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Journal of Biotechnology 67 (1999) 13 – 32

Review article
Lipophilic compounds in biotechnology—interactions with cells
and technological problems1,2

Blaga Angelova a, Hans-Peter Schmauder b,*


a
Institute of Microbiology, Bulgarian Academy of Sciences, Acad. G. Bonche6 Str., Bl. 26, BG-1113 Sofia, Bulgaria
b
Forschungszentrum für Medizintechnik und Biotechnologie e.V. (fzmb), Geranienweg 7, D-99947 Bad Langensalza, Germany

Received 16 February 1998; received in revised form 17 April 1998; accepted 23 April 1998

Abstract

Lipophilic compounds are of significant importance in modern biotechnology. Centerly of interest are the
biodegradation as well as the biotransformation of such lipophilic and often water-immiscible substances. Both whole
cells and/or enzymes are used for these processes. It is obvious that a wide range of problems arise in an application
of such complex systems consisting of biocatalysts substrate(s), product(s), water, (in some cases water-immiscible
organic solvents): (i) interactions between lipophilic compounds and the membranes resulting in the change of some
physiological characteristics of the living system; (ii) problems in the transport of these compounds (substrates and/or
products) within the complex structured reaction systems; (iii) the problem of increasing the solubility of the lipophilic
and mostly water-immiscible compounds with a minimum of inhibition effects on the processes; (iv) the presence of
lipophilic components may also cause changes of the transport processes within the system (e.g. immobilized cells)
resulting in changed yield or activity of the biological system. These problems are critically discussed in this review
in relation to the known modes of interaction of lipophilic compounds with membranes, the bioavailability of the
substrates, and the cases of steroid biotransformations. An outlook of future aspects in research, development and
application of such processes is given. © 1999 Elsevier Science B.V. All rights reserved.

Keywords: Bioavailability; Biodegradation; Biosurfactants; Biotransformation; b-Cyclodextrine; Immobilized cells;


Lipophilic compounds; Membranes; Steroids; Surfactants; Waste compounds

* Corresponding author.
1
Dedicated to Professor Dr Wolfgang Fritsche on the occasion of his 65th birthday.
2
Based on the symposium, ‘Interactions between cells and lipophilic substances’ held at the 8th European Congress on
Biotechnology (ECB8) in Budapest, Hungary, August 1997.

0168-1656/99/$ - see front matter © 1999 Elsevier Science B.V. All rights reserved.
PII S0168-1656(98)00139-4
14 B. Angelo6a, H.-P. Schmauder / Journal of Biotechnology 67 (1999) 13–32

1. Introduction 4. Stimulation of transport processes by increas-


ing the solubility of the water-immiscible com-
A huge variety of lipophilic compounds persists pounds and of oxygen by application of
in the environment both as a result of metabolic immobilized cells in the processes for biotrans-
activity of microorganisms, plants and animals formation or biodegradation of lipophilic
(steroids, terpenes, waxes etc.) and as a result of compounds.
human activities (e.g. different hydrocarbons or Unfortunately, there is still little knowledge on
related compounds derived from transports, the physiological response of microbial cells to the
chemical, and agrochemical industries). presence of lipophilic compounds. An attempt to
Both types of lipophilic compounds mentioned summarize the data concerning adaptation mech-
are of significant importance for modern biotech- anisms of microbial cells in the process of their
nology. Natural lipophilic compounds as sub- interaction with lipophilic compounds will be
strates for microbial transformations lead to made.
useful intermediates. On the other hand a lot of A discussion of such processes was in the centre
pollutants are best degraded by microorganisms of the special symposium No. 14 entitled ‘Interac-
or other biocatalysts. However, the low water tions between cells and lipophilic substances’ of
solubility of these substrates is a main limiting the ECB 8.
factor of microbial transformation and biodegra-
dation. This review focusses on the bioavailability
of lipophilic compounds to microbial cells as well 2. Interaction of lipophilic compounds with
as on the approaches suggested and/or already membranes
applied for improving the contact between the
biocatalysts and substrates. There are problems Investigations on the interaction between
with the solubility of the substrates or products in lipophilic compounds and microorganisms are of
most of the biotechnological processes of these central interest for the development in key areas
types. In consequence, the interaction between the of applied microbiology. The isolation of micro-
lipophilic and mostly water-immiscible com- bial strains resistant to toxic organic compounds
pounds and the cell membranes is important for led to new possibilities. These strains may im-
these processes. The effectiveness of the biotech- prove the efficiency of microbially mediated trans-
nological processes depends on the close contact formation and degradation processes of lipophilic
between the biocatalysts and the substrates/prod- compounds.
ucts. Thus, process control, yield and duration One of the main problems in the biotechnology
can be improved by increasing the bioavailability of lipophilic substrates is the transport into the
of the substrates or products by facillitating spe- system due to their water insolubility. These in-
cific surfactants. As a consequence the economy clude the passage through the cell membranes and
of the processes will be influenced more and more the temporary incorporation of the lipophiles in
by the following factors: the bilayers and the tendency for a film formation
1. Effect of lipophilic compounds on membranes on cell surfaces. This results in various forms of
and the consequences of such interactions on reduced bioavailability by limitation of their sup-
the cell physiology and related activities. ply with oxygen, substrates, ions etc. In the case
2. Transport of lipophilic compounds between of biotransformations of steroids—mostly hy-
the phases in the biotechnological processes droxylations—the hydroxylated products show a
(e.g. across the membranes, to the enzymes, to higher solubility so that transport out of the cell
different pools within the cells). may be facilitated.
3. Solubility and partition of such compounds, The main area for the first interactions of sub-
possibly improvable by chemical or other strate with cells are the bilayer membranes con-
methods (e.g. by using cyclodextrins, deter- sisting of an outer hydrophilic and an inner
gents of biological or chemical origin). hydrophobic part (for a detailed description see:
B. Angelo6a, H.-P. Schmauder / Journal of Biotechnology 67 (1999) 13–32 15

Van der Meer and Wieb, 1993). Shmeeda et al. 3. Production of biosurfactants promoting
(1994) discuss the ways of transport in such pro- microemulsions.
cesses: The compounds have to be transported via Microorganisms differ in their cell surface
lipoproteins (with a possible temporary mem- structure and composition and as a consequence
brane incorporation) into the cells. Inside the cell Gram-negative and Gram-positive bacteria were
the compounds are introduced in processes of de found to have different growth characteristics in
novo-synthesis, degradation and transformation. the presence of an organic liquid phase. Limita-
The products formed are transported out of the tions in the growth of microorganisms by high
cells and into the medium. All passages through concentrations of various organic solvents were
the phospholipid layers introduce the risk of (tem- also shown to be correlated with the solvent
porary) incorporation of the lipophilic com- parameter log P (Harrop et al., 1989; Inoue and
pounds into the bilayer. This is very well known, Horikoshi, 1991; Moriya and Horikoshi, 1993;
for example steroids and sterols. Peroxidation Vermue et al., 1993; Rajagopal, 1996). Some mi-
processes were used to minimize such incorpora- croorganisms are able to tolerate high concentra-
tions (see also: Shinitzky, 1993, 1994; Lichtenberg, tions of organic solvents (Inoue and Horikoshi,
1993). By construction of peroxistructures the bi- 1989; Inoue et al., 1991). Others possess both
layers became more rigid and did not allow incor- abilities, to tolerate and to use them as a single
poration of lipophilic compounds. However, source of carbon and energy (Guerin and Jones,
incorporation of the lipophiles and peroxidation 1988; Cruden et al., 1992; Weber et al., 1993;
change the physiological properties of the cells in Zaitsev et al., 1995; Paje et al., 1997). Tolerance
to some aromatic compounds (benzene, toluene
a more or less drastic way and also the biotechno-
and p-xylene) is suggested to be distinguished
logical process itself.
from microbial tolerance at least to n-alkanes
The main aspects of the discussion of interac-
(Moriya and Horikoshi, 1993).
tions of microorganisms with lipophilic com-
When n-alkanes were used as a single carbon
pounds include (e.g. de Bont, 1997; Schmauder,
source, some unusual components of bacterial cell
1997):
surfaces were reported: Trans-unsaturated fatty
1. Cell surface changes,
acids normally rare in bacteria were formed dur-
2. Mechanisms of adaptation to the presence of
ing continuos growth of Pseudomonas oleo6orans
lipophilic compounds, on octane (Chen et al., 1995). The concentration
3. Increasing the bioavailability of the substrates of sterols, monoacylglycerols and diacylglyc-
and their uptake by microbial cells, erophosphocholines increased in dodecane-grown
4. Metabolic routes of biodegradation, and cells of Rhodococcus rhodochrous (Sorkhoh et al.,
5. The genetical basis of microbial tolerance to 1990). Mycobacterium and Rhodococcus strains
toxic compounds as well as of the ability for incorporate aliphatic hydrocarbons into their
their biodegradation. lipids as a result of growth on n-alkanes (Guerin
The ability of microorganisms to degrade hy- and Jones, 1988). Octane-grown R. rhodochrous
drocarbons and to use them as a single source of NRRL B-2153 cells became more active in
carbon and energy is well known and used for cumene (2-phenylpropane) oxidation than glu-
obtaining a wide range of products (Rosenberg, cose-grown cells (Hou et al., 1994). Rhodococcus
1993). The mechanisms of hydrocarbon uptake as sp. cells grown on n-alkanes reveal higher steroid
a key process in their biotransformation and/or hydroxylating activity (Mutafov et al., 1997b).
biodegradation, however, are not well established. According to the current models only molecules
Nevertheless, three models of alkane uptake are of lipophilic compounds which are dissolved in
commonly discussed (e.g. Bouchez et al., 1997): the aqueous phase are available for intracellular
1. Dissolution in the aqueous phase, metabolism (Sikkema et al., 1995). Some prob-
2. Direct contact between the microbial cells and lems arise from the fact that hydrophobic organic
the hydrophobic phase, and pollutants are often present at waste disposal sites
16 B. Angelo6a, H.-P. Schmauder / Journal of Biotechnology 67 (1999) 13–32

in non-aqueous liquids (NAPLs) from which the the outer membranes of bacteria tested were quite
rate of partitioning to water is frequently low permeable to steroid compounds and that the
(Ortega-Calvo and Alexander, 1994). diffusion occurred mainly through the bilayer re-
The growth rate of Mycobacterium sp. strain gions of the membrane (Plesiat et al., 1991; Plesiat
BB1 was shown to be not dependent on the and Nikaido, 1992).
solubility of the substrates (phenanthrene, pyrene An important mechanism in the uptake of
and fluoranthene) when these compounds were lipophilic compounds is the partitioning of these
used as small (micro)particles and at high stirring molecules into the bilayer of the cytoplasmic
rates (Boldrin et al., 1993). membrane. Therefore, it is valuable to consider
According to Guerin and Boyd (1992) a gener- the different steps that are involved in the uptake
alisation regarding the bioavailability of sorbed of lipophilic compounds (Sikkema et al., 1995).
substrates is inappropriate due to organism-spe- Lipophilic compounds influence the overall
cific properties. This conclusion is based on exper- metabolism and growth characteristics of micror-
imental results obtained during the testing of the ganisms by accumulating preferentially in mem-
hypothesis that sorbed hydrocarbons (e.g. naph- branes and simultaneous altering their physico-
thalene) are unavailable to degradative microor- chemical properties. Consequently a correlation
ganisms. between the hydrophobicity of a compound and
The uptake of 4-chlorobenzoate by cells of the its partitioning into a lipid bilayer on the one
coryneform bacterium NTB-1 was shown to be hand and the toxicity for microorganisms on the
performed by an energy-dependent inducible other hand have been shown (Sikkema et al.,
transport system with a very narrow substrate 1994a,b, 1995; Weber and de Bont, 1996).
specificity (Groenewegen et al., 1990). Microorganisms react to changes in their envi-
Rapid uptake of the xenobiotic 4-toluene sul- ronment by modifying their surface composition
fonate (TS) by Comamonas testosteronii T-2 was and structure. For P. putida cells it has been
observed only in cells grown with TS or 4-methyl- shown by transmission electron microscopy that
benzoate as a carbon and energy source. The growth on p-xylene (up to 50%, v/v) results in
results provide the evidence that the TS uptake morphological variations as compared with those
system is a rather specific system which might cells grown on succinate. The outer cell mem-
have evolved from a transport system for natural brane becomes convoluted and membrane frag-
carboxylated aromatic compounds (Locher et al., ments are shed into the culture medium. The
1993). cytoplasmic membrane invaginates, forming vesi-
By using C14-labeled toluene the presence of an cles, and becomes disorganised. Electron-dense
energy dependent export system in Pseudomonas intracellular inclusions which are not present in
putida S12 was suggested. It was supposed that cells grown on succinate were observed in cells
this system is responsible for the resistance to grown on p-xylene (20%, v/v) and p-xylene va-
toluene. The whole-cell experiments alone do not pours (Cruden et al., 1992).
allow the authors conclusions about the nature of During the growth on octane P. oleo6orans cells
the efflux system. A confirmation by in vitro accumulate poly(b-hydroxyalkanoates) as intra-
experiments is necessary to assess unequivocally cellular inclusion bodies but do not incorporate
the existence of an active efflux system in solvent- substrate into the membranes (Preusting et al.,
resistant bacteria (Isken and de Bont, 1996). 1991). Loss of cell viability and membrane dam-
In an attempt to characterise the permeability age as observed by freeze-fracture electron mi-
properties of some Gram-negative bacteria to- croscopy in P. oleo6orans cells correlate with a
wards hydrophobic compounds the diffusion rates loss of hydrocarbon oxidation capacity, measured
of various steroids across the outer membranes as the conversion of 1-octene to 1,2-epoxyoctane
were determined quantitatively by coupling the (de Smet et al., 1983).
influx process with the subsequent enzymatic oxi- It was also shown that membranes of E. coli
dation of specific probes. It was concluded that were disrupted by toluene (de Smet et al., 1978),
B. Angelo6a, H.-P. Schmauder / Journal of Biotechnology 67 (1999) 13–32 17

and that membranes of P. oleo6orans (de Smet et In some cases application of an additional sub-
al., 1983) and E. coli (Favre-Bulle et al., 1991) strate such as succinate (Sikkema et al., 1992) or
were damaged by extended growth in octane. glutamate (Pinheiro and Cabral, 1991, 1992) pro-
For P. putida DOT-T1 cells grown in high vides cells with energy source that may fuel the
concentrations of toluene (up to 90%) a preserva- protection and adaptation of the organism.
tion of the outer membrane integrity and an It was suggested that envelope modifications
exhibition of a wider periplasmic space was observed in P. putida strains tolerant to o-xylene
shown. In some cases ribosomes appeared in the (increase in total fatty acids, in saturated fatty
periplasmic space. The damage occurred was sug- acids and modified lipopolisaccharides) aid in
gested to be at the level of cytoplasmic mem- their survival at high concentrations of organic
branes (Ramos et al., 1995). solvents (Pinkart et al., 1996).
The physiological response of the cells to com- As it could be seen a great deal of investiga-
pensate for direct influence of toluene and octane tions concerning interactions of microbial cells
by increasing the transition temperatures of cell with lipophilic compounds has been performed by
lipids was shown for P. putida and P. oleo6orans using different Pseudomonas strains. Another
cells, respectively (Weber et al., 1994; Chen et al., group of microorganisms used in such investiga-
1995). tions was the group of so called ‘hydrophobic’
Changes in the membranes are considered to be microorganisms including the genera Rhodococ-
a mechanism to keep them in the same fluidity cus, Mycobacterium, Arthrobacter and Nocardia.
In particular, the potential for using microorgan-
condition. It appears that a key process in the
isms of the genus Rhodococcus for transformation
membrane adaptations to lipophilic compounds is
and/or biodegradation of lipophilic compounds
the isomerization of cis- to trans-unsaturated
has been frequently discussed (Finnerty, 1992;
fatty acids (Heipieper et al., 1994; Weber et al.,
Klatte et al., 1994; Smith, 1994; Warhurst and
1994). Three strains of P. putida accumulated
Fewson, 1994; Warhurst et al., 1994; Zaitsev et
trans-unsaturated fatty acids in the membrane
al., 1995; Paje et al., 1997).
instead of the cis-isomers as reaction to toxic
The hydrophobic cell wall of Mycobacterium is
concentrations of toluene. This increase in trans/
considered to facilitate the contact between cells
cis ratio is supposed to have an rigidifying effect and hydrocarbon substrates. It was suggested that
(Weber et al., 1994). the hydrocarbon chains of the cell wall lipids in
In some cases preliminary adaptation is neces- Mycobacterium chelonae are arranged predomi-
sary for the growth in the presence of organic nantly in a direction perpendicular to the cell wall
solvents. P. putida DOT-T1 is able to grow in the surface probably producing an asymmetric bilayer
presence of 0.1 to 90% (v/v) toluene and the structure (Nikaido et al., 1993). This conclusion is
solvent tolerance is inducible. Bacteria grown in based on experimental investigations on the phys-
the absence of toluene required an adaptation ical arrangement of the lipids of the cell wall.
period before growth restarted (Ramos et al., Further investigation of cell interactions with
1995). Only acetate adapted cells of P. putida S12 lipophilic compounds are in progress. Evidences
are able to grow in the presence of supersaturat- obtained on their partitioning into cell mem-
ing concentrations of toxic non-metabolizable sol- branes could be used for predicting the concentra-
vents such as toluene (Weber et al., 1993). tion range at which microorganisms can be
Toxicity of toluene to E. coli is apparently due selected (Sikkema et al., 1992). The isolation of
to its interaction with the cytoplasmic membrane microorganisms able to survive in the presence of
leading to the loss of the cations Mg2 + and Ca2 + high concentrations of organic solvents is of con-
as well as other small molecules (de Smet et al., siderable interest because of the possibility of
1978). Mg2 + in the culture medium improved the their application for the elimination of high prior-
solvent tolerance of P. putida DOT-T1 to toluene ity pollutants such as toluene, xylene, styrene and
(Ramos et al., 1995). benzene (Keith and Telliard, 1979).
18 B. Angelo6a, H.-P. Schmauder / Journal of Biotechnology 67 (1999) 13–32

The discovery of a benzene tolerant bacterium for biodegradation of industrial pollutants is,
from the deep sea is also interesting for microbial however, limited by their toxicity and low
ecology. Deep-sea bacteria which showed the biodegradability. It was shown that synthetic sur-
characteristics of halotolerant growth in addition factants affect microbial surface structure, compo-
to organic solvent tolerance are considered benefi- sition, properties and functions. Tween 80® as a
cial in improving the degradation of polluting component of culture media causes changes in
petroleum-hydrocarbons in marine environment mycolic and fatty acid composition and the level
(Moriya and Horikoshi, 1993). of their saturation in Corynebacterium and
An application of biphasic (aqueous-organic) Rhodococcus sp. cells (Chevalier et al., 1988;
systems was suggested for the biodegradation of Avramova et al., 1997). Anionic surfactants en-
polynuclear aromatic hydrocarbons (Vanneck et hance cell surface hydrophobicity (Marchesi et al.,
al., 1994), for styrene degradation (El Aalam et 1994a,b) and increase membrane permeability
al., 1993) as well as for the selection of microor- (Laouar et al., 1996; Chan and Kuo, 1997). Syn-
ganisms able to grow at the expense of some thetic surfactants can facilitate the process of
xenobiotics (Ascon-Cabrera and Lebeault, 1993), aromatic hydrocarbon biodegradation by improv-
and for the degradation of naphthalene (Efroym- ing their bioavailability to microbial cells
son and Alexander, 1991). (Aronstein and Alexander, 1992, 1993; Marchesi
et al., 1994a,b; Tiehm, 1994; Liu et al., 1995;
Volkering et al., 1995; Stanley et al., 1997).
3. Bioavailability of lipophilic compounds Recently, a significant interest has been focused
on surfactants of microbial origin as an alterna-
For a biotransformation or biodegradation the
tive to the synthetic ones. This is due to their
availability of the in most cases practically water
environmentally friendly nature, the possibilities
insoluble compounds is of a high importance.
for their production from cheap and readily avail-
Organic solvents or surfactants have been used
able substrates (including waste materials and by-
mostly as mediators for increasing this solubility.
products) and their environmental compatibility
Organic solvents miscible or immiscible with wa-
(Fiechter, 1992; Rosenberg, 1993; Desay and Ba-
ter have been used, e.g. alcohols (mainly ethanol),
acetone, DMF, toluene, and xylene, but they are nat, 1997; Kosaric, 1997).
mostly toxic for the cells and this toxicity allows A great variety of biosurfactants produced by
only low ranges of concentrations and only an microorganisms were described and characterised
incomplete dissolution of the substrate/product. in detail with respect to their chemical composi-
The solubility of phenanthrene as a model com- tion and physico-chemical properties as well as
pound of the polycyclic hydrocarbons could be their possible application (Neu, 1996; Desay and
increased in several ways: The molecular com- Banat, 1997).
pound gives in most cases a dissolution too low Biosurfactants are extracellular compounds
for biotechnological processes. By adding surfac- with varying composition of polysaccharides and
tants the formed surfactant micelles allow a lipids or peptides. They are synthesized by grow-
higher degree of dissolution. In the aqueous phase ing as well as resting microbial cells on the ex-
an equilibrium partitioning of this hydrocarbon pense of both carbohydrates and hydrocarbons.
will result between micelles and the aqueous They remain attached to the cell surface or are
phase. Finally the cells have a higher concentra- excreted into the liquid medium. In some cases
tion of the substrate at their disposal (see also: hydrocarbons might be considered as inducers of
Guerin and Jones, 1988). biosurfactant production or at least as inducers of
Surfactants currently used in industry, agricul- their excretion. It was shown that the release of
ture, and medicine are mainly products of chemi- the surfactant produced by Corynebacterium lepus
cal synthesis. Their application in food and is stimulated by the presence of tetradecane (Du-
cosmetic industry as well as in the environment vnjak and Kosaric, 1985).
B. Angelo6a, H.-P. Schmauder / Journal of Biotechnology 67 (1999) 13–32 19

Mannosylerythritol lipids (MEL) production by tion—was described. It was supposed by the au-
Candida antarctica was suggested to be constitu- thors that this could be used for biosurfactant
tive whereas the excretion of MEL needed the production by resting cells (Davila et al., 1992;
presence of water-insoluble carbon substrates (Ki- Kitamoto et al., 1992a). A formation of a sepa-
tamoto et al., 1990, 1992a,b). rate organic phase containing sophorose lipids is
Biosurfactants are produced by microorganisms significant also for avoiding product inhibition
belonging to different taxonomical groups. Some and facilitating product separation (Kitamoto et
biosurfactants are produced by halotolerant (Be- al., 1992a).
jar et al., 1997) and thermotolerant microorgan- The mechanisms of action of biosurfactants on
isms (Banat, 1993; Makkar and Cameotra, 1997). lipophilic compounds and their application in
A thermo- and halotolerant Bacillus licheniformis biodegradation are widely described and discussed
strain was reported to produce biosurfactant un- (Banat et al., 1991; Rosenberg, 1993; Zhang and
der both, anaerobic and aerobic, conditions Miller, 1994; Banat, 1995a,b; Kosaric 1997). In-
(Yakimov et al., 1995, 1996). teresting results on applications were described in
Biosynthesis and excretion of biosurfactants 1997 by different groups. A spanish group (Bejar
into the medium are considered to be another cell et al., 1997) has compared the effectiveness of
mechanism aiming at an adaptation of the micro- several exopolysaccharides formed by different
bial metabolism for using external lipophilic (and microbial strains. They describe the varying influ-
also xenobiotic) compounds as carbon and energy ence against crude oil, mineral oil, n-octane, n-
sources (Bouchez et al., 1997). Biosurfactant pro- hexadecane and n-tetradecane. These results
duction at the expense of industrial wastes and pointed out that there is no ‘ideal’ surfactant.
oils (Espuny et al., 1995; Mercade et al., 1996; Individual optimization is therefor required. Page
Sudhakar et al., 1996; Galas et al., 1997; Lima et et al. (1997) describe an analogous result. Biosur-
al., 1997), polycyclic aromatic compounds (Deziel factants from P. aeruginosa (rhamnolipids) and
et al., 1996), animal fats (Deshpande and Daniels, Rhodococcus (glycolipids) were studied regarding
1995), and vegetable oils (Kitamoto et al., 1990, their role in increasing the solubility of hydrocar-
1992a,b, 1993; Davila et al., 1992; Mercade et al., bons of chain lengths between C10 and C24. In
1993) are of special interest. another paper Schmauder et al. (1997) describe
Microbial biosurfactant production is embed- the selection of mixtures of chemical surfactants
ded in the cell metabolism as a whole. Biosurfac- of the type AERES T22 and T27 for the washing
tant production by Pseudomonas aeruginosa is of oil-binding material contaminated with motor
induced by the shift in the phosphate metabolism or heating oil. The authors could reach efficient
(Milligan et al., 1989). Sophorose lipid production extraction followed by a normal biodegradation
by Candida bombicola was shown to take place of the hydrocarbons in the washing water.
under nitrogen limitation (Davila et al., 1992). A Besides practical application also basic microbi-
correlation between nitrogen metabolism and bio- ology and microbial ecology benefit from ex-
surfactant production was shown for P. aerugi- tended knowledge on biosurfactant production.
nosa (Milligan and Gibbs, 1989). The form of An enormous versatility of biosurfactant func-
nitrogen source in the culture medium affects the tions in natural environments could be expected
biosurfactant action on the petroleum oil taking into consideration the dependence of their
biodegradation by Pseudomonas sp. BP (Galas et chemical composition and structure on the nutri-
al., 1997). tional conditions. Biosurfactant production in
A kinetic description of both, sophorose lipid natural conditions is connected to the distribution
production by C. bombicola and MEL production of microorganisms and colonization of new sur-
by C. antarctica, shows that they are associated faces. Serrawettin W2, a surface-active exolipid
with the stationary phase of growth. The existence produced by Serratia marcescens is considered to
of two distinct phases — a phase of microbial be the potent extracellular product enhancing the
growth and a phase of biosurfactant produc- spreading growth of the bacteria in a surface
20 B. Angelo6a, H.-P. Schmauder / Journal of Biotechnology 67 (1999) 13–32

environment (Matsuyama et al., 1992). The exo- stituents, e.g. hydroxypropyl-groups, allow a bet-
polymer produced by Bacillus insolutus in the ter penetration of some guest molecules into the
process of copper bioleaching from copper sul- cavity and a better solubility of the resulting
fates may confirm and probably explain the mech- complexes. More details about the chemistry and
anism of interaction of microbial cells with the the structure of CD are given by Szejtli (1997),
hydrophobic surface of the substrates (Sklo- Croft and Bartsch (1983) and Larsen et al. (1997).
dowska and Mattlakowska, 1997). Leaf surfaces By forming such inclusion complexes different
represent another biological interface well known forms are possible:
as a habitat for microorganisms (bacteria, yeasts 1. The 1:1 inclusion complexes. Usually the con-
and filamentous fungi). Surface-active Pseu- centration of the included compound is linear
domonas strains were shown to affect leaf wet- with respect to the used CD and the guest
tability (Bunster et al., 1989). Interaction between molecule. This linearity follows a wide range
cells of some phytopatogenic Corynebacterium of concentrations. This allows in most cases an
strains and plant surfaces was suggested to be application for a (quantitative) estimation of
facilitated by biosurfactant production (Akit et different guest molecules, e.g. using the phe-
al., 1981). nolphthalein technique.
An interesting view on the bioavailability of 2. Inclusions complexes of a higher order with no
organic pollutants was given by Harms (1997). He linearity in the reaction.
has described in his lecture at the ECB 8 that The optimum applicability in biotechnology is
there is a closed connection between bioavailabil- realized by the first case. By this linear increase of
ity and spatial separation. Bioavalabilty will be the concentration of the guest molecule in the
influenced by the transport capacity within the cavities more substrate is in a soluble form. The
environment and the biochemical capacity of the yield and effectivity of the biotransformation or
cells. Advective flow, diffusive mass transfer, in- degradation could be increased. At higher concen-
traparticle diffusion, the length of the transport trations of the water-immiscible compounds in
pathways, the spatial distribution of both bacteria these complexes of the type 1) outside of the
and growth substrates, the complex structures of linearity a plateau is formed. Additional formed
the matrices and their resistance against diffusion complexes precipitate. Only for a small number of
processes influence all processes of degradation of organic substances an increase of solubility is not
pollutants in the soil. In brief, the complex system realizable.
of substrate bioavailability depends on the charac- Examples of different solubilities of steroid
teristics of the support matrix (e.g. organic mat- derivatives and phenanthrene in organic solvents
ter, soil, sand, water), the pollutant properties and in water after addition of b-CD or its deriva-
(adsorption, solubility, molecular weigth, other tives were described by Schlosser et al. (1993) and
organic contaminants) and the kinetic parameters Schmauder et al. (1992) and are summarized in
and maintenance needed by the microorganisms. Table 1.
For increasing the bioavailability of insoluble Some microorganisms can metabolize b-CD
substrates, e.g. sterols or steroids, the application and use the formed glucose as an additional
of cyclodextrines is of interest. The cyclodextrines source of carbon. A lot of organic pollutants can
(CD) are cyclic oligosaccharides formed from glu- only be degraded by feeding a cometabolic sub-
cose by microbial enzymes. They consist of differ- strate (e.g. glucose, phenol, lactate, toluene and
ent numbers of glucose moieties resulting in other compounds). There is an excellent possibil-
different sizes of the cyclic structure. The formed ity for application of the CD’s since they can
cavity sizes are between 4 and 8.5 3 Å. Guest function as a mediator for solubility and as a
molecules can be included such as a-CD benzene, cometabolic substrate. Such combinations could
b-CD substituted phenyl, and for g-CD naphthyl allow the development of new kinds of technolog-
compounds. Other substances can be included as ical applications in the biotechnology of lipophilic
well, e.g. b-CD steroids or sterols. Specific sub- compounds.
Table 1
Solubility of selected organic lipophilic compounds in aqueous methanol and after treatment with b-cyclodextrine

Methanol (%) b-Cyclodextrine (g/l) Hydroxypropyl-b-cyclodextrine (g/l)

0 2 4 6 0 1 2 4 8 16 0 1 2 4 8 16

13-Ethyl-gon-4-en-3,17-dione (GD) (mg/l) 15 30 55 100 15 100 151 276 490 897 n.e. n.e. n.e. n.e. n.e. n.e.
Phenanthrene (mg/l) n.e. n.e. n.e. n.e. 0.8 0.7 2.5 5.8 11.7 25 0.8 2.5 3 7 16 34

The compounds were solubilized at first in pure methanol and than added to water or culture liquid reaching the final concentration of 0, 2, 4, or 6% of methanol because higher concentrations
of methanol in the culture are toxic for the microbial cells. The concentration of cyclodextrins is given in water (Schmauder et al., 1992; Schlosser et al., 1993).
n.e.= not estimated.
B. Angelo6a, H.-P. Schmauder / Journal of Biotechnology 67 (1999) 13–32
21
22 B. Angelo6a, H.-P. Schmauder / Journal of Biotechnology 67 (1999) 13–32

4. Steroid biotransformation The inductive nature of the 15a-steroid hydrox-


ylase of Penicillium raistrickii was proved and it
The biotransformation of natural lipophilic was shown that steroids different from the sub-
compounds (sterols and steroids) is of great im- strate used for transformation have an inductive
portance in biotechnology because in these pro- effect (Irrgang et al., 1992).
cesses interesting products, e.g. for the pharma- The presence of distinct mechanisms controlling
ceutical industry, can be formed. Especially the 11a- and 11b-hydroxylation of exogenous
following three microbial steroid modifications steroids in Cunninghamella elegans and the possi-
are of importance for modern biotech- bility to stimulate separately the production of
nology and the chemical (pharmaceutical) indus- both derivatives were shown (Dlugonski et al.,
try (see also: Smith, 1984; Mahato and Banerjee, 1997).
1985; Mahato et al., 1989; Hocknull and Lilly, The induction of the 9a-steroid hydroxylating
1988; Flygare and Larson, 1989; Ahmad et al., activity followed by an induction of the D1-steroid
1992; Pinheiro and Cabral, 1992; Ahmad and dehydrogenating activity (degradation of the by-
Johri, 1993; Dias et al., 1994; Srivastava and product) was observed in Rhodococcus sp. cells.
Patil, 1995; Dlugonski et al., 1997; Mahato and The transformation of androstenedione by resting
Garai, 1997; Znidarsic and Pavko, 1997): cells with protein synthesis blocked after the de-
1. Selective side chain cleavage of natural sterols velopment of the 9a-steroid hydroxylating activity
2. D1-dehydrogenation, and and before the significant development of the
3. 11-hydroxylations. undesirable D1-dehydrogenating activity was
Microbial 9a-hydroxylation has opened an-
demonstrated as a possibility for achieving a com-
other means for the preparation of D9,11-interme-
plete substrate conversion (Mutafov et al., 1997a).
diates (which are subsequently transformed
A special focus was given recently on fungal
chemically to 9a-fluorocorticoids) eliminating tra-
steroid hydroxylases as site selective cytochrome
ditional microbial 11b-hydroxylation (Smith,
P-450 monooxygenases (Smith et al., 1993). Cy-
1984). Processes of future interests are also 7a-
tochrome P-450 content in intact protoplasts of
and 14a-hydroxylations because the products ob-
C. elegans was determined as an indicator of a
tained show a diuretic and anticancer activity in
medicine (Mahato and Majumdar, 1993). steroid hydroxylating system (Dlugonski et al.,
For carrying out the processes of microbial 1991).
transformation of steroid compounds for a long It was shown that the progesterone 11a-hy-
period of time free cells (growing and resting) droxylating system of Rhizopus nigricans consists
were preferentially used. As a result of these in- of four proteins: cytochrome P-450, cytochrome
vestigations a large amount of information is P-450 reductase, cytochrome b5 and cytochrome
available about the distribution of transforming b5-reductase. All four proteins have been shown
activities among different taxonomical groups of to be located on the cytoplasmic side of the
microorganisms. Many physiological characteris- endoplasmic reticulum (Osborne et al., 1990). The
tics of biocatalysts were established. The influence 15b-steroid hydroxylating system of Bacillus
of different factors on the processes were de- megatherium was chosen as a model system of
scribed. The microorganisms and steroid sub- monooxygenase catalysed reaction for studying
strates used as well as the products obtained were similar reactions in mammalian cells (Berg and
precisely listed in several reviews (Mahato and Rafter, 1991).
Banerjee, 1985; Mahato et al., 1989; Mahato and The reactions of microbial sterol side chain
Majumdar, 1993; Smith et al., 1993; Mahato and cleavage are of a considerable practical interest
Garai, 1997). New microbial reactions and novel because the manufacture of most pharmaceuti-
metabolites formed from C19 and C21 steroids cally active steroids proceeds from intermediates
were reported recently (Krischenowski and obtained from natural raw materials (Ahmad et
Kieslich, 1993; Madyastha, 1995). al., 1992).
B. Angelo6a, H.-P. Schmauder / Journal of Biotechnology 67 (1999) 13–32 23

In order to obtain physiologically important matis has been cloned using Tn611 transposon
steroid derivatives of widely available sterols by mutagenesis (Andor et al., 1997).
microorganisms cholic acid was used as a sub- A successful transfer of mammalian hydrocorti-
strate for Arthrobacter simplex cells (Mukherjee et sone producing enzyme system into yeast cells
al., 1993). Sterols with stigmastane side chains (Saccharomyces cere6isiae) was reported as a way
were used as starting material for 26-oxidized to achieve a one-pot (may be two-pot) production
metabolites by Mycobacterium cells obtained by of hydrocortisone by feeding cholesterol to the
spheroplast fusion (Ambrus et al., 1995). culture. But even in these case one of the factors
A neutral fraction and an upgraded neutral limiting the effectiveness of the process is the
fraction of Polish tall oil containing different initial substrate supply and uptake because the
amounts of b-sitosterol, sitostanol and campas- wild yeasts are impermeable to exogenous sterols
terol were used as substrates for Mycobacterium (Spagnoli, 1997).
sp. and the new metabolite 5a-andros-3,6,17-tri- Since the start of the application of immobi-
one was found (Szykula et al., 1991). lized cells for microbial transformation of steroid
Rhodococcus equi was used for transformations compounds, many different techniques for immo-
of single or mixed sterols into AD and ADD. The bilization as well as supports of natural and syn-
results indicated that the strain possesses the abil- thetic origin were developed. Physiological
ity to carry out conversion of 3-hydroxy-D5- to characteristics and biocatalytic activity of immo-
3-keto-D4-structures, D1-dehydrogenation, D4-de- bilized cells in comparison with the free ones were
hydrogenation and degradation of C17 side chains given in a lot of reports. Emphasis is put on the
varying in length and complexity (Ahmad et al.,
limitation of steroid transformation processes by
1991). The dependence of D1-dehydrogenation on
the oxygen availability to the immobilized biocat-
the C17 side chain was also found (Srivastava and
alysts (Schmauder et al., 1991; Irrgang et al.,
Patil, 1995).
1993a,b), cell immobilization techniques, includ-
Using some mutants blocked at different con-
ing coimmobilization of mixed microbial cultures
version steps it was found that four groups of
(O’Reilly and Scott, 1995), development of suit-
inductive enzymes working in 14 consecutive steps
able supports for repetitive and economic utilisa-
were involved in the sterol side chain degradation
pathway. It should be taken into consideration tion of biocatalysts and for cheaper product
that in the whole process more than 80 molecules recovery (Nikolova and Ward, 1993; Lilly, 1994),
of ATP could be formed during the sitosterol side and manipulation of culture media for improved
chain degradation process (Szentirnai, 1990). yields through the use of cyclodextrins (Szejtli,
For modern biotechnology one step biotrans- 1997). Some aspects for the future of biotransfor-
formations of natural sterols into useful end-prod- mations were discussed (Kieslich, 1992;
ucts are of great importance. Production of Schmauder, 1992). Despite the large amount of
testosterone from cholesterol using a single step experimental data published in scientific and
microbial transformation was described with My- patent literature, the problem of bioavailability of
cobacterium sp. cells. The oxidation of testos- steroid substrates to the biocatalysts, arising from
terone to androstenedione is prevented by the the low water solubility of steroid compounds,
addition of higher amounts of glucose (Liu and still remains to be solved. Attempts were made to
Lo, 1997). improve the steroid bioavailability in aqueous so-
The process of transformation of mixtures of lution by different techniques of derivatization,
natural steroids into 9a-hydroxy-AD by a mutant coimmobilization of microbial cells with the sub-
strain of Mycobacterium roseochromogenes was strate (Kaul et al., 1986) and using two-phase
patented by Jekkel et al. (1991). Recently, rapidly aqueous systems for carrying out the transforma-
growing mutant Mycobacterium strains were stud- tion reactions (Kaul and Matthiasson, 1986; Fly-
ied for their ability to degrade b-sitosterol and the gare and Larson, 1989; Andersson and
9a-steroid hydroxylase of Mycobacterium smeg- Hahn-Hagerdal, 1990).
24 B. Angelo6a, H.-P. Schmauder / Journal of Biotechnology 67 (1999) 13–32

Application of water-miscible organic solvents highly hydrophobic cell surface of most microor-
and especially tri-, di- and monovalent alcohols ganisms able to transform steroid compounds
has one promising advantage. As easily utilizable (and especially strains belonging to genera Nocar-
carbon sources they could be incorporated by dia, Rhodococcus, Mycobacterium, and Arthrobac-
resting cells in metabolic pathways for regenera- ter) transformations with whole cells in
tion of reducing cofactors (Strijewski, 1982; An- non-conventional media allow the achieving of a
gelova et al., 1996). homogenous dispersion of the biocatalyst. The
Different detergents are still applied in the pro- use of immobilized microbial cells for biotransfor-
cesses of natural sterols side chain degradation. mations in organic media can make them more
The influence of Tween 80® on growth and resistant to the organic solvents through the ap-
cholesterol oxidation by two rapidly growing My- plication of supports with varying hydrophilic-hy-
cobacterium strains was investigated. Any signifi- drophobic characteristics (Sonomoto et al.,
cant differences in ADD and AD accumulation 1983a,b; Fukui and Tanaka, 1984; Nikolova and
were not found. Although it was reported that Ward, 1993).
cyclodextrins improved product accumulation by It is considered that the toxic effect of organic
inhibiting side chain oxidations of sterol ring solvents on the microbial cells can arise from both
structures (Hesselink et al., 1987, 1989) it was also direct contact of the cells with the organic solvent
suggested that they improved yields by sequester- (phase toxicity) and from the interaction of the
ing ADD (Smith, 1984). cells with the solvent fraction dissolved in the
A liposomal medium made up of multilamellar aqueous phase (molecular toxicity) (Bar, 1987).
(MLVs) and small unilamellar vesicles (SUVs) of The logarithm of the partition coefficient of the
phosphatidylcholine was also applied for choles- solvent in the octanol-water system (log P) was
terol oxidation by Rhodococcus erythropolis proposed as a means of predicting the denaturing
(Goetschel and Bar, 1991; Goetschel et al., 1992). effect of the solvent on the biocatalytic systems
Because of the differences in water solubility of (Laane et al., 1985). The usefulness of the value of
both substrates (cholesterol and hydrocortisone) log P has been confirmed in the conversion of
cholesterol appeared to be a better example of a steroids with A. simplex cells in aqueous/organic
lipophilic water-insoluble substrate for bioconver- solvent two phase systems (Hocknull and Lilly,
sion in a liposomal medium. The SUVs promoted 1988, 1990; Pinheiro and Cabral, 1991) and R.
the biotransformation at a faster rate and to a nigricans in a range of solvent systems (Osborne
higher concentration than those obtained in an et al., 1990).
aqueous sterol dispersion. The mechanism of bio- The moisture content of organic solvents is
transformation in the liposomal medium was in- important in biocatalysis in non-conventional me-
terpreted as a direct interaction between the dia. It was suggested that the differentiation of
microbial cells and the SUVs (Goetschel et al., the states of water, such as freely dissolved water
1992). in the organic solvent in addition to the water
One of the most important technological ad- bound to the catalyst, should be estimated be-
vances with major impacts on biotransformation cause only the catalyst-bound water appear to
is the development of techniques for biocatalysis affect the catalytic activity (Yamane, 1988). In a
in organic media (Lilly, 1994). The first tests were comparative investigation of cholesterol to
made with the transformation of cholesterol into cholest-4-en-3-on bioconversion by A. simplex
cholest-4-ene-3-one by Nocardia sp. cells (Buck- cells it was confirmed that the water content in an
land et al., 1975). Among the main advantages of aqueous/organic two phase system is an impor-
carrying out biotransformations of steroid com- tant parameter of the process (Liu et al., 1994,
pounds in non-conventional media are the en- 1996). The amount of the water retained in the
hanced solubility of the substrates and the easy Cellite adsorbed cells of Mycobacterium sp. has
recovery of the product(s) and biocatalysts. It proven to be an important parameter for biologi-
should be taken into consideration that due to the cal activity retention in bis(2-ethylhexyl)phtalate
B. Angelo6a, H.-P. Schmauder / Journal of Biotechnology 67 (1999) 13–32 25

medium. The best results were obtained in the (Braun and Vecht-Lifshitz, 1991). It was found
range of 0.65 to 0.80 g water/g dry support. that R. nigricans growing in a pellet form hy-
Further drying of the matrix led to cell deactiva- droxylates progesterone at 11a-position with a
tion, a process which could be partially reversed higher activity. This morphological form was
through water addition to the bioconversion considered as a naturally immobilized biomass
medium (Fernandes et al., 1997). and suggested as an alternative in the process of
The process of D1-dehydrogenation of hydro- microbial biotransformation (Znidarsic and
cortisone and its derivatives by free and immobi- Pavko, 1997).
lized cells of A. simplex was studied for It was found that Mycobacterium 6accae cells
establishing the effect of solvent molecular toxic- transform more actively b-sitosterol when grown
ity and microenvironment composition on the on a medium containing egg-yolk lecithin. The
enzyme activity and stability. Chloroform and highest activity is related to the incorporation of
n-decan-1-ol were the best choice of solvent for a considerable amount of lecithin into cell envel-
both, solubility of the substrates and catalytic ope lipids during growth (Rumijowska et al.,
activity of free cells (Pinheiro and Cabral, 1991). 1997).
The system with polyurethane entrapped cells in One possible approach to enhance the
n-decan-1-ol provided the highest long-term ac- lipophilic substrate availability to the biocatalyst
tivity levels (Pinheiro and Cabral, 1992). In both is to increase the cell surface hydrophobicity. It
cases the addition of glutamate was found to was found that the greater hydrophobicity of
improve the retention of enzyme activity. Addi- cells and substrates results in greater attractive
tion of surfactants led to an increased initial
forces and higher level of adhesion (van Loos-
reaction rate but did not significantly change the
drecht et al., 1987; Stenström, 1988). It was
final conversion yields. First-order apparent ki-
shown that Rhodococcus sp. cells grown on n-
netics was observed in a menadione-saturated
alkanes have extremely hydrophobic surfaces
system for global substrate concentrations under
with a high content of unsaturated fatty acids.
10 mM (Fernandes et al., 1995).
The surface and membrane structures of these
Carbon tetrachloride was selected as a suitable
cells exhibit strong modifications in comparison
medium for conversion of cholesterol into AD
and ADD by R. equi cells. The increase in effi- with glucose grown cells and show many mem-
ciency was attributed to the high solubility of the brane surrounded lipid inclusions located in the
substrate and the products in the water-organic disrupted cell walls. It was suggested that the
co-solvent system chosen and to the rapid re- increased 9a-hydroxylating activity observed in
moval of AD and ADD which appeared to be n-alkane grown cells was due to both the en-
toxic for R. equi cells (Ahmad and Johri, 1993). hanced cell-substrate affinity caused by the in-
Several water immiscible organic solvents were creased cell hydrophobicity and the increased cell
tested also for biocompatibility capacities for b- wall permeability resulting from the cell surface
sitosterol side chain cleavage by free and celite® modifications (Mutafov et al., 1997b).
adsorbed cells and bis(2-ethylhexyl)phtalate was The ability of microorganisms to tolerate or-
selected (Dias et al., 1994). It was also shown ganic solvents was chosen as a criterion for se-
that specific activity is related to the carrier par- lecting cholesterol degrading microbial strains.
ticle size (Fernandes et al., 1996). For conversion The isolation of a Pseudomonas sp. strain was
of soybean sterols natural oils were described as described in medium overlaid with a mixture of
a better medium than organic solvents (Phase p-xylene and diphenylmethane containing choles-
and Patil, 1994). terol. It was assumed that the strain would
One interesting approach for improvement of provide an effective and convenient system to
bioavailability of steroid compounds to the bio- oxidize the C3 and C6 position of cholesterol by
catalyst is based on the ability of mycelial mi- introduction of a hydroxyl or keto group (Aono
croorganisms to grow in an aggregated form et al., 1994).
26 B. Angelo6a, H.-P. Schmauder / Journal of Biotechnology 67 (1999) 13–32

5. Outlook  biosurfactants
 modified compounds acting as mediators
As described before there is not enough infor- for solubility of the practically water insolu-
mation on the physiological response of microbial ble substrates (cyclodextrines, etc.)
cells to the presence of lipophilic compounds as a  estimation of the real bioavailability of
basis for a better control of biotechnological lipophilic waste compounds in natural habi-
processes. tats, e.g. soils
The reported data concerning different forms of  new technological variants for the applica-
adaptation mechanisms of microbial cells in the tion of biocatalysts in free or immobilized
process of their interaction with lipophilic com- form or in non-water solvent mixtures
pounds, of the problems in the transport of these 3. Development of systems/sensors/other analyti-
compounds (substrates and/or products) within cal techniques for a direct detection of traces
the complex reaction systems and of the regula- of waste compounds for controling the bio-
tion possibilities allow conclusions for the further technological or environmental processes in an
research and development. In the centre of inter- on-line-, in situ-, on-site-detection system, if
est should be the research on possibilities for an possible.
increasing of the solubility of the lipophilic and 4. Biological and technological safety aspects in-
mostly water-immiscible compounds, on an opti- cluding all questions on releasing of genetically
mal structure of the processes and on possibilities modified microorganisms, patenting etc.
and chances of an application of immobilized
biocatalysts or other technological variants in
these processes. An increase of the process yield,
and of the activity of the biological systems has to Acknowledgements
be more and more in the centre of interest for
researcher, engineers and industrial partners. The authors are thankful for partial support to
These problems are of interest in a broad area the European Commission (grants no. IC15CT
of biotechnology, e.g. in the pharmaceutical or 96-0710 and IC15CT 96-0716).
chemical industry or the environmental biotech-
nology. As a consequence of the critical discus-
sions at the special symposium No. 14 of the ECB References
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