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JOURNAL OF FERMENTATION AND BIOENGINEERING

Vol. 82, No. 2, 113-118. 1996

Autotrophic Growth and Carotenoid Production of Haematococcus


pluvialis in a 30 Liter Air-Lift Photobioreactor
MARK HARKER, ALEX J. TSAVALOS, AND ANDREW J. YOUNG*
School of Biological and Earth Sciences, John Moores University, Byrom Street, Liverpool, UK

Received 28 August 1995/Accepted 21 May 1996

Growth and ketocarotenoid (astaxanthin) by the fresh-water green unicellular alga Hae-
production
matococcus pluvialis has been evaluated in a 301 air-lift photobioreactor. Due to the different culture re-
quirements of the alga during the various stages of its development a two-stage batch production process
(effectively before and after addition of NaCl to the culture) was employed for (i) biomass and (ii) astaxanthin
by this alga. During the first stage, conditions within the reactor (light intensity, levels of nitrogen and phos-
phate) were maintained so as to achieve high rates of algal growth. When the algae in the reactor reached the
stationary phase of growth and levels of nitrogen and phosphate in the medium had become severely depleted
NaCl was added to stimulate the synthesis of ketocarotenoids (>95% astaxanthin, mainly in the form of mono-
and especially di-esters), partly overcoming the need to increase irradiance levels. H. pluvialis exhibited rela-
tively high rates of growth in the air-lift and accumulated up to 2.7% astaxanthin (of the dry cell weight of the
alga). This was, however, lower than could be achieved under laboratory-scale conditions (>5.5%). The use of
a hatch-production process in a air-lift reactor for the synthesis of algal carotenoids is discussed.

[Key words: astaxanthin, carotenoid, Haematococcus]

The occurrence of astaxanthin [(3S, 3S)-3,3-dihydro- This is in part due to several disadvantageous character-
xy-/3,/3-carotene-4,4-dione] in the freshwater microalga istics of the alga when compared to other micro-algae
Haematococcus pluvialis has been subject to increasing (e.g. the halotolerant microalga, Dunaliella spp.), includ-
experimental investigation in recent years due to great ing: (i) a relatively slow growth rate; (ii) a preference for
commercial interest in the pigmenting qualities of this low growth temperatures; (iii) a requirement of light for
carotenoid. Astaxanthin is used as a source in pigmenta- cultivation, and (iv) susceptibility to contamination and
tion of fish in aquaculture (especially salmonids; 1 and predation that may require the use of closed-growth sys-
2) and is also recognized as having superior antioxidant tem (cf. open-pond cultivation of Dunaliella spp. and
activity when compared to other carotenoids (e.g. p-caro- Spirulina spp.). Commercial cultivation of H. pluvialis
tene) and also to rw-tocopherol (3). Haematococcus spp. for the production of astaxanthin has, to date, been
accumulate astaxanthin when exposed to stress condi- limited to the growth of the alga in raceway ponds, con-
tions, generally high irradiances (4), usually in combina- taining 30,000 to one million liters of algal culture (10).
tion with nutrient deprivation (e.g. 5). The accumulation The major problem encountered when operating this
of astaxanthin in H. pluvialis (containing carotenoids open pond system is the contamination of the ponds by
typical of higher plants and the majority of green algae) bacteria, fungi and other faster growing algae, as well as
is associated with a morphological transformation from protozoan predators which have been reported to
green vegetative cells to deep-red, astaxanthin-rich, im- eliminate 90% of the algal biomass within 72 h (Spencer,
mobile aplanospores (6). These aplanospores are sur- K. G., US patent no. 4871551, 1989).
rounded by a thick cell wall composed of the bio-poly- The work reported in this study was conducted to
mer sporopollenin (7). The astaxanthin is accumulated in evaluate the use of a conventional, reliable autotrophic
lipid bodies which coalesce and eventually totally fill the system for the production of carotenoids from H. pluvia-
cell (8, 9). lis, which would minimise or eliminate the threat posed
In recent years there has been considerable interest in by potential predators and competitors. For this purpose
the synthesis of astaxanthin by Haematococcus, and a 30 I glass column, bubble airlift-photobioreactor (with
knowledge about the influence of abiotic factors such as conventional design features) was constructed (Fig. 1). A
irradiance levels, nutrient depletion and the use of ex- two-stage batch process was adopted for the production
ogenous compounds such as salt and ferrous forms of of astaxanthin from H. pluvialis designed to allow for
iron in controlling carotenogenesis in this alga is now biomass production followed by an astaxanthin accumu-
better understood. There is still, however, some debate lation stage. This involved manipulating the culture con-
as to the most suitable conditions (e.g. enrichment or ditions by the addition of NaCl at the onset of the sta-
depletion of nitrogen and phosphate in the culture medi- tionary phase of algal growth when levels of phosphate
um) which will allow for both algal growth and astaxan- and, especially nitrogen had been depleted, in order to
thin production (4, 5). Few studies have, however, consi- promote astaxanthin production in the alga.
dered the practical aspects concerned with the scale-up
of astaxanthin production in this freshwater microalga.
MATERIALS AND METHODS

* Corresponding author. Organism and growth media H. pluvialis 34/7 was


5 Present address: Dept. of Genetics, The Hebrew University of obtained from the Culture Collection of Algae and
Jerusalem, Givat Ram Campus, Jerusalem 91904, Israel. Protozoa, Windermere, U.K. The alga was cultivated in
113
114 HARKER ET AL. J.FERMENT.BIOENG.,

Bolds Basal Medium (11) modified to pH 7.0 (NaN03


2.9 mM; MgS04. 7Hz0 0.3 mM; NaCl 0.43 mM; K2HP04
0.43 mM; KH2P04 1.29 mM; CaCl*. 2H20 0.17 mM;
ZnS04.7Hz0 30.7 PM; MnC12.4H20 7.3 /-M; Moo3
4.9 /rM; CuS04.5H20 6.3 /IM; CoN03.6H20 1.7 /jM;
H1B03 0.18mM; EDTA 0.17mM; KOH 0.55 mM;
FeS04. 7Hz0 17.9 /lM; HzS04 10 /*M).
The photobioreactor In the closed photobioreactor
the alga was grown under semi-axenic conditions
throughout the batch-run. Prior to inoculation of the
algae in the photobioreactor, the reactor was sterilised
by means of a 0.2% sodium hypochlorite solution or
high-pressure steam. The photobioreactor (30 I medium
volume) was of a cylindrical construction and made of
glass (see Fig. 1 for a schematic representation and
dimensions). The photobioreactor was equipped with an
external illumination system composed of four fluores-
cent tubes, which supplied 50 ,nmol rnp2 ssl (PAR) to
the surface of the culture vessel (Fig. 1). The photo-
bioreactor was surrounded by a polythene insulating
sheet in an effort to maintain a relatively constant tem-
perature within the environment closely surrounding the
reactor. No other means of temperature control within
the vicinity of the photobioreactor were available.
Mixing and aeration were achieved by bubbling am-
bient air through a sparger unit located in the base of
the photobioreactor (Fig. 1). Air, sterilised using filters
(0.2 Im, Millipore), was passed via the air inlet tube
(Fig. 1) into the sparger, from which the air penetrated
into the culture medium through a series of capillary
holes. The turbulence caused by air passing up through
the culture vessel was enough to mix the medium Scale drawing of the pilot photobioreactor
sufficiently to keep the cells in constant suspension in the FIG. 1. Schematic representation of the 30 I air-lift photo-
culture medium. The aeration supplied the cells with a bioreactor constructed for the evaluation of growth and carotenoid
continuous source of ambient levels of COz. The air was production in the unicellular microalgae H. pluvialis. Numbers: @ air
pumped through the sparger at a rate of 1.5-3.01 per outlet; 2, air outlet filter; :T media, antifoam and inoculum inlet; 2
min, depending upon the age of the culture so that aera- condenser; :.:I air inlet filters; #G one of four fluorescent tubes sur-
tion was increased during the formation of the non- rounding the bioreactor; 121 growth medium; C.$ air inlet; f.9 sparger;
motile astaxanthin-rich aplanospores in order to keep them 10 sample line and bottle.
in suspension. A silicon-based antifoam (Basildon Chemi-
cals Ltd.) was added to the culture at 0.2ml per liter dry cell weight after centrifugation to separate the cells
of algal culture when required. from the media followed by washing and drying at
When the alga had reached the stationary phase of 100C for 24 h. Pigments (chlorophyll and carotenoid)
growth NaCl was added to the culture medium. Equal were extracted in 100% acetone using a tissue homoge-
amounts of NaCl were added at 30, 35 and 40d to nizer (Mickle Engineering Co. Ltd., UK) and quantified by
achieve a final concentration of 40mM NaCl in the reac- reversed-phase HPLC as described previously (13). A
tor on day 40. The application of NaCl in this manner Spherisorb 0DS2 column (25.0~4.6cm) operating on a
(three separate stages over a period of ten days to yield a solvent gradient of O-100% (over 30 min) ethyl acetate in
final concentration of 40 mM) was used as the results of acetonitrile/water (9/l v/v) at 1.0 ml/min was used to
earlier experiments demonstrated that this would induce separate carotenoids and chlorophylls. Nitrate levels
the culture to produce higher yields of astaxanthin per were determined using selective ion probes (Orion) and
unit volume of culture medium rather than if the salt appropriate standards. Phosphate assays were carried
was added in a single application by minimising high out using the heteropoly blue/ascorbic acid method (14).
rates of cell death (12).
When the astaxanthin levels of the cells in the reactor
RESULTS AND DISCUSSION
had reached the stationary phase of accumulation (as
determined by HPLC analysis-see below), aeration of the The carotenoid composition of the H. pluvialis cells in
culture vessel was stopped. The algal culture was allowed the reactor at different stages of the batch-run as shown
to settle on the base of the reactor and then harvested in Table 1 indicate that the green vegetative cells contain
as a thick algal suspension by allowing the concentrated those pigments characteristic of chloroplasts of the
algal slurry to flow out of the reaction via the sample majority of green algae and higher plants. Such cells
line (Fig. 1). The thick algal suspension was rapidly frozen (examined during exponential growth of the culture) com-
at ~20C and kept for subsequent analysis. pletely lack any ketocarotenoid (e.g. astaxanthin) and
Analytical methods Algal cell growth was deter- contain only the so-called primary carotenoids (;3-caro-
mined either by cell number as determined using a tene, neoxanthin, lutein and the xanthophyll cycle
Improved Neubauer haemocytometer, or by determining carotenoids-violaxanthin, antheraxanthin and zeaxan-
VOL. 82, 1996 ASTAXANTHIN SYNTHESIS BY HAEMATOCOCCUS 115

TABLE 1. Carotenoid composition (expressed as a percentage of total carotenoid) of three different strains of H. pluvialis during
different stages of the life cycle as determined by reversed-phase HPLC (present study)

Percentage of total carotenoid


Carotenoid Vegetative Cells Aplanospores Aplanospores Palmella stage
CCAP 34/l CCAP 34/7 MUR 145 (15) NIVA CHL 9 (16)
Beta-Carotene 8 <l 5 1
Echinenone <l 4
Canthaxanthin - 1 4
Adonirubin - <l 3
Astaxanthin di-ester - 65 34 7
Astaxanthin mono-ester 31 46 76
Astaxanthin - <l 1 1
Lutein 70 1 6 7
Violaxanthin 10 - - 2
Neoxanthin 12 - 1
Total carotenoid ,% of dry wt. 0.5-1.0 2.1 0.7 -

The vegetative cells were analysed early during the exponential phase of growth and the aplanospores towards the end of the stationary phase.
The carotenoid composition (especially esterification) of aplanospores changes with time (see Fig. 3C for additional information). The data for
the palmella stage refer to adult cells collected early in the stationary phase immediately prior to encystment (16).

thin), of which lutein was the most abundant carote- culture are substantially reduced compared to either the
noid. In contrast, highly encysted cells (aplanospores) of control (no NaCl) or the optimum value of 40 mM NaCl
W. pluvialis (harvested during the stationary phase) were (12). The effect of NaCl has been shown to be one of
primarily composed of astaxanthin (in esterified form) the most important factors in governing astaxanthin syn-
but also traces of canthaxanthin and echinenone (the
secondary carotenoids)-up to a maximum carotenoid
content of 2.7% of algal dry weight. The carotenoid
composition of these aplanospores was in agreement
with that reported previously from laboratory-scale cul-
tures of aplanospores (15) or non-motile palmella cells
(taken from early in the stationary phase-prior to
aplanospore formation) (16).
Growth and pigment levels of cells of H. pluvialis
during the course of the bioreactor batch run are shown _._
0 20 40 60 80 100
in Fig. 2. During the early stages of the batch-run the
number of cells (Fig. 2A) increased at a relatively rapid Day
rate which corresponded to high rates of chlorophyll 50
B
synthesis. However during this growth phase the level of

E
total carotenoid increased only slightly (Figs. 2A, B) 40
and represented - 1.O% of algal dry weight. The addition 30
of NaCl (between day 30-40) to the culture had a sig-
nificant effect on the growth characteristics of the culture 20
and the rate of synthesis of carotenoids. NaCl was added
10
to the reactor when the cells had reached the stationary
phase of growth and had the effect of causing an in- a- n
crease in the cell mortality rate within the culture (Fig. 0 20 40 60 80 loo-
2A). This caused a significant reduction in the levels of Day
chlorophyll but stimulated a rapid increase in carotenoid
synthesis (Figs. 2A, B). Chlorophyll levels in the bioreac- 70
tor only began to fall after the addition of NaCl on day
30 (the level on day 29 was the highest recorded and that 60
on day 35-after NaCl addition-was lower than that
measured on day 14). Previous studies on a lab-scale 50
had shown that NaCl addition was an effective way of
accelerating the process of encystment and concomitant 40
synthesis of astaxanthin in H. pluvialis (and in a number
of other fresh-water microalgae, e.g. Oocystis spp.), 30
although its addition will result in some cell death (12).
Those cells which survive the addition of 40mM NaCl Day
are capable of increasing rates of astaxanthin synthesis
FIG. 2. Growth and carotenoid accumulation of H. pluvialis cells
per cell (3-4 x greater than the rates in the absence of in the photobioreactor. Symbols: (A) 0 , cell number (S.E. C4.9?4;
NaCl; see Ref. 12). Levels in excess of 40 mM NaCl (the n=3); 0, dry cell weight (S.E.k3.3,; n=3); (B) 0, chlorophyll
concentration used in this study) will further increase the (S.E.f3.6%; n=3); 0, carotenoid (mg/l) (S.E.i4.7!4; n=3); (C)
rate of astaxanthin synthesis (on a per cell basis) but will q , chlorophyll (S.E. *3.9x; n=3); 0, carotenoid (pg per cell)
also induce higher rates of cell death in the culture and (S.E.+3.1%; n=3). Arrows indicate first and final applications of
therefore the levels of astaxanthin per unit volume of NaCl to the photobioreactor.
116 HARKER ET AL. J. FERMENT.BIOENG.,

thesis in Haematococcus and can nearly be as effective as


exposure to high irradiances in production per algal cell
(exposure to high irradiances can also induce very high
levels of cell death; Ref. 12).
Effectively, two different stages can be promoted in
the culture by the addition of NaCl: in the first (before
NaCl is added) the vegetative cells lack astaxanthin
whilst in the second (after NaCl addition) growth ceases
(indeed cell numbers actually decline), aplanospores are
formed which continue to increase in size and astaxan-
thin content (non-motile adult palmella cells are only
transiently observed after the initial addition of NaCl). 0 20 40 60 80 100
Thus, the addition of NaCl simply serves to accelerate Day
the natural transition to the second (stationary) stage of
the culture and subsequent
Haematococcus (12).
astaxanthin synthesis in 2 4o
The change in the carotenoid composition of the alga 3 30
during the full course of the batch-run is shown in Fig. 8
3. A reduction in the levels of primary carotenoids B
B 20
(especially lutein which declined steadily throughout the U
experiment) after the first addition of NaCl (Fig. 3A) was c
accompanied by an increase in levels of total secondary 4 10
8
carotenoid (especially di-esters of astaxanthin). The in- h:
crease in carotenoid content during the batch run is rn 0
primarily due to the synthesis of astaxanthin (in es- 0 20 40 60 80 100
terified form; Figs. 3B, C) which correlated well with Day
an increase in dry cell weight (Fig. 2A). During the 30 ,
batch-run the total carotenoid content increased by 8-10
fold when expressed on a per unit column or per cell
basis (Figs. 2B, C, respectively). In comparison, total
carotenoid per dry cell weight only increased from -1,Od
to 2.7x, reflecting (at least in part) the synthesis of the
sporopollenin-based cyst wall. Indeed, throughout the
course of the batch-run the dry cell weight of the culture
(Fig. 2A) increased at a constant rate, indicating that
cells of H. pluvialis which survived the addition of NaCl
continued to increase in size as they accumulated astaxan-
thin esters. 0 20 40 60 80 100
Table 1 shows that mono-esters of astaxanthin were Day
the dominant pigments in the previous study by FIG. 3. Change in carotenoid content and composition of cells of
Renstr$m et al. (14), who analysed adult palmella cells H. pluvialis during the course of the batch-run. (A) Changes in the
(in nitrate-depleted cultures) immediately prior to encyst- levels of lutein ( n ), $-carotene (O), neoxanthin (a) and the xan-
ment. In the present study the data clearly shows that thophyll cycle carotenoids (violaxanthin + antheraxanthin + zeaxan-
esterification of astaxanthin and of the astaxanthin mono- thin: q ); (B) increase in total secondary carotenoid content of the
esters continued during the process of secondary carote- culture; (C) increase in levels of astaxanthin mono-esters ( l ), astax-
noid accumulation with the ratio of di-esters; mono- anthin di-esters (0) and canthaxanthin (0). Levels of free
(unesterified) astaxanthin accounted for <2% of total carotenoid.
esters of astaxanthin increasing after NaCl treatment (Fig.
Arrows indicate first and final applications of NaCl to the
3C; see also Ref. 15). Only relatively small amounts of photobioreactor.
both canthaxanthin and echinenone were measured, even
in highly encysted cells and free, or unesterified, astax- been reported for other algal production systems, includ-
anthin accounted for <2% of total carotenoid in encyst- ing ,3-carotene synthesis in Dunaliella spp. On scale-up
ed cells. Not only does the carotenoid content and com- the level of environmental control that can be exerted
position of aplanospores of Haematococcus change with over the algal culture decreases and sub-optimal condi-
the age of the culture, but there also appears to be con- tions may therefore result. In the present study, for
siderable inter-strain variation in the relative amounts example, irradiance levels could not be efficiently main-
of the minor keto-carotenoids (e.g. canthaxanthin) in tained at the high levels (approximately 1,500 /Imol m 2
encysted cells. The ratio of chlorophyll a : b during the s i) previously identified as leading to maximum rates
exponential and stationary phases of the batch run was of astaxanthin accumulation (17).
constant at a relatively low value of 1.5 : 1 (data not The pattern of cell growth and carotenoid accumula-
shown). tion was as expected for an algal batch production
The highest carotenoid content obtained in this reac- system, thus it can be concluded that one or more com-
tor was 2.7% (cell dry weight) which is markedly lower ponents of the system was a limiting factor in cell
than the levels regularly obtained in laboratory-scale growth/carotenoid production during the batch-run.
cultures (>5.5% carotenoid on a dry wt. basis; data not Figure 4 indicates that the alga soon depleted the phos-
shown). Such a reduction upon scale-up of algal cultiva- phate and particularly the nitrate present in the medium
tion is not altogether unexpected and similar trends have during the exponential growth phase which resulted in a
VOL. 82, 1996 ASTAXANTHIN SYNTHESIS BY HAEMATOCOCCUS 117

5 a
4 100
a
._
80
.Z
.c 60
0
# 40
; 20
L
.Z 0
z 0 10 20 30 40 50 60 g
Day 0 20 40 60 80 100
FIG. 4. Effect of nitrate and phosphate concentrations on H.
pluviulis growth in the photobioreactor. (A) A, cell number
(S.E.t4.9%; n=3); (B) q , nitrate; 0, phosphate. Arrows indicate FIG. 5. Temperature and pH of the H. pluvialis culture medium
first and final applications of NaCl to the photobioreactor. in the photobioreactor. (A) A, pH; (B) 0, maximum temperature; A,
minimum temperature (C). Arrows indicate first and final applica-
tions of NaCl to the photobioreactor.
reduction and finally cessation of the growth of the
algae. The depletion of the nitrate content of the medium tion and to obtain the best light regime, high levels of
coincided with the onset of the stationary growth phase mixing were required to reach a turbulent flow of the cul-
and the increased synthesis of carotenoids (which was ture. In microalgal mass culture, productivity is affected
accelerated by the addition of NaCl-see above). Nitrate by the mixing system and when light is considered as a
levels were depleted by day 20 (down to <20x of the limiting factor of growth, the effective self shading of a
starting level-see medium composition above) by which cell has a significant effect on the growth rateicarotenoid
time the number of cells had reached the stationary production. This means that an efficient turbulent flow
phase but the total biomass continued to increase (Figs. (high liquid velocity) must be maintained within the cul-
2A and 4). The depletion of nitrogen does not specifically ture, so each cell is able to receive enough light in order
appear to be a causative agent in the reduction of total to improve the photosynthetic efficiency of the culture
chlorophyll. The level of Fe also declined during the (20). The mixing system employed in the present study
exponential phase falling from 1.79~ 10m5 M to 0.6 X had to take into account the current phase in the life
10e5 M (data not shown). cycle of the alga, so that the culture of green vegetative
During the exponential growth period the density of cells in the exponential phase of growth required a lower
the culture greatly increased restricting the amount of liquid velocity because of their fragility and the relatively
light each cell received. The bioreactor constructed for low cell concentration. The aplanospores are more
the present study was of a typical air-life design with a robust than the green vegetative cells due to their thick
relatively small ratio of surface area (available for illumi- cell wall and their culture in the stationary phase
nation): reactor volume. This is an important implica- required a higher liquid velocity to prevent cell sinking,
tion considering the importance of light for algal growth and to ensure the best light regime for carotenogenesis.
and carotenoid production (1). Green, vegetative, cells Nevertheless the high cell densities of Haematococcus
of EL pluviafis require a relatively low light intensity produced in the batch-run would affect the subsequent
[-40-50pmol rn-* SK (PAR)] to achieve high rates of ability to optimise high irradiance levels for carotenoid
growth, whilst very high light intensities [--l,SOO- synthesis in the same reactor vessel. An alternative light
2,00O/*mol m-* s-l (PAR)] significantly improve the induction system would be preferable, perhaps incor-
rate of astaxanthin synthesis in the aplanospores (17). porating a reactor design that, unlike a conventional air-
Rather than considering the total light energy impinging lift reactor, maximises light interception and is combined
on the culture surface, the most important factor is the with dilution of the culture prior to the stage of astaxan-
quantity of energy available at the cellular level. These thin accumulation.
concepts of light per cell and light regime expressed The results also pose the question of when is the best
by Richmond (18), describe the duration of each ex- time to harvest such a fermentation process? It has been
posure of the average cell in the photic zone, below the suggested that it is commercially viable to harvest Hae-
compensation point, or in darkness. Soeder (19) was matococcus once the carotenoid content of the culture
among the first to elaborate the idea of area1 density, has reached -2.0% of the dry cell weight of the culture
thereby taking into account the mutual shading of cells. (2, 18). In the present study this would have meant
Thus, an efficient growth system for large-scale cultiva- harvesting the bioreactor earlier in the batch-run.
tion of algal biomass (requiring relatively low irradiances During the batch-run the pH of the medium in the
and producing high cell densities) will generally be bioreactor did not deviate significantly from the optimal
unsuited to a process such as carotenogenesis which is pH for cell growth (pH 7.0) (Fig. SA) and no attempt
optimised at high irradiances. was therefore made to control this. Similarly the temper-
To partly overcome the phenomenon of light limita- ature of the culture medium was not subject to any
118 HARKER ET AL. J. FERMENT.BIOENG.,

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17. Harker. M., Tsavalos, A. J., and Young, A. J.: Use of
100f bioreactor of similar design is used as a draw/fill response surface methodology to optimise carotenogenesis in
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ACKNOWLEDGMENT 19. Soeder, C. J.: Massive cultivation of microalgae: results and
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This work was supported by a John Moores University Research 20. Gudin, C. and Chaumont, D.: Cell fragility: the key problem
Grant and by the European Agriculture and Fisheries Research of microalgae mass production in closed photobioreactors.
Programme (AIR2 CT94-1283). Bioresource Technol., 38, 145-151 (1991).