**10. Supercritical fluid extraction in bioprocess technology**

Recent investigations on the applications of SCE from post fermentation biomass or *in situ*  extraction of inhibitory fermentation products as a promising method for increasing yield are reviewed (Khosravi-Darani and Vasheghani-Farahani 2005). Although SC-CO2 is unfriendly and toxic, for some living cells, which precludes direct fermentation in dense CO2, it does not rule out other useful applications for *in situ* extraction of inhibitory fermentation products and fractional extraction of biomass constituents due to the potential of system modification by physical parameters and addition of co-solvents to selectively extract compounds of varying polarity, volatility and hydrophilicity with no contamination.

#### **10.1 Advantages and disadvantages of SCE especially for the biotechnology industries**

The advantages of utilizing SCE have been well documented (Schultz et al., 1991). The application of SCF is simple, inexpensive, non- injurious to the structure and function of some enzymes (Lin et al., 1992) and protein activities (Juhasz et al., 2003; Kamat et al., 1995; Zheng and Tsao, 1996). Nowadays, SCE is a well-known unit operation, with some industrial as well as many lab and pilot scale applications. Introduction of SC-CO2 to fermentation broth decreases the overall viscosity, facilitates the handling of the broth and enhances mass transfer from the liquid to the SC-phase. Randolph has summarized special advantages of SCE, especially for the biotechnology industries (1990):


Reverchon 1999; Subramanian et al. 1997), such as microspheres (Falk et al. 1997) threads, fibers, networks (Dixon and Johnstone 1993), sponges, foams, and films. One of the advantages of using SCF in polymer processing is the possibility of producing different solid shapes and structures at low temperature with a minimum amount of residual organic solvents. Also the process is environmentally safe and economic (Elvassore et al. 2001). A

The conformation of monomeric enzyme trypsin has been reported in SC-CO2 (Zagrobelny and Bright 1992). To follow in situ conformation of trypsin (as a function of CO2 density), steady state fluorescence spectroscopy was used. Zagrobelny showed that protein denaturation can occur during the fluid compression step and that the native trypsin is only

Conventional purification methods are not specific and must be repeated or combined for highly purification. Although, (immuno) affinity-based procedures are rapid and specific; but they are expensive, and reagents from biological origin are needed. Also the interactions involved between the product and the support are often strong and imply the use of rather denaturing reagents (either for the product or the support) to attain an efficient desorption yield (Lemay 2002). SCE has introduced as a more suitable method for purification of natural products. This technique helps to remove trace impurities in the synthetic active

Recent investigations on the applications of SCE from post fermentation biomass or *in situ*  extraction of inhibitory fermentation products as a promising method for increasing yield are reviewed (Khosravi-Darani and Vasheghani-Farahani 2005). Although SC-CO2 is unfriendly and toxic, for some living cells, which precludes direct fermentation in dense CO2, it does not rule out other useful applications for *in situ* extraction of inhibitory fermentation products and fractional extraction of biomass constituents due to the potential of system modification by physical parameters and addition of co-solvents to selectively extract compounds of varying polarity, volatility and hydrophilicity with no contamination.

biocopolymers from maleic anhydride and pinene (Jarzebski and Malinowski 1995).

**10.1 Advantages and disadvantages of SCE especially for the biotechnology** 

• High diffusivity reduces mass transfer limitations from porous solid matrices • Low surface tension allows penetration and wetting of pores to extract from cell • selectivity of extraction due to sensitivity of solubility to changes in P and T

The advantages of utilizing SCE have been well documented (Schultz et al., 1991). The application of SCF is simple, inexpensive, non- injurious to the structure and function of some enzymes (Lin et al., 1992) and protein activities (Juhasz et al., 2003; Kamat et al., 1995; Zheng and Tsao, 1996). Nowadays, SCE is a well-known unit operation, with some industrial as well as many lab and pilot scale applications. Introduction of SC-CO2 to fermentation broth decreases the overall viscosity, facilitates the handling of the broth and enhances mass transfer from the liquid to the SC-phase. Randolph has summarized special

**10. Supercritical fluid extraction in bioprocess technology** 

advantages of SCE, especially for the biotechnology industries (1990):

basic description of these techniques is reported by Bertucco and Pallado (2000).

slightly more stable (1.2 kcal/mol) than the unfolded form.

**9.6 Purification of natural active copolymers** 

**industries** 


The main disadvantages of SCE processes include low solubility of biomolecules in SCF and high capital costs. Furthermore, insufficient data exist on the physical properties of many bio-molecules, making prediction of phase behavior difficult. The addition of co-solvents may obviate the advantage of minimal solvent residues in the final product.

#### **10.2 Supercritical extraction (SFE) from biomass 10.2.1 Post fermentation extraction of products**

There are only a few reports using SFE on bacterial cell. SCFs are found to be useful in extracting desired materials from animal tissues, cells, and organs (Kamarei and Arlington, 1988). By varying the choice of SCF, experimental conditions, and biological source materials, one may obtain lipids, proteins, nucleotides, saccharides, and other desirable components or remove undesirable components (Kamarei and Arlington, 1988). Processing of lipid natural products by SCF has been reviewed (King, 2004). SCF can be applied for obtaining aromatic and lipid components from plant tissues (Kamarei and Arlington, 1988), lignin conversion (Avedesian, 1986), carotenoids extraction from carrots (Bath et al., 1995), tomato paste waste (Baysal et al., 2000) and microalgae (Mendes et al., 1995). The CO2 extraction process is selective in the presence of chlorophyll a.

Moreover, there are some reports which describe the SFE of bacterial (Gharaibeh and Voorhees, 1996) and fungal lipids (Cygnarowicz et al., 1992) for use in the classification of them by fatty acid profiles. A simple two–step process was developed to extract and purify medium chain length polyhydroxyalkanoates (MCL-PHA) from bacterial cells (*Pseudomonas resinovorans*) grown on lard and tallow (Hampson and Ashby, 1999). The process consists of SCE of the lyophilized cells with CO2 to remove lipid impurities, followed by chloroform extraction of the cells to recover the MCL–PHA. SFE conditions were varied as to T 40 – 100°C, P (13.78 – 62.05 MPa), and CO2 flow rate (0.5 – 1.5 L/min, expanded gas). The results show that the two- step process saves time, uses much less organic solvent, and produces a purer MCL-PHA biopolymer than previous extraction and purification methods. Khosravi-Darani et al. (2003) have reported the equilibrium solubility of poly(hydroxybutyrate) (PHB) in SC-CO2. The effects of the main parameters such as P, T, and solvent density on solubility were determined at different T (35 – 75°C) and P (12.2 – 35.5) MPa. Hejazi et al. (2003) reported the effects of process variables such as exposure time, P, T, volume of methanol as a modifier, and culture history on PHB recovery from suspended *R. eutropha* in buffer solution. In another report, Khosravi-Darani et al. extended this work to obtain maximum recovery with minimum energy consumption (2004). In this work PHB recovery was examined using a combination of supercritical disruption and chemical (salt and alkaline) pretreatments. Bacterial cells, treated in growth phase, exhibited less resistance to disruption than nutrient limited cells in the stationary phase. It was also found that the wet cells could be utilized to recover PHB, but purity of the product was lower than that obtained from freeze-dried cells. Pretreatment with a minimum of 0.4% wt NaOH was necessary to enable complete disruption with two repetitions of P release. Salt pretreatment was less effective; however, disruption was improved by the application of alkaline shock.

The use of SCE of biologically active compounds (chaetoglobosin A, mycolutein, luteoreticulin, 7,8–dihydro–7,8–epoxy–1–hydroxy–3–hydroxymethylxanthone–8–carboxylic

Supercritical Fluid Application in Food and Bioprocess Technology 567

phase at saturation levels. The disadvantage of liquid-liquid extraction is the residual of toxic solvent, which presents significant separation, purification, and environmental challenges (Job et al., 1989). Also membrane fermentation and adsorption vacuum fermentation are not cost-effective. Guvenc et al. (1998) demonstrated the feasibility of ethanol extraction from a post–fermentation broth using SC-CO2. However, application of SC-CO2 for *in situ* extractive fermentation has been limited by its inhibitory effect on the metabolism of a variety of yeasts and bacteria (Isenschmid et al., 1995; Van Eijs et al., 1988). This toxicity is attributed, in part, to the acidic pH (Toews et al., 1995) that results from the increased solubility of CO2 at high partial Ps (Knutson et al., 1999). By buffering the medium and carefully controlling the compression and expansion conditions, the survival rate of cells increases. Van Eijs et al. developed an extraction procedure in which the *Lactobacillus* 

The impact of dense gases and SCF (N2, CO2, and ethane) on the carbohydrate consumption and ethanol formation by *Clostridium thermocellum* has been reported. Non–growing cells capable of metabolism were incubated at 60°C with cellobiose as a substrate in the presence of the three pressurized fluids. The rate and extent of ethanol production were similar in cell suspensions maintained at atmospheric and 6.9 MPa P under nitrogen (conventional method). Ethane at 6.9 MPa reduced the extent of ethanol production by less than 20% relative to the atmospheric control, whereas CO2 at the same P reduced ethanol formation. The results suggest that pressurized hydrocarbons have benefits over SC-CO2 for the *in situ* 

*In situ* extraction of acetone, butanol and ethanol from synthetic media, simulating the downstream processing of a *Clostridium acetobutylicum* fermentation broth has been described (Van Eijs et al., 1988). It was also observed that extraction yield is a close function of the extraction time. Also increased P helps to achieve higher yields (Guvenc et al., 1998). The extractive fermentation of 2-phenylethyl alcohol, the rose aroma, coupling fermentation with *Kluyveromyces marxianus* and SC-CO2 extraction has been reported (Fabre et al., 1999). Similar results show enhancement of 2-phenylethanol productivity by *Saccharomyces cerevisiae* in two-phase fed batch fermentation using solvent immobilization (Serp et al., 2003). Stark and coworkers reported the extractive bioconversion of 2-phenylethanol by *Saccharomyces cerevisiae* (2002). It has further been reported that furfural, a growth inhibitory byproduct, was successfully removed during fermentation of *clostridium* on sugars by

Selection of biocompatible solvents is critical when designing bio-processing applications for the *in situ* biphasic extraction of metabolic end-products. The prediction of the biocompatibility of supercritical and compressed solvents is more complicated than that of liquid solvents, because their properties can change significantly with P and T. The activity of the anaerobic thermophilic bacterium, *Clostridium thermocellum*, was studied when the organism was incubated in the presence of compressed nitrogen, ethane, and propane at 333

SC and near critical fluids are used to fractionate biomass materials such as microbial cells in two steps. In the first step, the biomass is exposed to elevated pressure SC or near critical fluid to bring about disruption of the biomass to liberate structural biomass constituents. In the second step, the disrupted biomass is subjected to a multiplicity of SC or near critical fluid extraction steps, with different solvation conditions used for each fraction. Thus,

*plantarum* cell death was minimized (Van Eijs et al., 1988).

recovery of volatile microbial products (Knutson et al., 1999).

introducing liquefied CO2 at room T and 5.9 MPa (Sako et al., 1992).

K and multiple pressure (Jason et al., 2000)

**10.2.3 Fractionation of cellular biomass** 

acid methyl ester, sydowinin B and elaiophylin) from the biomass has been compared with organic solvents extraction (methanol and dichloromethane). The extraction strength of SC-CO2 alone appeared to be lower than that of dichloromethane. All the components of interest that were extractable with dichloromethane and methanol were also extractable with methanol-modified CO2 (Cocks et al., 1995). A technique for the SC-CO2 extraction of the fungal metabolite ergostrol in its free (non-conjugated) form was developed and applied to samples of flour moldy bread and mushrooms. The overall method showed an 83% recovery of free ergostrol for spiked bread flour (Young and Games, 1993).

Citric acid has successfully been separated from fermentation broth by a novel and unique purification process, which is characterized by organic solvent extraction and precipitation with compressed CO2 as a poor solvent. Compressed CO2 was then dissolved in acetone solution of crude citric acid to remove the residual impurities as precipitates using the antisolvent effect of CO2. Citric acid crystals could be obtained by the anti-solvent crystallization with CO2 (Shishikura et al., 1992). Dry mouldy bran resulting from solid state fermentation of *Gibberella fujikuroi* were subjected to SCE. The extraction of the sterol by SCE was found to improve with the use of ethanol as entrainer. The solid material retained the gibberellic acid activity without any loss (Kumar et al., 1991). The solubility of cholesterol in SCFs have also been studied and the solubility is correlated by using equation of states (Hartono et al., 2001).

Extraction of ethanol from aqueous phase of a yeast fermentation broth has been described and a lower energy cost as compared to distillation has been reported (De Filippi and Moses, 1983). Shimshick reported the extraction of carboxylic acids from dilute aqueous media with SC-CO2. The specific advantage of this application is the pH decrease of the aqueous phase, which results in a higher concentration of the free acids. This shift is necessary for effective extraction of the carboxylic acids (Shimshick, 1981). SC-CO2 extraction has been reported to be more suitable for extraction of non-polar compounds with molecular weights less than 400. Griseofulvin is an antifungal antibiotic having a molecular weight of 353, making it amenable to SC-CO2 extraction. The optimized conditions for SCE of griseofulvin from dried media after solid state fermentation were obtained (Saykhedkar and Singhal, 2004). Furthermore, SCF has been developed mainly for unit operation to recover intracellular enzymes, recombinant-DNA proteins and nucleic acids from microbial cell cultures (Khosravi-Darani, 2005, Castor and Hong, 1995).

#### **10.2.2 In situ extraction from the biomass of microbial fermentation**

*In situ* product removal is the fast removal of product from a producing cell thereby preventing its subsequent interference with cellular or medium components. Freeman and coworkers indicated future directions including application in situ extraction to a wider range of products and the developed methodologies, applicable under sterile conditions in the immediate vicinity of the producing cells (Freeman et al., 1993). End-product inhibition occurs in many fermentation processes and *in situ* removal of them typically enhances product formation rates, yields, and specificity (Christen et al., 1990; Gyamerah and Glover, 1996; Qureshi et al., 1998). Techniques that have been employed for *in situ* removal of fermentation products include liquid-liquid extractive fermentation (Adrian et al., 2000), use of selective membranes (Chang et al., 1992), cell recycling (Roca and Olsson, 2003), adsorption (Millitzer et al., 2002), microcapsule application (Stark et al., 2003) and vacuum fermentation (Qureshi et al., 1998). However, the intimate contact of an organic phase with the broth implies that the organic components of this phase may be present in the aqueous

acid methyl ester, sydowinin B and elaiophylin) from the biomass has been compared with organic solvents extraction (methanol and dichloromethane). The extraction strength of SC-CO2 alone appeared to be lower than that of dichloromethane. All the components of interest that were extractable with dichloromethane and methanol were also extractable with methanol-modified CO2 (Cocks et al., 1995). A technique for the SC-CO2 extraction of the fungal metabolite ergostrol in its free (non-conjugated) form was developed and applied to samples of flour moldy bread and mushrooms. The overall method showed an 83%

Citric acid has successfully been separated from fermentation broth by a novel and unique purification process, which is characterized by organic solvent extraction and precipitation with compressed CO2 as a poor solvent. Compressed CO2 was then dissolved in acetone solution of crude citric acid to remove the residual impurities as precipitates using the antisolvent effect of CO2. Citric acid crystals could be obtained by the anti-solvent crystallization with CO2 (Shishikura et al., 1992). Dry mouldy bran resulting from solid state fermentation of *Gibberella fujikuroi* were subjected to SCE. The extraction of the sterol by SCE was found to improve with the use of ethanol as entrainer. The solid material retained the gibberellic acid activity without any loss (Kumar et al., 1991). The solubility of cholesterol in SCFs have also been studied and the solubility is correlated by using equation of states (Hartono et al.,

Extraction of ethanol from aqueous phase of a yeast fermentation broth has been described and a lower energy cost as compared to distillation has been reported (De Filippi and Moses, 1983). Shimshick reported the extraction of carboxylic acids from dilute aqueous media with SC-CO2. The specific advantage of this application is the pH decrease of the aqueous phase, which results in a higher concentration of the free acids. This shift is necessary for effective extraction of the carboxylic acids (Shimshick, 1981). SC-CO2 extraction has been reported to be more suitable for extraction of non-polar compounds with molecular weights less than 400. Griseofulvin is an antifungal antibiotic having a molecular weight of 353, making it amenable to SC-CO2 extraction. The optimized conditions for SCE of griseofulvin from dried media after solid state fermentation were obtained (Saykhedkar and Singhal, 2004). Furthermore, SCF has been developed mainly for unit operation to recover intracellular enzymes, recombinant-DNA proteins and nucleic

acids from microbial cell cultures (Khosravi-Darani, 2005, Castor and Hong, 1995).

*In situ* product removal is the fast removal of product from a producing cell thereby preventing its subsequent interference with cellular or medium components. Freeman and coworkers indicated future directions including application in situ extraction to a wider range of products and the developed methodologies, applicable under sterile conditions in the immediate vicinity of the producing cells (Freeman et al., 1993). End-product inhibition occurs in many fermentation processes and *in situ* removal of them typically enhances product formation rates, yields, and specificity (Christen et al., 1990; Gyamerah and Glover, 1996; Qureshi et al., 1998). Techniques that have been employed for *in situ* removal of fermentation products include liquid-liquid extractive fermentation (Adrian et al., 2000), use of selective membranes (Chang et al., 1992), cell recycling (Roca and Olsson, 2003), adsorption (Millitzer et al., 2002), microcapsule application (Stark et al., 2003) and vacuum fermentation (Qureshi et al., 1998). However, the intimate contact of an organic phase with the broth implies that the organic components of this phase may be present in the aqueous

**10.2.2 In situ extraction from the biomass of microbial fermentation** 

recovery of free ergostrol for spiked bread flour (Young and Games, 1993).

2001).

phase at saturation levels. The disadvantage of liquid-liquid extraction is the residual of toxic solvent, which presents significant separation, purification, and environmental challenges (Job et al., 1989). Also membrane fermentation and adsorption vacuum fermentation are not cost-effective. Guvenc et al. (1998) demonstrated the feasibility of ethanol extraction from a post–fermentation broth using SC-CO2. However, application of SC-CO2 for *in situ* extractive fermentation has been limited by its inhibitory effect on the metabolism of a variety of yeasts and bacteria (Isenschmid et al., 1995; Van Eijs et al., 1988). This toxicity is attributed, in part, to the acidic pH (Toews et al., 1995) that results from the increased solubility of CO2 at high partial Ps (Knutson et al., 1999). By buffering the medium and carefully controlling the compression and expansion conditions, the survival rate of cells increases. Van Eijs et al. developed an extraction procedure in which the *Lactobacillus plantarum* cell death was minimized (Van Eijs et al., 1988).

The impact of dense gases and SCF (N2, CO2, and ethane) on the carbohydrate consumption and ethanol formation by *Clostridium thermocellum* has been reported. Non–growing cells capable of metabolism were incubated at 60°C with cellobiose as a substrate in the presence of the three pressurized fluids. The rate and extent of ethanol production were similar in cell suspensions maintained at atmospheric and 6.9 MPa P under nitrogen (conventional method). Ethane at 6.9 MPa reduced the extent of ethanol production by less than 20% relative to the atmospheric control, whereas CO2 at the same P reduced ethanol formation. The results suggest that pressurized hydrocarbons have benefits over SC-CO2 for the *in situ*  recovery of volatile microbial products (Knutson et al., 1999).

*In situ* extraction of acetone, butanol and ethanol from synthetic media, simulating the downstream processing of a *Clostridium acetobutylicum* fermentation broth has been described (Van Eijs et al., 1988). It was also observed that extraction yield is a close function of the extraction time. Also increased P helps to achieve higher yields (Guvenc et al., 1998).

The extractive fermentation of 2-phenylethyl alcohol, the rose aroma, coupling fermentation with *Kluyveromyces marxianus* and SC-CO2 extraction has been reported (Fabre et al., 1999). Similar results show enhancement of 2-phenylethanol productivity by *Saccharomyces cerevisiae* in two-phase fed batch fermentation using solvent immobilization (Serp et al., 2003). Stark and coworkers reported the extractive bioconversion of 2-phenylethanol by *Saccharomyces cerevisiae* (2002). It has further been reported that furfural, a growth inhibitory byproduct, was successfully removed during fermentation of *clostridium* on sugars by introducing liquefied CO2 at room T and 5.9 MPa (Sako et al., 1992).

Selection of biocompatible solvents is critical when designing bio-processing applications for the *in situ* biphasic extraction of metabolic end-products. The prediction of the biocompatibility of supercritical and compressed solvents is more complicated than that of liquid solvents, because their properties can change significantly with P and T. The activity of the anaerobic thermophilic bacterium, *Clostridium thermocellum*, was studied when the organism was incubated in the presence of compressed nitrogen, ethane, and propane at 333 K and multiple pressure (Jason et al., 2000)

#### **10.2.3 Fractionation of cellular biomass**

SC and near critical fluids are used to fractionate biomass materials such as microbial cells in two steps. In the first step, the biomass is exposed to elevated pressure SC or near critical fluid to bring about disruption of the biomass to liberate structural biomass constituents. In the second step, the disrupted biomass is subjected to a multiplicity of SC or near critical fluid extraction steps, with different solvation conditions used for each fraction. Thus,

Supercritical Fluid Application in Food and Bioprocess Technology 569

possible. Non polar compounds can be extracted at low energy costs by this procedure. The process is cost effective due to carrying out at fermentation temperature. If whole fermentation broth put in contact with SC-CO2, may inactivate the microorganisms. These results offer the opportunity of in situ extraction of fermentation products with SC or sub critical (liquid) CO2. Use of SCF for both the disruption and extraction simplifies the procedure, and minimizes equipment and labor needs, time, contamination and loss of yield. In fact, the entire process can be readily automated. The use of super or near critical fluids allows for easy removal of the solvent by depressurization. The use of SCF allows the control of extraction condition by variation of temperature, pressure or modifier solvents. The finding that fermentation conditions influence the resistance of microbial cells to disruption should be further investigated. Studies of disruption kinetics and of the influence of cell morphology on kinetics of disruption are needed, and not information is available on disruption of mycelial organisms. The effects of thermal deactivation on cell properties and pre-incubation temperature on cell resistance to heat shock have received less attention. Further work is therefore required to characterize this interaction and relate it to changes in

Aaltonen, O. & Rantakyla, M. (1991). Biocatalysis in supercritical CO2, *Chemical Technology,*

Adrian, T., Freitag, J. & Maurer, G. (2000). A novel high-pressure liquid-liquid extraction

Athes, V., Lange, R. & Combes, D. (1998). Influenec of polyols on the structural properties of

Avedesian, M.M. (1986). Apparatus and method involving supercritical fluid extraction;

Baysal, T., Ersus, S. & Starmans, D.A.J. (2000). Supercritical CO2 extraction of beta-carotene

Bertucco, A. & Pallado, P. (2000). Micronization of polysaccharide by a supercritical

Bertucco, A. & Spilimbergo, S. (2001). Treating microorganisms with High Pressure. In: *High* 

(Ed.), pp. (626-630), Elsevier Science, Amsterdam. ISBN 139780444504982 Bertucco, A. & Vetter, G. (2001). *High Pressure Technology Fundamentals and Applications*,

Bleich, J. & Mueller, A. (1996). Production of drug based microparticles by the use of

*Journal of Microencapsulation,* Vol.13, pp. 131-139. ISSN 0265-2048

Elsevier, Amsterdam, 626-640. ISBN 139780444504982

juice, *Journal of Food Science*, Vol.56, pp. 1030-1033. ISSN 0022-1147

*Journal of Biochemistry,* Vol.255, pp. 206-212. ISSN 0021-924X

process for downstream processing in biotechnology: Extraction of cardiac glycosides. *Biotechnology and Bioengineering,* Vol.69, pp. 559-65. ISSN 0006-3592 Arreola, A.G., Balaban, M.O., Marshall, M., Peplow, A., Wei, C.I. & Scornell, J. (1991).

Supercritical carbon dioxide effects on some quality attributes of single orange

*Kluyveromyces lactis* β-galactosidase under high hydrostatic pressure, *European* 

and lycopene from tomato paste waste. *Journal of Agricultural Food Chemistry*,

antisolvent techniques, In: *Supercritical fluid methods and protocols*: *Methods in biotechnology,* J.R. Williams & A. A. Clifford, (Ed.), pp. (193-200), Humana Press,

*Pressure Process Technology: Fundamentals and Applications*, A. Bertucco & G. Vetter

supercritical gases with the aerosol solvent extraction system (ASES) process.

cell and broth properties.

USP:4,714,591.

Vol.21, pp. 240-248. ISSN 1744-1560

Vol.48, pp. 5507-11. ISSN 1684-5315

ISBN 0896035719, Totowa.

**12. References** 

fractionation of the biomass to obtain one or more compounds is effected (Castor and Hong, 1995). Different solvation properties are obtained using different Ts, Ps and/or modifier concentrations. Industrial applications are designed to take benefit of the very high selectivity of SCFs with attractive costs related to continuous operation: polymer fractionation, aroma production from fermented and distilled beverages, polyunsaturated fatty acids, active compounds from fermentation broth, pollution abatement on aqueous streams, etc (Perrut, 2000). SC and near critical CO2 have been used to fractionate cellular biomass isolated from soil, air, water, swamps, hot springs, sea water, animal or plant (Castor et al., 1998).

A SCE procedure and a chromatographic separation/detection method were developed for the detection of earth-based microorganisms. The analytical results demonstrated the feasibility of using the reported techniques to detect the chemical signature of life in barren desert sand samples (Lang et al., 2002).

Another interesting application of SCF in biotechnology is detecting the presence of a microorganism in an environmental sample. In this strategy, after exposure of sample to SCF nucleic acid will be isolated from the microorganism and detecting the presence of a particular sequence of nucleic acid by hybridization and PCR method, the contamination will be identified (Nivens and Applegate, 1996).

#### **11. Conclusion**

Application of supercritical is a promising alternative method for the pasteurization and sterilization of foodstuff, thermo sensitive substances, as well as thermally and hydrolytically sensitive polymeric materials, e.g. polymeric particles for drug delivery or implants. Furthermore, application of SC-CO2 seems to be attractive for its economical feasibility, as it needs very low pressure (lower than 20 MPa) compared to the so–called ultra high pressure treatment (200–700 MPa). Another special applications of SCFs in food processing include the decaffeination of green coffee beans, the production of hops extracts, the recovery of aromas and flavors from herbs and spices, the extraction and fractionation of edible oils and the removal of contaminants. These applications are now extended to new areas like formulation or specific chemical reactions, due to lightening environmental regulations; concern over the use of chemical solvents in food manufacturing; increased demand for higher quality products; increased cost of energy.

In the future, two areas of SCF applications in food industry are forecast for growth; the treatment of industrial wastes and the high value added products. So new application will developed e.g. novel processes for the disruption of microorganisms of therapeutic interest, the production of liposomes with implication to the cosmetic and pharmaceutical industries, and even a process to destroy and remove viruses effectively. The emergence of such a process brings real excitement and suggests that in the field of pharmaceutical and bioprocess industries, commercial applications may find their way to its implementation in the next decade.

Future trends in industrial development of SCFs include; legal issues which require banning organic solvents, quality consideration (raw material decontamination) for instance, pests from tropical products; the extraction of residues and toxins from food materials; as well as the deodorization and removal of fat, cholesterol, caffeine.

From the results of a number of extractions reported in literature can be concluded that by application of SC-CO2 selective, extraction of several compounds from fermentation broth is possible. Non polar compounds can be extracted at low energy costs by this procedure. The process is cost effective due to carrying out at fermentation temperature. If whole fermentation broth put in contact with SC-CO2, may inactivate the microorganisms. These results offer the opportunity of in situ extraction of fermentation products with SC or sub critical (liquid) CO2. Use of SCF for both the disruption and extraction simplifies the procedure, and minimizes equipment and labor needs, time, contamination and loss of yield. In fact, the entire process can be readily automated. The use of super or near critical fluids allows for easy removal of the solvent by depressurization. The use of SCF allows the control of extraction condition by variation of temperature, pressure or modifier solvents. The finding that fermentation conditions influence the resistance of microbial cells to disruption should be further investigated. Studies of disruption kinetics and of the influence of cell morphology on kinetics of disruption are needed, and not information is available on

disruption of mycelial organisms. The effects of thermal deactivation on cell properties and pre-incubation temperature on cell resistance to heat shock have received less attention. Further work is therefore required to characterize this interaction and relate it to changes in cell and broth properties.

### **12. References**

568 Mass Transfer - Advanced Aspects

fractionation of the biomass to obtain one or more compounds is effected (Castor and Hong, 1995). Different solvation properties are obtained using different Ts, Ps and/or modifier concentrations. Industrial applications are designed to take benefit of the very high selectivity of SCFs with attractive costs related to continuous operation: polymer fractionation, aroma production from fermented and distilled beverages, polyunsaturated fatty acids, active compounds from fermentation broth, pollution abatement on aqueous streams, etc (Perrut, 2000). SC and near critical CO2 have been used to fractionate cellular biomass isolated from soil, air, water, swamps, hot springs, sea water, animal or plant

A SCE procedure and a chromatographic separation/detection method were developed for the detection of earth-based microorganisms. The analytical results demonstrated the feasibility of using the reported techniques to detect the chemical signature of life in barren

Another interesting application of SCF in biotechnology is detecting the presence of a microorganism in an environmental sample. In this strategy, after exposure of sample to SCF nucleic acid will be isolated from the microorganism and detecting the presence of a particular sequence of nucleic acid by hybridization and PCR method, the contamination

Application of supercritical is a promising alternative method for the pasteurization and sterilization of foodstuff, thermo sensitive substances, as well as thermally and hydrolytically sensitive polymeric materials, e.g. polymeric particles for drug delivery or implants. Furthermore, application of SC-CO2 seems to be attractive for its economical feasibility, as it needs very low pressure (lower than 20 MPa) compared to the so–called ultra high pressure treatment (200–700 MPa). Another special applications of SCFs in food processing include the decaffeination of green coffee beans, the production of hops extracts, the recovery of aromas and flavors from herbs and spices, the extraction and fractionation of edible oils and the removal of contaminants. These applications are now extended to new areas like formulation or specific chemical reactions, due to lightening environmental regulations; concern over the use of chemical solvents in food manufacturing; increased demand for

In the future, two areas of SCF applications in food industry are forecast for growth; the treatment of industrial wastes and the high value added products. So new application will developed e.g. novel processes for the disruption of microorganisms of therapeutic interest, the production of liposomes with implication to the cosmetic and pharmaceutical industries, and even a process to destroy and remove viruses effectively. The emergence of such a process brings real excitement and suggests that in the field of pharmaceutical and bioprocess industries, commercial applications may find their way to its implementation in

Future trends in industrial development of SCFs include; legal issues which require banning organic solvents, quality consideration (raw material decontamination) for instance, pests from tropical products; the extraction of residues and toxins from food materials; as well as

From the results of a number of extractions reported in literature can be concluded that by application of SC-CO2 selective, extraction of several compounds from fermentation broth is

(Castor et al., 1998).

**11. Conclusion** 

the next decade.

desert sand samples (Lang et al., 2002).

will be identified (Nivens and Applegate, 1996).

higher quality products; increased cost of energy.

the deodorization and removal of fat, cholesterol, caffeine.


Supercritical Fluid Application in Food and Bioprocess Technology 571

Debs-Louka, E., Louka, N., Abraham, G., Ghabot, V. & Allaf, K. (1999). Effect of compressed

Degraeve, P. & Lemay, P. (1997). High pressure-induced modulation of the activity and

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**25** 

*VTT Finland* 

Hannu Viitanen

**Moisture and Bio-Deterioration** 

**Risk of Building Materials and Structures** 

During the service life of buildings, natural aging and eventual damage of materials due to different chemical, physical, and biological processes can take place. Ageing of the materials is one aspect of the environmental processes and involve different chemical, mechanical and biological reactions of the materials. Bio-deterioration, e.g. mould, decay and insect damage in buildings, is caused when moisture exceeds the tolerance of structures which may be a

Modelling of the development of mould growth and decay development is a tool for evaluate the eventual risk of ambient humidity or moisture conditions of materials for biodeterioration of materials. The modelling can be used in combination of hygro-thermal

Moisture availability is the primary factor controlling mould growth and decay development, but the characteristics of the substrate and environmental conditions determine the dynamics of the growth. However, moist materials may also dry and become wet again thus, resulting in fluctuating moisture conditions. Mould and decay problems in buildings are most often caused by moisture damage: water leakage, convection of damp air and moisture condensation, rising damp from the ground and moisture accumulation in the structure. Repeated or prolonged moisture penetration into the structure is needed for

There are several biological processes causing aging and damage to buildings and building components. This is due to natural ageing of materials but also caused by excessive moisture and damage of materials. For mould development, the minimum (critical) ambient humidity requirement is shown to be between RH 80 and 95 % depending on other factors like ambient temperature, exposure time, and the type and surface conditions of building materials (Table 1) For decay development, the critical humidity is above RH 95 %. Mould typically affects the quality of the surfaces and the adjacent air space with volatile compounds and spores. The next stage of moisture induced damage, the decay development, forms a serious risk for structural strength depending on moisture content, materials, temperature and time. The worst decay damage cases in North Europe are found in the floors and lower parts of walls, where water accumulates due to different reasons.

critical factor for durability and usage of different building materials.

**2. Critical environmental conditions for bio-deterioration** 

analyses of building and building components.

**1. Introduction** 

damage to develop.

