Mohammad J. Taherzadeh

Pretreatment of Lignocellulosic Wastes to Improve Ethanol and Biogas Production: A Review

Lignocelluloses are often a major or sometimes the sole components of different waste streams from various industries, forestry, agriculture and municipalities. Hydrolysis of these materials is the first step for either digestion to biogas (methane) or fermentation to ethanol. However, enzymatic hydrolysis of lignocelluloses with no pretreatment is usually not so effective because of high stability of the materials to enzymatic or bacterial attacks. The present work is dedicated to reviewing the methods that have been studied for pretreatment of lignocellulosic wastes for conversion to ethanol or biogas. Effective parameters in pretreatment of lignocelluloses, such as crystallinity, accessible surface area, and protection by lignin and hemicellulose are described first. Then, several pretreatment methods are discussed and their effects on improvement in ethanol and/or biogas production are described. They include milling, irradiation, microwave, steam explosion, ammonia fiber explosion (AFEX), supercritical CO2 and its explosion, alkaline hydrolysis, liquid hot-water pretreatment, organosolv processes, wet oxidation, ozonolysis, dilute-and concentrated-acid hydrolyses, and biological pretreatments.

Inhibition effects of furfural on alcohol dehydrogenase, aldehyde dehydrogenase and pyruvate dehydrogenase

The kinetics of furfural inhibition of the enzymes alcohol dehydrogenase (ADH; EC 1.1.1.1), aldehyde dehydrogenase (AlDH; EC 1.2.1.5) and the pyruvate dehydrogenase (PDH) complex were studied in vitro. At a concentration of less than 2 mM furfural was found to decrease the activity of both PDH and AlDH by more than 90%, whereas the ADH activity decreased by less than 20% at the same concentration. Furfural inhibition of ADH and AlDH activities could be described well by a competitive inhibition model, whereas the inhibition of PDH was best described as non-competitive. The estimated K(m) value of AlDH for furfural was found to be about 5 microM, which was lower than that for acetaldehyde (10 microM). For ADH, however, the estimated K(m) value for furfural (1.2 mM) was higher than that for acetaldehyde (0.4 mM). The inhibition of the three enzymes by 5-hydroxymethylfurfural (HMF) was also measured. The inhibition caused by HMF of ADH was very similar to that caused by furfural. However, HMF did not inhibit either AlDH or PDH as severely as furfural. The inhibition effects on the three enzymes could well explain previously reported in vivo effects caused by furfural and HMF on the overall metabolism of Saccharomyces cerevisiae, suggesting a critical role of these enzymes in the observed inhibition.

Physiological effects of 5-hydroxymethylfurfural on Saccharomyces cerevisiae

Abstract The physiological effects of 5-hydroxymethylfurfural (HMF) on Saccharomyces cerevisiae CBS 8066 in the presence and absence of furfural were studied. Experiments were carried out by pulse addition of HMF (2–4 g/l) as well as HMF (2 g/l) together with furfural (2 g/l) to batch cultivations of S. cerevisiae. Synthetic medium with glucose (50 g/l) as carbon and energy source was used. Addition of 4 g/l of HMF caused a decrease (approx. 32%) in the carbon dioxide evolution rate. Furthermore, the HMF was found to be taken up and converted by the yeast with a specific uptake rate of 0.14 (±0.03) g/g · h during both aerobic and anaerobic conditions, and the main conversion product was found to be 5-hydroxymethylfurfuryl alcohol. A previously unreported compound was found and characterized by mass spectrometry. It is suggested that the compound is formed from pyruvate and HMF in a reaction possibly catalysed by pyruvate decarboxylase. When HMF was added together with furfural, very little conversion of HMF took place until all of the furfural had been converted. Furthermore, the conversion rates of both furfural and HMF were lower than when added separately and growth was completely inhibited as long as both furfural and HMF were present in the medium.

Acetic acid-friend or foe in anaerobic batch conversion of glucose to ethanol by Saccharomyces cerevisiae?

The permissible region of growth of Saccharomyces cerevisiae on glucose under anaerobic conditions was determined as a function of both pH and the concentration of added acetic acid to the medium. In the absence of acetic acid, growth was possible at a pH as low as 2.5, whereas a total acetic acid addition of 10 gl−1 increased the minimum allowable pH for growth to 4.5. The results showed that the concentration of the undissociated form of acetic acid should not exceed 5 gl−1 in the medium for growth to occur. The addition of acetic acid had a profound effect on growth energetics, thereby leading to an increased ethanol yield on glucose. At a concentration of 3.3 gl−1 of undissociated acetic acid, the ethanol yield was 20% higher than without added acetic acid. Furthermore, the biomass and glycerol yields decreased by 45 and 33%, respectively.

Conversion of furfural in aerobic and anaerobic batch fermentation of glucose by Saccharomyces cerevisiae.

The effect of furfural on aerobic and anaerobic batch cultures of Saccharomyces cerevisiae CBS 8066 growing on glucose was investigated. Furfural was found to decrease both the specific growth rate and ethanol production rate after pulse additions in both anaerobic and aerobic batch cultures. The specific growth rate remained low until the furfural had been completely consumed, and then increased somewhat, but not to the initial value. The CO(2) evolution rate decreased to about 35% of the value before the addition of 4 g x l(-1) furfural, in both aerobic and anaerobic fermentations. The decrease of the CO(2) evolution rate was rapid at first, and then a more gradual decrease was observed. The furfural was converted mainly to furfuryl alcohol, with a specific conversion rate of 0.6 (+/-0.03) g (furfural) x g(-1) (biomass) x h(-1) by exponentially growing cells. However, the conversion rate of furfural by cells in the stationary phase was much lower. A previously unidentified compound was detected during the conversion of furfural. This compound was characterized by mass spectrometry and it is suggested that it is formed from furfural and pyruvate.

The effects of pantothenate deficiency and acetate addition on anaerobic batch fermentation of glucose by Saccharomyces cerevisiae

Abstract Physiological effects of deficiency of pantothenate, a necessary precursor in the synthesis of coenzyme A, were studied using the yeast strain Saccharomyces cerevisiae CBS 8066. Cells were grown on defined media in anaerobic batch cultures with glucose (50 g/l) as the carbon and energy source. Batch cultures containing more than 60 μg/l pantothenate showed no significant differences with respect to growth rates and product yields. However, with an initial pantothenate concentration of 30 μg/l, the average glucose consumption rate was 50% lower than in rich medium and, at even lower concentrations of pantothenate, the culture did not consume all the glucose in the medium. Furthermore, pantothenate deficiency caused the acetate and pyruvate yields to increase and the biomass yield to decrease, compared to the yields in pantothenate-rich medium. The increased acetate formation could be counteracted by initial addition of acetate to the medium, and thereby the glycerol yield could be decreased. An initial addition of acetate of 1.6 g/l to pantothenate-deficient medium (30 μg/l) caused a 35% decrease in glycerol yield and a 6% increase in ethanol yield. Furthermore, the time required for complete conversion of the glucose decreased by 40%. Acetate addition affected the acetate and glycerol yields in a similar way in pantothenate-rich medium (1000 μg/l) also.

Production of biofuels, limonene and pectin from citrus wastes.

Production of ethanol, biogas, pectin and limonene from citrus wastes (CWs) by an integrated process was investigated. CWs were hydrolyzed by dilute-acid process in a pilot plant reactor equipped with an explosive drainage. Hydrolysis variables including temperature and residence time were optimized by applying a central composite rotatable experimental design (CCRD). The best sugar yield (0.41g/g of the total dry CWs) was obtained by dilute-acid hydrolysis at 150 degrees C and 6min residence time. At this condition, high solubilization of pectin present in the CWs was obtained, and 77.6% of total pectin content of CWs could be recovered by solvent recovery. Degree of esterification and ash content of produced pectin were 63.7% and 4.23%, respectively. In addition, the limonene of the CWs was effectively removed through flashing of the hydrolyzates into an expansion tank. The sugars present in the hydrolyzates were converted to ethanol using bakers yeast, while an ethanol yield of 0.43g/g of the fermentable sugars was obtained. Then, the stillage and the remaining solid materials of the hydrolyzed CWs were anaerobically digested to obtain biogas. In summary, one ton of CWs with 20% dry weight resulted in 39.64l ethanol, 45m(3) methane, 8.9l limonene, and 38.8kg pectin.

Effects of furfural on the respiratory metabolism of Saccharomyces cerevisiae in glucose-limited chemostats,

ABSTRACT Effects of furfural on the aerobic metabolism of the yeast Saccharomyces cerevisiae were studied by performing chemostat experiments, and the kinetics of furfural conversion was analyzed by performing dynamic experiments. Furfural, an important inhibitor present in lignocellulosic hydrolysates, was shown to have an inhibitory effect on yeast cells growing respiratively which was much greater than the inhibitory effect previously observed for anaerobically growing yeast cells. The residual furfural concentration in the bioreactor was close to zero at all steady states obtained, and it was found that furfural was exclusively converted to furoic acid during respiratory growth. A metabolic flux analysis showed that furfural affected fluxes involved in energy metabolism. There was a 50% increase in the specific respiratory activity at the highest steady-state furfural conversion rate. Higher furfural conversion rates, obtained during pulse additions of furfural, resulted in respirofermentative metabolism, a decrease in the biomass yield, and formation of furfuryl alcohol in addition to furoic acid. Under anaerobic conditions, reduction of furfural partially replaced glycerol formation as a way to regenerate NAD+. At concentrations above the inlet concentration of furfural, which resulted in complete replacement of glycerol formation by furfuryl alcohol production, washout occurred. Similarly, when the maximum rate of oxidative conversion of furfural to furoic acid was exceeded aerobically, washout occurred. Thus, during both aerobic growth and anaerobic growth, the ability to tolerate furfural appears to be directly coupled to the ability to convert furfural to less inhibitory compounds.

A critical review of analytical methods in pretreatment of lignocelluloses: Composition, imaging, and crystallinity.

Lignocelluloses are widely investigated as renewable substrates to produce biofuels, e.g., ethanol, methane, hydrogen, and butanol, as well as chemicals such as citric acid, lactic acid, and xanthan gum. However, lignocelluloses have a recalcitrance structure to resist microbial and enzymatic attacks; therefore, many physical, thermal, chemical, and biological pretreatment methods have been developed to open up their structure. The efficiency of these pretreatments was studied using a variety of analytical methods that address their image, composition, crystallinity, degree of polymerization, enzyme adsorption/desorption, and accessibility. This paper presents a critical review of the first three categories of these methods as well as their constraints in various applications. The advantages, drawbacks, approaches, practical details, and some points that should be considered in the experimental methods to reach reliable and promising conclusions are also discussed.

Strategies for enhancing fermentative production of glycerol - A review

The present paper reviews the metabolic basis of different methods for fermentative glycerol production. The most important microbial production organism is the yeast Saccharomyces cerevisiae but other yeast species, as well as molds, algae, and bacteria are of potential interest for glycerol production. A large variety of methods have been applied to increase the fermentative glycerol yield. The first methods were based on physiological control, e.g. chemically induced overproduction of glycerol through NADH entrapment by the addition of chemical steering agents (such as bisulfite). More recently, genetic engineering of the glycolytic pathway has been used to improve production, involving modulated function of e.g. triose phosphate isomerase, phosphoglycerate mutase, PDC or alcohol dehydrogenase. Direct intervention in the glycerol pathway, such as overexpression of G3P dehydrogenase, has also been tried. The applied strategies can be divided into three principal groups; (a) deactivation or down-regulation of NADH oxidation sites alternative to G3P dehydrogenase, (b) increase of NADH generation or, (c) direct changes in the carbon flux to glycerol.