Unstable oil markets with rising environmental concerns have revived widespread interest in production of fuel ethanol from renewable materials. Cellulosic materials are abundant and prominent feedstocks for cheap ethanol production. However, due to recalcitrant structure of these materials, pretreatment is a prerequisite. Depending on the biomass, pretreatment and hydrolysis conditions, a number of degradation products and/or toxic components may be released that show strong inhibitory effects on the fermenting microorganisms. This thesis deals with application of encapsulation technology to ferment the highly toxic hydrolyzates without further pretreatment. Free cells could not tolerate presence of 5 g/l furfural in defined medium, and inhibitors in wood and peel hydrolyzates in batch mode of operation and fermentation failed. Continuous cultivation of wood hydrolyzate was only successful at 0.1 h−1 and the majority of cells lost their viability after 5 retention times. Encapsulated cell system could successfully ferment the synthetic medium containing 5 g/l furfural during sequential batch cultivations with ethanol yield of 0.41-0.42 g/g. Cultivation of undetoxified hydrolyzates was also carried out, where glucose and mannose were converted within 10 h without significant lag phase. However, a gradual decrease in cell activity was observed in sequential batches. Continuous cultivation was more successful, and wood hydrolyzate was fermented to ethanol by encapsulated S. cerevisiae at dilution rates up to 0.5 h−1. More than 75% of the encapsulated cells were viable in the worst conditions. Ethanol was produced with yield 0.44 g/g and specific productivity 0.14–0.17 g/g•h at all dilution rates. Contrary to wood hydrolyzate, where there is no preference for permeation of sugars or inhibitors through the capsules’ membrane, encapsulation technology was applied to eliminate inhibition of limonene in fermentation of orange wastes to ethanol. The capsules’ membrane, of hydrophilic nature, is practically impermeable to hydrophobic compounds such as limonene while allowing penetration of nutrients and products. While presence of 0.1% v/v limonene in the medium results in strong inhibition or even failure of cultivation with free cells, using this technique allowed fermentation of a medium containing 1.5% v/v limonene. The impact of encapsulation on the anaerobic growth pattern, morphological and physiological changes of S. cerevisiae over long-term application was investigated. The growth rate, total RNA and protein content of the encapsulated cells decreased gradually over repeated batch cultivations, while stored carbohydrates content increased. Within 20 batch cultivations, total RNA and protein content of encapsulated cells decreased by 39% and 24%, whereas glycogen and trehalose content increased by factors of 4.5 and 4, respectively.

Optimization study of citrus wastes Saccharification by dilute acid hydrolysis

Optimization study of citrus wastes Saccharification by dilute acid hydrolysis

The effects of time, acid concentration, temperature and solid concentration on dilute-acid hydrolysis of orange peels were investigated. A central composite rotatable experimental design (CCRD) was applied to study the individual effects of these hydrolysis factors and also their interdependence effects. The enzymatic hydrolysis of the peels by cellulase, beta-glucosidase, and pectinase enzymes resulted in 72% dissolution of the peels, including 18.7% galacturonic acid and 53.3% of a total of glucose, fructose, galactose, and arabinose. Dilute-acid hydrolysis up to 210 C was not able to hydrolyze pectin to galacturonic acid. However, the sugar polymers were hydrolyzed at relatively low temperature. The optimum results were obtained at 116 degrees C, 0.5% sulfuric acid concentration, 6% solid fraction, and 12.9 min retention time. Under these conditions, the total sugars obtained at 41.8% dry peels and 2.6% of total hexose sugars were further degraded to hydroxymethylfurfural (HMF). No furfural was detected through these experiments from decomposition of pentoses.

The impact of encapsulation on the anaerobic growth pattern of S. cerevisiae CBS 8066 in a defined synthetic medium over 20 consecutive batch cultivations was investigated. In this period, the ethanol yield increased from 0.43 to 0.46 g/g, while the biomass and glycerol yields decreased by 58 and 23%, respectively. The growth rate of the encapsulated cells in the first batch was 0.13 h-1, but decreased gradually to 0.01 h-1 within the 20 sequential batch cultivations. Total RNA content of these yeast cells decreased by 39% from 90.3 to 55 mg/g, while the total protein content decreased 24% from 460 to 350 mg/g. On the other hand, the stored carbohydrates, i.e. glycogen and trehalose content, increased by factors of 4 and 4.5 within 20 batch cultivations, respectively. Higher biomass concentrations inside capsules led to a lower glucose diffusion rate through the membrane, and volumetric mass transfer coefficient for glucose was drastically decreased from 6.28 to 1.24 (cm3/min) by continuing the experiments. Most of the encapsulated yeast existed in the form of single and non-budding cells after long-term application.

This work deals with application of encapsulation technology to eliminate inhibition of D-limonene in fermentation of orange wastes to ethanol. Orange peel was enzymatically hydrolyzed with cellulase and pectinase. However fermentation of the released sugars in this hydrolyzate by freely suspended S. cerevisiae failed due to inhibition of limonene. On the other hand, encapsulation of S. cerevisiae in alginate membranes was a powerful tool to eliminate inhibition of limonene. The encapsulated cells were able to ferment the orange peel hydrolyzate in 7 h, and produce ethanol with yield 0.44 g/g fermentable sugars. Cultivation of the encapsulated yeast in defined medium was successful, even in the presence of 1.5% (v/v) limonene. The capsules’ membranes were selectively permeable to the sugars and the other nutrients, but not limonene. While 1% (v/v) limonene was present in the culture, its concentration inside the capsules was not more than 0.054% (v/v).