI 10.1007/978-3-642-32735-3_2, © Springer-Verlag Berlin Heidelberg 2013

Polysaccharides are natural, non-toxic, and biodegradable polymers that cover the surface of most cells and play important roles in various biological mechanisms such as immune response, adhesion, infection, and signal transduction [2, 3]. In­vestigations on the alternative treatments applied by different cultures throughout the history revealed the fact that the utilized plants and fungi were rich in bioac­tive polysaccharides with proven immunomodulatory activity and health promoting effects in the treatment of inflammatory diseases and cancer. Hence considerable research has been directed on elucidating the biological activity mechanism of these polysaccharides by structure-function analysis [4].

Besides the interest on their applications in the health and bionanotechnology sectors, polysaccharides are also used as thickeners, bioadhesives, stabilizers, pro­biotic, and gelling agents in food and cosmetic industries [5-7] and as emulsifier, biosorbent, and bioflocculant in the environmental sector [8].

Polysaccharides are either extracted from biomass resources like algae andhigher — order plants or recovered from the fermentation broth of bacterial or fungal cultures. For sustainable and economical production of bioactive polysaccharides at industrial scale, rather than plants and algae, microbial sources are preferred since they en­able fast and high yielding production processes under fully controlled fermentation conditions. Microbial production is achieved within days and weeks as opposed to plants where production takes 3-6 months and highly suffers from geographical or seasonal variations and ever increasing concerns about the sustainable use of agricul­tural lands. Moreover, production is not only independent of solar energy which is indispensible for production from microalgae but also suitable for utilizing different organic resources as fermentation substrates [5].

According to recent reports, the global hydrocolloid market dominated by algal and plant polysaccharides like starch, galactomannans, pectin, carrageenan, and al­ginate is expected to reach 3.9 billion US dollars by 2012 [9]. Superseding these traditionally used plant and algal gums by their microbial counterparts requires in­novative approaches and considerable progress has been made in discovering and developing new microbial extracellular polysaccharides (exopolysaccharides, EPSs) that possess novel industrial significance [6, 7]. A recent review pointed out to four EPSs, namely, xanthan, pullulan, curdlan, and levan, as biopolymers with outstand­ing potential for various industrial sectors [5]. However, when compared with the synthetic polymers, natural origin polymers still represent only a small fraction of the current polymer market, mostly due to their costly production processes. Therefore, much effort has been devoted to the development of cost-effective and environmen­tally friendly production processes such as investigating the potential use of cheaper fermentation substrates.

In this chapter, after a brief description of microbial polysaccharides, various microbial production processes utilizing cheap biomass resources as fermenta­tion substrates are discussed with a special focus on the employed pretreatment methods.

To realize multiple products conversion from stalk with fractionation technique, a single technique is unavailable. At first, the single technique reported could not fractionate stalk into different parts. Dilute acid or alkali pretreatment could only remove lignin and hemicelluloses [24, 25]. Steam cooking and steam explosion just make hemicellulose hydrolyzed and whole stalk loosen [26]. Ionic liquid dissolves cellulose component [27]. Pyrolysis degrades each component into small molecule first [28], and then, small molecules are fractionated with petroleum refining equip­ment. Secondly, advanced biological, chemical, and physical processes would be integrated to convert different fractions into different products.

Therefore, it is necessary to integrate various refining and conversion technologies in stalk conversion process to realize fractionation oriented by multiple products.

MW pretreatment has a potential to induce stress reactions in plant systems or oil seeds by the rupture of their cell membranes. This results into high mass transfer coefficients, thus obtaining higher extraction yield. Cheng et al. [30] applied MW to the pretreatment of palm fruits prior to extraction of its oil. Applying MW for 3 min, an extraction yield comparable to the conventional and commercial palm oil milling process with an average of 20 % was achieved. The resulting palm oil also exhibits desirable and very low FFA content of about 0.26 % and moisture content of 0.05 %.

Moreover, most of the oil feedstocks for biodiesel syntheses contain relatively high amount of FFAs especially the waste cooking oil. This has become a big hurdle for industrialization of the proposed process, because the presence of fatty acids significantly affects the solubility of Ca-based or alkali catalysts in the products. Government quality standards for biodiesel require the level of Ca to be below 5 ppm, while the fatty acid content should not exceed 1 wt%. Thus, pretreatment of FFAs in oil is necessary prior to transesterification of the triglyceride components. In this regard, MW irradiation could also be applied to convert FFAs into biodiesel. Our previous studies showed that about 88 % conversion of FFAs in waste cooking oil could be obtained in 1 min of MW irradiation at a power of 700 W using ion exchange resin (Amberlyst 15) as catalysts [12] as shown in Fig. 6.3. With these results, a two-step process is proposed for the conversion of waste oil, or any other types of oil feedstock containing high amount of FFAs, to biodiesel fuel. The process consists of a first step of esterification of fatty acids followed by a second step of transesterification of the triglyceride. While the two-step process seems ideal for the treatment of FFAs in oil, this also minimizes the solubility of Ca-based catalysts as a result of the reduction of fatty acid contents.

Falkowski et al. [48] reported that lignin may accumulate in terrestrial ecosystems for decades, on longer time scales most of these molecules are oxidized, so that the accumulation of organic carbon in soils is a miniscule fraction of the total carbon fixed by the ecosystem. Lakes may also store substantial amounts of organic matter in sediments. Various microorganisms have been used by many researchers and this zero pollution approach has received good attention as it helps to enhance the fermentation and enzymatic saccharification rate without much capital investment.

Carob (Ceratonia siliqua L.), which has long been regarded as just a nitrogen-fixing tree grown in the Mediterranean region, has recently found its place in the food industry as a biomass substrate due to its very high sugar content [100]. Moreover, it has been established as a viable biomass resource for bioethanol production [101]. Carob extracts have also been used for microbial production of xanthan [52] and pullulan [44] polysaccharides. Roseiro et al. [52] developed a multistep pretreatment process for carob-based feedstocks that involves aqueous extraction of carob pulp followed by pressing. By recycling of press liquor, the final sugar content of the carob extract was improved however, as a result of esterasic activities, the syrup was found to contain increasing concentrations of isobutyric acid with time in a pH-dependent manner. Though accumulation of isobutyric acid could be controlled by an additional heat treatment step, its presence was found to inhibit the growth of X. campestris cells [52]. When carob extracts with 25g/L initial sugar content were used for pullulan production by a pigmented strain of A. pullulans (SU-M18), a pullulan productivity of 2.16 g/L/day could be reached at pH 6.5 and 25 °C [44]. For dextran production, carob pod residues obtained from the galactomannan industry were milled and the sugars were extracted at 70 °C by use of an acetate buffer. A dextran yield of 8.56 g/L could be reached by L. mesenteroides NRRL B512 cultures within 12 h of fermentation period [37].

Condensed corn solubles (CCS) is a by-product of bioethanol industry. While ethanol is separated from the fermentation broth via distillation, the remaining solids are first recovered by centrifugation and then concentrated using evaporators. The final product CCS contains changing levels of carbohydrates, proteins, vitamins, and nutrients [102]. CCS obtained from a dry-mill ethanol plant has been diluted and used for the cost-effective production of scleroglucan by S. glucanicum [51, 103]; however, the yields were lower than those of S. rolfsii cultures grown on sugarcane juice ormolasses [32], coconutwater [32], and waste loquat kernel [50]. In another study, CCS was diluted, neutralized, clarified by centrifugation and filtration, and then used for the poly-(3-hydroxybutyrate-co-3-hydroxyvalerate) production by Rhodospirillumrubrum cultures [102]. CCS from a wet-mill ethanol production plant has been clarified by centrifugation and then used as substrate for pullulan production by Aureobasidium sp. strain NRRL Y-12974 cultures and the yields (4.5 g/L in 9 days) were found to be comparable with those of soluble starch (5.4 g/L in 9 days), however, much lower than glucose containing medium (10.1 g/L in 9 days) [43]. CCS has also been used for curdlan production by Agrobacterium sp. ATCC 31749 shake flask cultures. A maximum curdlan yield of 7.72 g/L was recovered after 120 h of fermentation in media containing 400 g/L CSS [36].

Ram horn hydrolysates were also reported to be a suitable enhancer for xanthan production by X. campestris EBK-4 because of their high amino acid and mineral content. To obtain the hydrolysates, ram horns, which are usually discharged as waste in slaughterhouses, were subjected to acid hydrolysis with sulfuric acid followed by heat treatment at 130 °C. After neutralization, the hydrolysates were clarified by filtration and then added to the fermentation medium [58].

Waste loquat kernel is another potential biomass resource for EPS production due to its high protein and carbohydrate content. Waste kernels are dried and milled and then subjected to acid hydrolysis with 2 M HCl using an autoclave. Then the hydrolysates were detoxified with Ca(OH)2, neutralized, and then used for scleroglu — can production by S. rolfsii MT-6 [50] and EPS production by Morchella esculenta [104].

Other biomass residues used for microbial EPS production include corn-steep, spent grain and spent sulfite liquors, hydrolyzed potato starch, peach pulp, and peat hydrolysate [28, 30].

Size reduction is an important step in preparing biomass for pelletization. It is energy intensive. Depending on the original form of feedstock, there are one or two steps of size reduction necessary prior to pelletization. Woody biomass can be transported from the forest to the end user (e. g., power plant) in the chips form. They were processed from logs into chips. Logs have to be debarked, and the clean wood logs are size reduced to chip shape using a chipper. Wood chips with 25-50 mm length and width are usually further size reduced to 3.0-6.4 mm ground particles before compacting the material into pellets using either a hammer mill or a knife mill. Often chipping is carried out in the forest, and the chips are transported to the pellet plant where they are ground.

Size reduction is a sensitive step for controlling the ground particles size, dis­tribution is done by installing screen with different sizes in the mill. For the wood pellet industry, a hammer mill with 3.0-6.4 mm screen is mostly preferred for pellet production. A larger particle size (greater than 1 mm) will also act as predetermined breaking points in the pellet, and therefore the optimum particle size range between

0. 5 and 0.7 mm is sometimes suggested [28]. In reality, a mixture of different parti­cle size gives the optimum durability of the pellet as they have a better inter-particle bonding with less interspaces [29-31]. Therefore, there is a need to study the particle size distribution on the durability of the wood pellets.