Process Biochemistry Vol. 33, No. 3, pp.

323-329, 1998
© 1998 Published by Elsevier Science Ltd
All rights reserved. Printed in Great Britain
0032-9592/98 $19.00 + 0.00
ELSEVIER
PII: S0032-9592(97)00086-1

Cranberry processing waste for solid state

fungal inoculant production

Zuoxing Zheng & Kalidas Shetty*

Department of Food Science, University of Massachusetts, Amherst, MA 01003, USA

(Received 7 July 1997; revised version received 28 August 1997; accepted 1 September 1997)

Abstract

Cranberry pomace is a primary by-product of the traditional cranberry juice processing industry and its
disposal presents economic and environmental problems. Microbial conversion of cranberry pomace into
various value-added products is a practical approach for solving such disposal problems. The present
research was undertaken to test the growth of several agriculturally and industrially important fungi on
cranberry pomace substrate through solid-state fermentation. Fungi, such as Trichoderma viride If-26, Tricho-
derma harzianum ATCC 24274, and Trichoderma pseudokoningii ATCC 26801, a novel polymeric dye
decolorizing PeniciUium isolate, and a food-grade Rhizopus strain isolated from Tempeh, that produce
industrially important extracellular enzymes were grown on a cranberry pomace-based medium at 25°C for
4 days. The glucosamine content of the heterogeneous fermented mixture was a good indicator of fungal
growth. The maximum growth of all fungi was established on cranberry pomace supplemented with 0.05 g
of C a C O 3 , 2"0 ml of water, and 0"05 g of N H 4 N O 3 o r 0"2 ml of fish protein hydrolysate per gram of pomace.
It was concluded that bioconversion of cranberry processing waste by industrially beneficial fungi through
solid-state fermentation was feasible. This potential can be coupled with the utilization of fish processing
waste as an organic nitrogen source to develop mutually complementary products benefiting both the fishery
and cranberry processing industries. © 1998 Elsevier Science Ltd
Keywords: cranberry waste, fungal inoculants, glucosamine content, solid-state fermentation.

Introduction orange waste [7], sugar-cane pressmud [8], and kiwi-

Approximately 420 million pounds of cranberries are
produced annually in the USA, about 90% of which
are used for processing purposes [1]. The primary
by-product of traditional cranberry juice processing is
cranberry pomace. It consists of the processed skins,
seeds and stems, and constitutes about 5% of the wet
weight of the original fruit. Freshly pressed cranberry
pomace contains a large amount of insoluble carbo-
hydrates with small amounts of protein, minerals and
some remaining juice with sugars and other soluble
substances. Owing to its high moisture content, freshly
pressed cranberry pomace is susceptible to rapid
microbial growth. Like other fruit processing wastes,
such as apple pomace [2], grape pomace [3], tomato
pomace [4], citrus waste [5], pineapple waste [6],
*To whom correspondence should be addressed. Tel.: (+1)
413 545 1022; f a x : (+1) 413 545 1262; e-mail:
kalidas@foodsci.umass.edu
323
fruit peel waste [9], cranberry processing waste is com-
monly used as animal feed or fertilizer. However, its
value as animal feed is very limited because of its low
protein content and its use as fertilizer may not be
economically competitive. Further, direct disposal of
pomace waste to soil or in a landfill poses significant
environmental problems. Thus, the exploration of
novel uses for cranberry waste is needed.
Biological conversion of fruit processing wastes into
various value-added products through solid-state fer-
mentation (SSF) has been of major interest to many
laboratories around the world. SSF deals with the utili-
zation of water-insoluble materials for microbial
growth and metabolism, and it is usually carried out in
solid or semi-solid systems in the near absence of free
water or reduced water content compared with sub-
merged fermentation [10]. Many of the potential
products from fruit pomace wastes have been
developed using the SSF technique, and such products
include ethanol [11], methane [7], lactic acid [8], citric
324 Bioconversion of cranberryprocessing waste
acid [6], protein [5], mushrooms [12], enzyme [13], and
food ingredient [3]. However, no report has been
found dealing with the utilization of cranberry pro-
cessing waste.
Trichoderma is notably capable of producing various
polysaccharide-degrading enzymes which enable it to
grow on cranberry pomace substrate. It has been
reported that some Trichoderma species are widely
used to produce various industrially important enzymes
[14-16]. Trichoderma species can also be effective as
biological control agents against pathogenic organisms
which usually cause many plant root diseases [17]. Tr/-
choderma harzianum also has the ability to degrade
organochlorine pesticides, such as DDT, dieldrin,
endosulfan, pentachloronitrobenzene, and pentachlor-
ophenol, and hence has potential applications for bio-
remediation [18].
Several fungal species belonging to the genus Rhi-
zopus have been used in SSF for several centuries,
especially in Asia for preparing many fermented food-
stuffs. Rhizopus not only enhances the digestibility and
protein content of foodstuffs, but also prevents the
formation of toxic substances such as aflatoxin B1.
Some Rhizopus species can also produce anti-carcino-
genic substances and antibiotics [19,20]. A strain of
Rhizopus oligosporus, which was isolated from commer-
cial Tempeh in our laboratory, was used in this study in
order to develop a potential protein-enhanced value-
added product from cranberry pomace for use as
animal feed.
Another fungus used in this study is a novel Pen-
icillium isolate which is capable of decolorizing the
polymeric dyes Poly R-478 and Poly S-119 in liquid
media [21]. This isolate has potential applications in
bioremediation of aromatic pollutants since it could be
used to remove some dyestuffs from dye-contaminated
water or soil.
Fish offal is a major fishery by-product which is
usually disposed of on land or offshore as waste every
year. Since it has a high nitrogen content [22], its acid
hydrolysate could be supplemented to cranberry
pomace medium to enrich the organic nitrogen for
fungal growth. The objective of this research was to
develop novel approaches to utilize cranberry pomace,
coupled with utilization of fishery waste, to generate
value-added products, like microbial inoculants, using
the beneficial fungi mentioned above. Cranberry
pomace could not only serve as an excellent carbon
source, but in addition it could be used as an organic
carrier for fungal inoculants for food, agricultural and
environmental applications.
Materials and methods
Microorganisms
T. viride IF-26, T. harzianum ATCC 24274, and T. pseu-
dokoningii ATCC 26801 were obtained from the
American Type Culture Collection (Rockville, MD); a
Penicillium sp. ATCC 74414 that decolorized polymeric
dyes was isolated in our laboratory [21]; a strain of
Rhizopus oligosporus was isolated from unpasteurized
Tempeh product. The Tempeh product was kindly pro-
vided by Life-Life Foods Co., Greenfield, MA.
Media and cultivation condition
The microorganisms were maintained on potato dex-
trose agar (PDA) slants and petri plates at 4°C and
subcultured monthly. All fungi were cultured at room
temperature for 7 days before use. 125 ml Erlenmeyer
flasks containing 10 g of cranberry pomace, 0.5 g of
CaCO3, 20 ml water, and 0"5 g of NHaNO3 or 2 ml fish
protein hydrolysate (FPH) as the supplemental
nitrogen source were used for SSF. The freshly pressed
cranberry pomace was obtained from Veryfine, Inc.,
Westford, MA, and was dried and ground and stored
in a refrigerator before use. The water content of cran-
berry pomace used in the experiment was 5.8% (w/w,
wet basis). FPH was obtained from Ocean Crest
(Gloucester, MA) as herring waste containing
0.6575 g m1-1 of soluble solids. The spores from one
PDA plate were inoculated into about 20 flasks. The
flasks were incubated at 25°C for 4 days. The cultiva-
tion of all fungi was also extrapolated for 100 g of
cranberry pomace with proportional addition of other
supplements calculated from the 10 g level.
Protein assay
100 ml of distilled water was added into the fungus-
pomace-containing flasks and the culture was homoge-
nized using a Waring blender, then centrifuged at
1500g for 15 min. The supernatant was used for protein
assay. Soluble protein was determined using a commer-
cial assay kit (Bio-Rad Protein Assay Kit II, Bio-Rad
Laboratory, Hercules, CA) with bovine serum albumin
as standard, according to the procedure described by
Bradford [23]. The soluble protein produced by fungal
strains in the cranberry pomace medium was expressed
as milligrams per gram of pomace (original dry
weight).
Moisture content (MC) and water activity determination
The MC of cranberry pomace medium was determined
by measuring both the wet weight and dry weight of
the sample. After measuring the wet weight, the
sample was dried in an oven at 105°C for 2 days, or
until the weight was constant, before recording the dry
weight. The water activity aw of the cranberry pomace
medium was determined according to the method
described by McCune et al. [24]. A reference material
(circle of filter paper) of known sorption isotherm was
obtained by equilibrating for 24 h to each of six salt
slushes and was equilibrated for 24 h to the sample; the
 Z. Zheng and K. Shetty
water activity of the equilibrated filter paper was cal-
culated using the linear equation of the isotherm curve
from its MC, which was determined by measuring the
weight gain of the filter paper during equilibration. The
water activity of the sample was equal to that of the
equilibrated filter paper. The relationship between MC
(wet basis) and aw of the sample was expressed by the
equation
MC = A + B log (1 -aw)
where, at 25°C,A = 0.0033 and B = -0.1155.
Glucosamine assay
The glucosamine content of the fermented culture
mixture consisting of fungal mycelia and cranberry
pomace medium was used to estimate fungal biomass
during the SSF as the growth indicator of Trichoderma,
Rhizopus, and Penicillium strains. It was determined by
the modified method of Sakurai et al. [25]. After culti-
vation, the culture mixture in each flask was mixed with
100ml of distilled water. The mixture was then
homogenized using a Waring blender. A 1 ml suspen-
sion of homogenized sample was mixed with 2 ml of
H2504 (98%) in a test tube. After standing for 24 h at
25°C, it was diluted with 47 ml of water and autoclaved
at 120°C for 1 h. The hydrolysate was then neutralized
with NaOH to pH7-0 and diluted to 100ml, from
which 0.5 ml was mixed with 0.5 ml of NaNO2 (5%)
and 0-5 ml of KHSO4 (5%) in a centrifuge tube. After
shaking occasionally for 15 min, it was centrifuged at
1500g for 2 min. 0.6 ml of supernatant was then mixed
with 0"2 ml of NHaSO3NH2 (12"5%) and shaken for
3 min. To the mixture, 0.2 ml of 3-methyl-2-benzothia-
zolinone hydrazone hydrochloride (MBTH, 0.5%, pre-
pared daily) was added and then the mixture was
boiled for 3 min. The reaction mixture was immediately
cooled to room temperature following boiling and
0.2 ml of FeCI3 (0"5%, prepared within 3 days) was
added. After standing for 30min, the absorbance at
650nm was measured spectrophotometrically. The
glucosamine content was calculated as milligrams per
gram of pomace (original dry weight) according to the
standard curve.
Results and discussion
The effect of CaC03 supplementation on the growth of
selected fungi
Since the cranberry pomace contains organic acids, the
pH of cranberry-pomace-based medium was relatively
low (approximately 3.0 to 3-2) and hence inhibited the
growth of fungi. In order to obtain the maximal
growth, it was necessary, therefore, to neutralize the
medium before inoculation. In general, most fungi are
able to grow in a pH range of neutral to slightly acidic.
Considering the desired pH range, CaCO3 was con-
325
8.
0.
i i I i i i i i I
1 2 3 4 5 6 7 8 9 10
CaC03 (g/lOOgpomace)
Fig. l. The effect of CaCO3addition on the growth of Tricho-
derma, Rhizopus and Penicillium strains on cranberry pomace.
sidered as an ideal neutralizer because it can increase
the pH of cranberry pomace medium to 5.8-7.0 and it
is inexpensive for large-scale use. Again, Trichoderma
viride, Rhizopus isolate, and Penicillium G-1 strains
were used to examine the effect of CaCO3 addition in
cranberry pomace on fungal growth using 2 mlg
water and 0.05 g NH4NO3. It was demonstrated that
cranberry pomace medium supplemented with
0.04-0.05 g of CaCO3 per gram of pomace would be
satisfactory for the growth of all three selected fungi
(Fig. 1).
The effect of water addition in cranberry pomace on the
growth of selected fungi
The moisture level of the medium is a critical factor
influencing the growth of fungi in SSF. In general, a
higher moisture level results in decreased porosity or
intracellular spaces, lower oxygen diffusion and gas
exchange and enhanced formation of aerial mycelium.
In contrast, a low moisture level will lead to decreased
substrate swelling and decreased microbial growth. In
this study, partially dried and ground cranberry pomace
with an MC of 5"8% (w/w) was used. Since it had very
low water content, additional water was required to
increase the moisture level of the medium. To deter-
mine the optimal amount of water for maximum
growth, the growth of T. viride, T. pseudokoningii, T.
harzianum, Penicillium G-l, and the Tempeh Rhizopus
isolate were examined using the basal cranberry
pomace medium with inorganic nitrogen as described
in the Material and methods section with varying water
contents. The soluble protein produced by various
fungi in the pomace medium was measured as the indi-
cator of growth (Fig. 2). All fungi tested in the experi-
ment grew very poorly if no additional water was
added to the medium; they exhibited very similar
growth patterns as the amount of water supplemented
to the medium increased up to 4 m l g -~ before
326 Bioconversion of cranberry processing waste

solid matrix and cannot be quantitatively separated

from it; so, direct measurement of fungal biomass is
impossible. Many authors have described methods of
indirect biomass estimation including measuring: (a)
| fungal cell constituents, such as ergosterol, nucleic
E
g 2 acids, protein, nitrogen and chitin; (b) primary metabo-
lites, such as CO2, ATP, or enzymatic activity; (3)
nutrient consumption [26, 27].
a. 1 As glucosamine is an essential and stable compo-
nent in chitin of mycelial cell walls, the glucosamine
¢//. /o - * - r . ~,,',~o,,m
content seems to be a useful parameter for the estima-
tion of the total sum of the growing mycelium and its
0 l I I I
changes may correspond to the development of the
0 100 200 300 400 500
mycelium, although the values cannot be converted to
H20 (ml/100g pomace) mycelial weight quantitatively [25]. Roche et al. [27]
Fig. 2. The effect of water addition in cranberry pomace reported that a linear correlation existed between
medium on the growth of Trichoderma, Penicillium and Rhi- cumulative biomass and cumulative glucosamine of fila-
zopus strains. mentous fungi during SSF. Desgranges et al. [26] also
suggested that the glucosamine measurement gave a
good indication of fungal biomass development, but
declining slightly at higher water addition the biomass indicator could only be used to compare
(4.5-5-0 ml g-I). Addition of about 2 ml of water per different media having the same constituents, even if
gram of cranberry pomace was sufficient for the the C/N ratios were different.
maximum growth of all selected fungal strains. In our study, two methods for biomass estimation
were compared when additional nitrogen source was
The relationship between MC and water activity of added to the medium. In the case when NH4NO3 was
cranberry pomace medium supplemented as a nitrogen source, as shown in Figs 3
and 4, the measurement of soluble protein content was
A better way of expressing the MC of cranberry consistent with the measurement of glucosamine
pomace medium is the water activity aw, which indi- content, as both gave a similar growth pattern. Under
cates the availability of water for the growth of fungi. such conditions, therefore, either soluble protein or
The MC and water activity of the cranberry pomace glucosamine content could be used as the growth indi-
medium corresponding to each level of water supple- cator of the selected strains in SSF.
mented to the medium were determined using the
methods described in the Materials and methods The effect of NH4N03 on the growth of selected fungi
section. It appeared that the optimal MC in the
pomace for the growth of all selected fungi was about An important indicator of nutritional regulation of
67% (wet basis), whereas the corresponding optimal growth in SSF is the C/N ratio. The optimal C/N ratio
water activity was 0.99 (Table 1).

Biomass estimation

In SSFs, one of the most important problems encoun-

i.-.,--~
tered is the biomass measurement. Unlike submerged
cultivation, fungal mycelia are intimately bound to the
E
g

Table 1. The effect of water addition on the MC and aw of -J

cranberry pomace medium
n 1
Water addition MC in the pomace aw
to the medium (%, wet basis)
(ml/g pomace)
I I I I
0 5"79 0-4643 0 2 4 6 8 10
1 51"49 0-9747
2 67"34 0"9904 NI-14NOa(g/100g Ix)mace)
3 75-38 0"9938
4 80-05 0-9945 Fig. 3. The effect of NH4NO3 addition on the growth of
5 83"50 0-9960 Trichoderma, Rhizopus and Penicillium strains on cranberry
pomace.
 Z. Zheng and K. Shetty
10
E FPH on the growth of Trichoderma viride, Rhizopus
8.
g pomace medium. Fungal growth was expected to be
E
!
(.9
/ "--X--R/~o/x~
---l.-Pet~
I I I I
2 4 6 8
NH4NOa (g/100g pomace)
Fig. 4. The effect of NH4NO3 supplementation on the glucos-
amine production by Trichoderma, Rhizopus and Penicillium
strains grown on cranberry pomace.
varies in a wide range from 10 to 100 or higher in
various SSF processes, but the availability of C and N
can be more important than the ratio. In most SSF
systems, the carbon source comes from the natural
soluble and insoluble carbohydrates while the nitrogen
source is added. Cranberry pomace contains higher
carbohydrates ( ~ 50% on a dry weight basis), some of
which are fermentable and could be used as a carbon
source, but a relatively low content of nitrogen source
available for fungi became the limiting factor for fungal
growth. Therefore, in order to obtain the optimal
growth of fungi on cranberry pomace, supplementation
of nitrogen source was needed. Two kinds of nitrogen
source, NHaNO3 and FPH, were used in this study.
The growth of T. viride, Rhizopus isolate, and Peni-
cillium G-1 was determined by measuring both the
soluble protein content and glucosamine content of
fermented culture. The effect of NH4NO3 on the
growth of the three selected fungi is shown in Fig. 3 (in
terms of soluble protein production) and Fig. 4 (in
terms of glucosamine content). Addition of about
0-05g of NH4NO3 per gram of pomace gave the
maximal growth of all selected fungal species (Figs 3
and 4).
The effect of FPH on the growth of selected fungi
Fish offal is another important food processing waste,
and its disposal has not yet been solved satisfactorily
[22]. By a combination of papain and acid hydrolysis,
the fishery by-product was converted into FPH in
which a high concentration of nutrients, such as
nitrogen, was expected to enhance the fungal growth
on cranberry pomace medium. Martin and Chintalapati
[22] demonstrated that the growth of Scytalidium acid-
ophilum on acid peat hydrolysate was enhanced when it
327
grew in fish offal-peat compost. We proposed that
FPH could be an alternative nitrogen source for cran-
berry pomace medium enrichment.
We tested the effect of supplementation of herring
isolate, and Penicillium G-1 strains on cranberry
expressed in terms of both soluble protein and glucos-
amine contents. However, it was problematic to use
soluble protein content as the growth indicator because
the partially hydrolyzed products of fish protein, such
as polypeptides in herring FPH supplemented in the
medium, contributed to the total soluble protein
10 content of the fermented medium. This would explain
the misleading and confusing result that the soluble
protein was constantly increasing as the level of FPH
supplemented to the medium increased (data not
shown). In this case, therefore, soluble protein content
was not a reliable fungal growth indicator as it tended
to give an erroneous result. It could be used as a fungal
growth indicator only if the medium contained an ign-
orable amount of soluble proteins. Instead, it was more
reliable to use the glucosamine content as the growth
indicator. It is shown in Fig. 5 that the addition of
about 0.2-0.3ml of FPH per gram of cranberry
pomace resulted in the optimal growth of all selected
fungi.
The growth of all tested fungi on an optimized cranberry
pomace medium
With the goal of obtaining a general cranberry-based
medium for growth of selected fungi, an optimized
medium formula was developed as described in the
Material and methods section. The optimization of the
medium was based on the hypothesis that all the fungi
selected had similar growth patterns in response to
E 6
g
4
E
!
o 2
J .-X--~
I I I I
I0 20 30 40 50
FPH (ml/100g pomace)
Fig. 5. The effect of FPH addition on glucosamine produc-
tion by Trichoderma, Rhizopus and Penicillium strains on cran-
berry pomace.
328
•- ' O - - T. ~ k / e
5 I I J
4
E grade Rhizopus oligosporus grown on cranberry and fish
R wastes could be targeted as an animal feed.
3
2
a.
1
0 I I I I I
0 1 2 3 4 5
Culture time (days)
Fig. 6. Growth curves of Trichoderma, Rhizopus and Pen-
icillium strains on cranberry pomace supplemented with 5%
CaCO3, 5% NH4NO3 and 200% water.
changes of the medium composition; this was corrobo-
rated by the results obtained (Figs 1-5). All five fungal
strains tested were grown on an optimized cranberry
pomace medium supplemented with NHaNO3 as the
nitrogen source and the soluble protein content was
used as the growth indicator, as the amount of soluble
protein in cranberry pomace was ignorable. All
exhibited a similar growth pattern over culture time
(Fig. 6). It appeared that the optimized medium
formula could be used for the growth of all selected
fungi, and under such conditions maximum growth was
reached after 4 days of cultivation. These experimental
conditions will be now used for studies on enzyme and
secondary metabolite production as well as fungal
inoculant testing for various applications. Further
improvement of growth of specific fungal species
chosen in this study is possible if variation in soluble
and insoluble protein contents in the mycelium of each
species is optimized. It was also evident from this
investigation that a better estimate of total fermentable
carbohydrates and phosphorus is required; this may be
limiting the growth of some fungal inoculants for
specific uses envisioned in this study. These issues will
be clarified in our subsequent studies.
Conclusions
Among all possible approaches for disposal of cran-
berry pomace wastes from traditional juice processing,
SSF provides a practical way to convert the waste into
various potential value-added products such as fungal
inoculants. The growth studies on several beneficial
fungi have provided a foundation and novel possibili-
ties for the utilization of cranberry pomace. For
instance: many organochlorine pesticides are persistent
in the soil environment, thus it is feasible to make the
T. harzianum inoculant grown on cranberry pomace
Bioconversion of cranberryprocessing waste
and to apply it into the soil environment for the
purpose of enhancing pesticide degradation; in the case
- of polymeric dye pollution a novel Penicillium sp.
inoculant could be used. On the other hand, food-
The soluble protein content and glucosamine
content of fermented culture could be used for the
estimation of fungal biomass in SSF. However, if the
medium contained a certain amount of soluble proteins
or peptides, glucosamine measurement could be used
as the growth indicator.
6 7
To obtain the maximal growth of the selected fungi
a formula for the cranberry-pomace-based medium was
developed, and further improvements are possible. The
optimized medium was extrapolated and tested and
consisted of cranberry pomace supplemented with
4-5 g of CaCO3, 200 ml water, 5 g of NH4NO3 or
20 ml FPH per 100 g of cranberry pomace.
It should be noted that FPH, a high nitrogen-con-
taining fishery by-product, can serve as a good supple-
mental nitrogen source for cranberry pomace medium.
In this direction, the utilization of cranberry processing
waste can be coupled with the utilization of fishery
waste. Such strategies will integrate two different
natural resource sectors in a beneficial manner.
Acknowledgements
This research was supported by a grant from Veryfine,
Inc., Westford, Massachusetts.
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