Hydrolysis of Carbohydrate Polymers

The hydrolysis of glucans is a significant source of fermentable sugars for fuel ethanol production. The most important glucans in ethanol industry are starch and cellulose. In this chapter, the features of starch and cellulose hydrolyses are discussed. In particular, the difficulties related to the enzymatic hydrolysis of cellulose are analyzed taking into consideration the enzyme complexes used and the presence of solid particles in the reaction mixture. Some process engineering aspects emphasizing the kinetic modeling of these processes are also discussed.

5.1 STARCH SACCHARIFICATION

In Chapter 4, the use of a-amylase during starch liquefaction was mentioned. It is worth emphasizing here some general issues related to the enzymatic hydrolysis of starch. The utilization of enzymes to break down the starch has some advantages over the hydrolysis using acids. In the latter case, strong conditions are required to achieve the degradation of starch (150°C, pH of 1.5 to 1.8). The amylolytic enzymes work under milder conditions (temperatures lower than 110°C, neutral pH) with the corresponding energy savings. In addition, the enzymatic process does not generate compounds resulting from degradation or oxidation of sugars due to the very high specificity of the enzymes (Lopez-Munguia, 1993). For these reasons, the sweeteners industry does not use the acid hydrolysis of starch. This process has been taken over by the fuel ethanol industry in order to obtain the fermentable sugars needed for yeast cultivation.

Main amylases employed for starch hydrolysis at the industrial level are from bacterial and fungal origin though some plant enzymes are eventually used (Table 5.1). The enzyme a-amylase obtained from thermophilic bacteria or produced by a recombinant microorganism using the gene obtained from a thermophilic organism is one of the most used amylases. This enzyme randomly hydrolyzes the a(1,4) glycosidic bonds within the chains of both amylose and amylopectin. For this enzyme to attack the starch, the previous gelatinization of starch should have been carried out. Thus, the broken starch granules release the amylose and amylopectin and the enzymatic action can be started. The a-amylase can support high temperatures of up to 110°C while keeping its activity, thus it is ideal for starch liquefaction process. The molecular weights and optimum temperatures of enzymatic activity of the a-amylases most commonly employed for starch processing are shown in Table 5.2. Apar and Ozbek (2004) provide information about the effects of operating conditions on the enzymatic hydroly­sis of corn starch using commercial a-amylase. In general, the optimum pH of

TABLE 5.1 Main enzymes

used for starch Hydrolysis

Bond

enzymes

source

hydrolyzed

Products

Mechanism

a-amylase

Bacillus licheniformis B. subtilis

a(1,4)

Dextrines,

maltodextrines,

maltose

Endoenzyme

p-amylase

Asergillus oryzae Bacillus cereus Barley

a(1,4) from nonreducing ends

Dextrines,

maltose

Exoenzyme

Glucoamylase

(amyloglucosidase)

A. niger 1 Rhizopus sp.

a(1,4) from nonreducing ends and a(1,6)

Glucose

Exoenzyme

Pullulanase

B. acidopullulyticus Klebsiella pneumoniae

a(1,6)

Maltodextrines

Endoenzyme

Origin and Properties of Different a-Amylases and Glucoamylases

TABLE 5.2

Microbial source

Molecular

Weight

Optimum

Temperature/°C

Optimum/pH

References

a-amylases

Bacillus subtilis

54,780

80

5.6

Nigam and Singh

(1995); Pandey et

Bacillus

49,000

70

al. (2000a)

amyloliquefaciens

Bacillus licheniformis

62,000

90

6.0-6.5

Glucoamylases

Aspergillus awamori

83,700-88,000

60

4.5

Nigam and Singh (1995); Pandey et

Aspergillus niger I

99,000

60

4.5-5.0

al. (2000a)

Aspergillus niger II

112,000

60

4.4

Aspergillus oryzae I

76,000

60

4.5

Aspergillus oryzae II

38,000

50

4.5

Aspergillus oryzae III

38,000

40

4.5

Aspergillus saitoi Cephalosporium

90,000

26,850

45-62

4.5

eichhormonie

Lipomyces

811,500

50

kononenkoae

Mucor rouxianus I

59,000

55

4.7

Mucor rouxianus II

49,000

55

4.7

Penicillium oxalicum I

84,000

55-60

4.6

Penicillium oxalicum II

86,000

60

4.6

Rhizophus delemar

100,000

40

4.5

the a-amylase is about 6. For this reason, the pH of the cooked starch should be adjusted before the enzyme addition during the liquefaction process.

Glucoamylase (amyloglucosidase) is the other most employed enzyme in starch — to-ethanol process. This enzyme is generally obtained from Aspergillus niger or a species of Rhizopus genus (Labeille et al., 1997; Nigam and Singh, 1995; Shigechi et al., 2004). The glucoamylase is an exo-enzyme that hydrolyzes the a(1,4) bonds from the nonreducing ends of amylose or amylopectin chains forming glucose. Unlike a-amylase, most glucoamylases have the ability to hydrolyze the a(1,6) bonds in branching points of the amylopectin, though the hydrolysis rate of this bond is 15 times lower than for the a(1,4) bonds (Lopez-Munguia, 1993; Pandey et al., 2000b). This feature allows this enzyme to convert the dextrins formed dur­ing the liquefaction step into glucose. The optimum temperatures of enzymatic activity for glucoamylases are lower than those of a-amylases employed during starch liquefaction (see Table 5.2). Consequently, the cooling of liquefied starch is needed to ensure an appropriate conversion toward glucose. In some commercial formulations of amylases, the enzyme pullulanase, which specifically hydrolyzes the a(1,6) bonds, is also employed. Finally, the P-amylase is mostly employed in the brewing industry and for production of maltose syrups.

The process of hydrolysis (or saccharification) of the stream exiting the lique­faction tank is aimed at obtaining a glucose-rich solution for its later fermenta­tion. This stream is adjusted at a pH of 4.5 and cooled down to 65°C in order to ensure the optimum conditions of the hydrolysis process (Bothast and Schlicher, 2005). The saccharification product is called corn mash if this cereal is the feed­stock employed or saccharified starch if starch is the feedstock employed, as in the case of wet milling.

The steps related to the starch degradation are responsible for 10 to 20% of the energy consumption of the ethanol process in the case of fuel ethanol produced from starchy materials. One potential option to minimize this high amount of energy is the substitution of enzymatic hydrolysis technologies in liquid media at high temperatures with technologies involving the starch hydrolysis using amy­lases working at low temperatures in solid phase. This approach would make pos­sible the “cold hydrolysis” of the native starch (Cardona and Sanchez, 2007). For this, the discovery and characterization of new enzymes displaying these prop­erties are required (Robertson et al., 2006). Some microorganisms, such as the bacterium Clostridium thermohydrosulfuricum, have the ability to digest nonge — latinized starch and convert it into ethanol at 66°C. However, the productivities attained are too low (Mori and Inaba, 1990).

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