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BEST_A_328061 bestxml-als-v1.cls March 26, 2010 14:4
BEST #328061, VOL 40, ISS 5
Use of Filamentous Fungi for Wastewater
Treatment and Production of High Value
Fungal Byproducts: A Review
Sindhuja Sankaran, Samir Kumar Khanal, Nagapadma Jasti, Bo Jin,
Anthony L. Pometto III, and J. (Hans) van Leeuwen
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TABLE OF CONTENTS LISTING
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below:
Use of Filamentous Fungi for Wastewater Treatment and Production
of High Value Fungal Byproducts: A Review
Sindhuja Sankaran, Samir Kumar Khanal, Nagapadma Jasti, Bo Jin, Anthony
L. Pometto III, and J. (Hans) van Leeuwen
0
BEST_A_328061 bestxml-als-v1.cls March 26, 2010 14:4
Critical Reviews in Environmental Science and Technology, 40:1–49, 2010
Copyright © Taylor & Francis Group, LLC
ISSN: 1064-3389 print / 1547-6537 online
DOI: 10.1080/10643380802278943
Use of Filamentous Fungi for Wastewater
Treatment and Production of High Value
Fungal Byproducts: A Review
SINDHUJA SANKARAN,1SAMIR KUMAR KHANAL,2
NAGAPADMA JASTI,2BO JIN,3ANTHONY L. POMETTO III,4
and J. (HANS) VAN LEEUWEN5
1North Dakota State University
Q1
2Department of Civil, Construction and Environmental Engineering, Iowa State University
3School of Earth and Environmental Sciences, The University of Adelaide
4Department of Food Science and Human Nutrition, Iowa State University
5Department of Civil, Construction and Environmental Engineering and Department of
Agricultural and Biosystems Engineering, Iowa State University
Conventional biological wastewater treatment generates large
amounts of low value bacterial biomass. The treatment and dis-
posal of this excess bacterial biomass, also known as waste activated
sludge, accounts for about 40–60% of the wastewater treatment
plant operation cost. A different form of biomass with a higher value
5
could significantly change the economics of wastewater treatment.
Fungi could offer this benefit over bacteria in wastewater treatment
processes. The biomass produced during fungal wastewater treat-
ment has, potentially, a much higher value than that from the bac-
terial activated sludge process. The fungi can be used to derive valu-
10
able biochemicals and can also be used as a protein source. Various
high-value biochemicals are produced by commercial cultivation
of fungi under aseptic conditions using expensive substrates. Food-
processing wastewater is an attractive alternative as a source of
low-cost organic matter and nutrients to produce fungi with con-
15
comitant wastewater purification. This review summarizes various
findings in fungal wastewater treatment, particularly focusing on
byproduct recovery during wastewater treatment. This review also
provides an overview on performance of fungal treatment systems
Address correspondence to J. (Hans) van Leeuwen, Department of Civil, Construction
and Environmental Engineering and Department of Agricultural and Biosystems Engineering,
Iowa State University, Ames, IA 50011, USA. E-mail: leeuwen@iastate.edu
Q2
1
BEST_A_328061 bestxml-als-v1.cls March 26, 2010 14:4
2S. Sankaran et al.
under various operational conditions. Important factors such as 20
pH, temperature, hydraulic and solids retention time, nonaxenic
and axenic operation, and others that affect the fungal treatment
system are discussed. Moreover, certain important practical issues
such as bacterial contamination under nonaseptic operation are
also covered. The goal of the review paper is to evaluate the feasi- 25
bility of cultivating fungi during wastewater treatment for deriving
valuable biochemicals.
KEY WORDS: fungal wastewater treatment, fungal byproducts
resource recovery, yeast, bacterial contamination
INTRODUCTION 30
Microbial communities play an important role in biodegradation of organic
compounds in wastewater—in both natural and engineered systems. Bacteria
are primarily responsible for organic pollutant removal in a typical biolog-
ical wastewater treatment system. Conventional aerobic treatment (e.g., the
activated sludge process) mineralizes organic compounds into carbon diox- 35
ide and water and generates a huge amount of bacterial biomass that is
the predominant component of so-called secondary sludge. On average, the
activated sludge process produces about 0.4 g biomass per 1 gram chem-
ical oxygen demand (COD) removed (Metcalf & Eddy et al., 2003). Thus,
nearly half of the COD removed from wastewater is actually transformed 40
into new bacterial cells. Sludge processing, treatment, and disposal consti-
tute one of the major environmental problems in many countries (Weemaes
& Verstraete, 1998). In fact, the costs associated with treatment and disposal
of excess sludge account for up to 60% of the total wastewater treatment
plant operating costs (Canales et al., 1994). Anaerobic digestion, often used 45
for reducing excess sludge volumes, is energy efficient with lower biomass
production and converting wastewater organics into methane, does not pro-
duce high-value byproducts and is capital intensive (Metcalf & Eddy et al.).
Wastewater treatment places a considerable burden on food-processing in-
dustries, resulting in no other benefits than environmental protection. 50
On the other hand, filamentous fungi are often cultivated in food
industries as a source of byproducts such as protein and biochemicals,
among others, on relatively expensive substrates such as starch or molasses
(Barbesgaard et al., 1992). The use of filamentous fungi to treat high-strength
wastewater is an attractive option. Fungal treatment not only converts the 55
wastewater organics into high-value fungal protein and valuable biochem-
icals, but it also produces highly dewaterable fungal biomass, which can
be used as a source of animal feed and potentially in human diets (Guest &
BEST_A_328061 bestxml-als-v1.cls March 26, 2010 14:4
Fungal Wastewater Treatment 3
Smith, 2002; Stevens & Gregory, 1987; Zheng et al., 2005). Fungi can produce
a wide range of fine biochemicals and enzymes, and are more effective than
60
bacteria in metabolizing complex carbohydrates such as starch (Jin et al.,
1998; Jin et al., 1999d; van Leeuwen et al., 2003).
In addition to food supplements, fungi are often cultured in industry
for use in deriving a variety of beneficial substances such as amino acids,
enzymes, dyes, organic acids, organic alcohols, and others (van Leeuwen
65
et al., 2003). Food-processing wastewater is a rather promising substrate for
the production of fungal protein because it requires minimal supplements to
allow microbial growth. Additional nutrients required for fungal growth are
often minimal during the treatment process. The possibility of wastewater
purification using yeasts and molds for microbial biomass protein (MBP) pro-
70
duction has been investigated for some time (Jin et al., 1998; Jin et al., 1999c;
Zheng et al., 2005). Yeast bioconversion of wastewater has been attractive to
many researchers for ease of cultivation, ability to grow at pH values lower
than 5, and growth rates faster than those of molds (Bergmann et al., 1988;
Gonzalez et al., 1992). In addition, yeasts are less susceptible to contamina-
75
tion by other microorganisms and produce biomass with high nutritive value
(Satyawali & Balakrishnan, 2007). Molds have been considered less suitable
than yeasts for MBP production. However, the filamentous nature of these
fungi simplifies separation and recovery of the MBP from culture media.
Moreover, the obligatory acidophilic properties of these organisms suggest
80
that the fungi would not act as opportunistic pathogens (Jin et al., 1999d;
Nigam, 1994). Mycotoxins produced by pathogenic fungi through secondary
metabolic processes, usually to eliminate other microorganisms competing
in the same environment, could be a concern (Adams, 2001). However, such
fungi can be eliminated as candidates for cultivation. Thus, filamentous fungi
85
could have interesting benefits for industrial wastewater treatment processes.
Fungi have several other advantages over bacteria in biological wastew-
ater treatment, in addition to being a source of valuable fungal byproducts
such as amylase, chitin, and lactic acids. First, fungi contain a group of extra-
cellular enzymes that facilitate the biodegradation of recalcitrant compounds
90
such as phenolic compounds, dyes, and polyaromatic hydrocarbons (PAH),
among others, through nonspecific oxidation reactions (D’Annibale et al.,
2004; Giraud et al., 2001; Jaouani et al., 2005). Bacterial cells, by contrast,
can produce target-specific enzymes for degrading these recalcitrant contam-
inants (Riser-Roberts, 1998; Chr´
ost & Siuda, 2002). Fungi also have a greater
95
resistance to inhibitory compounds than do bacterial species. The hyphal
growth of fungi provides a greater protection for their sensitive organelles.
The cell walls of fungi, a layer of extra-polysaccharide matrix, protect them
from inhibitory compounds through adsorption. Moreover, fungi are eu-
karyotes, having considerably more genes than bacteria, which make them
100
more versatile in tolerating inhibitory compounds (Guest & Smith, 2002). The
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4S. Sankaran et al.
higher number of genes in fungi imparts greater reproductive selectivity,
which might result in better adaptations to the environment (Bennett &
Lasure, 1991).
One of the major reasons for using bacterial biomass in biological 105
wastewater treatment is its well-understood growth kinetics. More research
is needed to examine the kinetics of fungal cultivation and in wastewater
treatment processes. It is necessary to establish a better understanding of
fungal systems to gain acceptance as a wastewater treatment technology.
The purpose of this review is to (a) summarize the technical literature 110
pertaining to fungal wastewater treatment, (b) identify the unique merits
and demerits of the fungal process, and (c) examine the pertinent operating
constraints of the fungal process. Additionally, the review also summarizes
important features related to fungal treatment not previously discussed, and
outlines future research directions. 115
HISTORICAL DEVELOPMENT
The earliest documented research on fungi in wastewater was conducted
by Curtis (1969). This author examined different fungal species commonly
present in domestic wastewater and their effects on wastewater treatment.
Cooke (1976) advocated the use of fungi in wastewater treatment because 120
fungi appeared to show higher degradation rates of organic matter than did
bacteria, with a better ability to degrade complex polymers such as cellulose,
hemicellulose, and lignin. A survey of fungal species indicated around 950
species present in domestic wastewater and polluted waters; however, the
taxonomic assignment was not extended to all fungal isolates. Cooke (1976) 125
suggested the addition of fungal species to the existing microbial community
could aid the wastewater treatment process. The research, however, did
not progress beyond a survey of the populations to create a fungal-based
wastewater treatment system (Guest & Smith, 2002).
Among the different aerobic biological wastewater treatment systems, 130
fungi were found to play an important role in trickling filters (Curtis, 1969;
Cooke, 1976; Taber, 1976). Unlike in the activated sludge process, lichens
(symbiotic association of filamentous fungi and algae) occupy a significant
part of the active biomass in trickling filters. The biomass is typically at-
tached to support media (e.g., gravel or plastic) and is known as biofilm, a 135
natural form of immobilized cells. The biofilm in the trickling filter consists
of bacteria, fungi, and algae. Fungal biomass is habitually present in larger
proportions in wastewater treatment systems containing lignocellulosic ma-
terial and lignin because they are more effective degraders of these materials
than bacteria (Crawford et al., 1983). In contrast to Cooke, Taber stated that 140
although filamentous fungi occurred naturally in wastewater, they did not
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Fungal Wastewater Treatment 5
play a significant role in biochemical oxygen demand (BOD) removal. Taber
suggested that the addition of yeasts might lead to an enhancement in BOD
removal.
Research on microorganisms and their uses in different substrates for
145
the production of single cell protein (SCP) began in the 1960s (Casas, 1993).
On the other hand, the application of fungal systems for deriving valuable
byproducts started only in the early 1970s. Several batch-scale studies have
been conducted using different fungal species to produce animal feed and
SCP from wastewater (Hiremath et al., 1985; Thanh & Simard, 1973a, 1973b).
150
Thanh and Simard (1973b) screened 27 yeast strains for their biomass yield,
while maximizing removal of phosphate, ammonia, and organic compounds
from sterilized wastewater. The removal of COD, total nitrogen, ammonium
nitrogen, and phosphate removal varied from 0 to 72%, 22 to 93%, 27 to 90%,
and 12 to 100%, respectively. In another study, Thanh and Simard (1973a)
155
screened different fungal strains capable of degrading dissolved and sus-
pended organic matter from the wastewater. The authors also made efforts
to obtain the optimum pH and temperature for fungal growth in domestic
wastewater during nutrient removal. Trichothecium roseum was found to be
ideal for wastewater treatment based on its protein content and its ability to
160
remove nitrogen and phosphorus. Hiremath et al. (1985) conducted similar
batch studies using seven fungal species isolated from a wastewater stabiliza-
tion pond to maximize fungal production for animal/human consumption.
The experiments were conducted using a filter-sterilized wastewater at room
temperature for a period of 10 days. The organic removal efficiency was
165
lower than the nutrient removal efficiency, but not significantly different
from the study conducted by Thanh and Simard (1973b). The BOD, ammo-
nium nitrogen, and phosphate removal efficiencies were 53–72%, 49–77%,
and 34–77%, respectively. Because the culture flasks were agitated twice
daily, oxygen availability for biodegradation might have been a limiting fac-
170
tor. Although the results were promising, they were inconclusive because
important parameters such as agitation speed and pH were ignored. Guest
and Smith (2002) stated that the importance of experimental design and enu-
meration techniques were ignored in previous studies in comparing bacterial
and fungal treatment systems. Most importantly, many earlier studies were
175
conducted under aseptic conditions, which make it extremely difficult to
adopt these data for nonaseptic evaluation of fungal wastewater treatment
systems, as would be required in practice.
MICROBIOLOGY OF FUNGI
Fungi are eukaryotes varying in size and shape (Gravesen et al., 1994). The180
size of fungi varies from individual cells (e.g. yeasts) to long chains of cells
BEST_A_328061 bestxml-als-v1.cls March 26, 2010 14:4
6S. Sankaran et al.
that stretch for miles. Fungal cells are oblong (3–8 µm×5–15 µm) with
very large trichomes containing organelles and large intracellular granules
and structures. Some fungi are saprophytes (grow on dead organic matters),
whereas others are parasites. Fungi are absorptive heterotrophs as they break 185
down organics by secreting digestive enzymes onto the substrates and then
absorbing easily accessible organic molecules. Fungal hyphae have small
volumes but large surface areas, which enhance the absorptive capacity of
fungi.
Classification of Fungi 190
Fungi are broadly classified as macrofungi and microfungi according to the
size of their fruiting bodies. Macrofungi form fruiting bodies readily visible
to the naked eye, with a diameter of at least 1 mm (e.g., mushrooms), and as
much as 30 cm in puff balls. Microfungi, on the other hand, are microscopic,
having minute fruiting bodies that cannot be seen by the naked eye (e.g., 195
Penicillium). Their mode of reproduction is through spore formation. Most
fungal spores range from 2 to 20 µm in size and are of different shapes and
colors (Gravesen et al., 1994). Fungi are basically classified by their mode of
reproduction (both sexual and asexual) and the nature of their multinucleate
or multicellular hyphal filaments. Historically, true fungi are classified into 200
five taxonomic divisions. The characteristics of each division are given in
Table 1.
TABLE 1. Characteristics of Major Fungal Divisions (Farabee, 2001; Futcher, 2005)
Division Characteristics
Chytridiomycota The fungi produce zoospores capable of moving on their own through
a liquid medium by simple flagella.
Zygomycota The hyphae do not have one nucleus per cell, but rather have long
multinucleate, haploid hyphae that comprise their mycelia. Asexual
reproduction is by spores produced in stalked sporangia.
Ascomycota They contain more than 30,000 species of unicellular (yeasts) to
multicellular fungi. Yeasts reproduce asexually by budding and
sexually by forming a sac/ascus.
Basidiomycota Mushrooms, toadstools, and puffballs are commonly encountered
basidiomycetes. These conspicuous features of the fungi are the
reproductive structures. Sexual reproduction involves the formation
of basidiospores on club-shaped cells known as basidia.
Deuteromycota A group of fungi that either lack the perfect stage (i.e., sexual
reproduction) or whose perfect stage is as yet undiscovered. They
reproduce most frequently by conidia or conidia-like spores. Many
forms of deuteromycota are pathogenic, affecting man, animals, or
plants.
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Fungal Wastewater Treatment 7
Fungal Composition
Fungi, being eukaryotes, have a cellular chemistry similar to that of other
eukaryotic organisms. The DNA base ratio (percentage guanine +cytosine)
205
of eukaryotic cells is reported to be 38–63%, although the DNA content of
fungi has been found to be as low as 0.15–0.30% (Kendrick, 2000). Most
fungal carbohydrates are polysaccharides, such as chitin, chitosan, mannan,
glucan, starch, and glycogen. Chitin, a principal cell wall component in
Dikaryomycota (group of fungi characterized by hyphae with perforated
210
septa, which usually occur in the dikaryotic phase), is a polymer of β-
1,4 N-acetylglucosamine. Although glycogen is the main storage polymer,
disaccharides such as trehalose and sugar alcohols such as mannitol are
also used. Lipids include long-chain fatty acids (such as palmitic, oleic, and
linoleic acids), phospholipids, and sphingolipids (Kendrick, 2000).
215
Growth Requirements of Fungi
Fungi are free-living heterotrophic osmotrophs in which assimilation of di-
gested food material takes place through the cell wall (Dick, 1997). Fungi
absorb nutrients from the substrates on which they grow. They absorb sim-
ple, easily soluble nutrients, such as sugars through their cell walls via ac-
220
tive and passive transport. They excrete digestive enzymes to break down
complex nutrients into simpler forms that they can absorb. Fungi derive
their energy and intermediates for synthesis from oxidation of compounds
through respiration and fermentation. Fungi produce large quantities of or-
ganic compounds, including acids such as itaconic acids, oxalic acids, lactic
225
acid, pigments (e.g., β-carotene, astaxanthin) and aromatic alcohols (e.g.,
resorcinol and phenol; Moore-Landecker, 1990).
CARBON SOURCE AND NUTRIENTS
Carbohydrates constitute the major carbon source. Fungi differ widely in
their abilities to use different carbon sources, and the utilization efficiency
230
of a defined carbon source may be affected by the medium composition
and the culture conditions, such as pH. Various substrates such as cellu-
losic and lignocellulosic wastes, starch wastes, cheese whey, spent sulfite
liquor (black liquor from sulfite process), molasses, and sugar beet pulp
can be used for MBP and fungal byproduct production. These substrates
235
differ in their compositions that are suitable for growing different types of
fungi. For example, the white- and brown-rot fungi are known for their abil-
ity to degrade lignin and cellulose. The ecological group of white-rot fungi
includes both simultaneous and selective lignin-degrading species. In the
latter group, lignin degradation takes place with minimized consumption of
240
cell wall polysaccharides (i.e., cellulose and hemicellulose). Therefore, the
degradation efficiency towards cellulose is highly diversified with white-rot
BEST_A_328061 bestxml-als-v1.cls March 26, 2010 14:4
8S. Sankaran et al.
fungi and is species-specific. As a consequence, special applications such
as biomechanical pulping require the use of selective lignin degraders. By
contrast, brown-rot fungi include most efficient cellulose-degrading species 245
(Tanesaka et al., 1993; Tuomela et al., 2000). The white-rot fungi have been
studied for treating pulp and paper mill wastewater, cork-boiling wastew-
ater, and other similar wastewaters (Guimar˜
aes et al., 2005; Mendonca
et al., 2004; Raghukumar et al., 2004; Wu et al., 2005; van der Westhuizen &
Pretorius, 1998). Among different species of white-rot fungi, Phanerochaete 250
chrysosporium is the most effective species in degrading lignin and cellu-
lose; it grows slowly and synthesizes low quantities of extracellular enzymes
(Friedrich et al., 1986; Shrestha et al., 2007).
In addition to carbohydrates, fungi also utilize lipids and fatty acids
such as oleate and palmitate as their carbon source (Walker & White, 2005). 255
Roux-van der Merwe et al. (2005) treated oil-processing plant effluent with
different fungal species. The fungi were able to utilize the edible oil as
their sole carbon source. A maximum COD reduction of 98% was achieved
using fungal species, Cunninghamella echinulata. In addition, Emerisella
nidulans produced 408.7 mg/L of gamma-linolenic acid (GLA), a high-value 260
byproduct. The lipase-producing fungi can utilize sunflower oil as a main
carbon source (Ratledge, 1989). The fungal lipases degrade the fats and
oils by converting the triacylglycerols to free fatty acids and glycerol. These
hydrolyzed products are transported by the cell through simple diffusion and
reassembled within the cell (Finnerty, 1989; Roux-van der Merwe et al.). 265
In conventional biological wastewater treatment plants, the microbial
energy source determines the dominant microorganism in the mixed liquor.
Each microorganism consumes specific substrates at different rates due to
their diverse metabolisms (Wainwright, 1992). For example, Graham et al.
(1976) determined the yield of Rhizopus oligosporus,Rhizopus formis,and 270
Aspergillus corymbifera on different substrates and found that the yield varied
based on the microbial preferences (see Table 2).
Jin et al. (2005) studied the production of lactic acid and fungal biomass
from different waste streams by the fungal species Rhizopus arrhizus and
Rhizopus oryzae. Simultaneous saccharification and fermentation processes 275
TABLE 2. Effect of Substrate on 48-hr Yield (g/L) of Dry Mycelia (Graham et al., 1976)
Carbon source Rhizopus oligosporus Rhizopus Formis Aspergillus corymbifera
Glucose 1.452 ±0.12 1.410 ±0.05 1.720 ±0.09
Galactose 1.202 ±0.01 0.688 ±0.07 0.922 ±0.06
Sucrose 0.050 ±0.00 0.043 ±0.02 0.056 ±0.00
Lactose — — 0.060 ±0.01
Raffinose — — 0.082 ±0.00
Stachyose — — 0.146 ±0.03
Soluble starch 1.183 ±0.00 1.100 ±0.01 0.871 ±0.02
BEST_A_328061 bestxml-als-v1.cls March 26, 2010 14:4
Fungal Wastewater Treatment 9
were studied in potato, corn, wheat, and pineapple waste streams. These
media had starch or sugar concentrations of approximately 20 g/L. The
highest rates of lactic acid production and biomass formation were found
in pineapple waste, as glucose was easily accessible for fungus consump-
tion. The results indicated that carbon sources had some impact on the
280
lactic acid production by R. oryzae and fungal biomass production of
R. arrhizus.
The nitrogen source for fungi can be nitrates, nitrites, ammonium, or
organic nitrogen substances, such as yeast extract or peptone, depending on
the type of fungi. Numerous fungi consume nitrates: exceptions are Blasto-
285
cladiales sp.,Saprolegniaceae sp., a few types of yeast, and few higher ba-
sidiomycetes (Cantino, 1955; Liu & Sundheim, 1996; Moore-Landecker, 1990;
Siverio, 2002; Whitaker, 1976). Nitrite is utilized by certain fungal species.
However, it tends to be toxic to most fungi as it can deaminate amino acids
or interfere in sulfur metabolism (Moore-Landecker, 1990).
290
Many fungal species can also utilize organic nitrogen because of their
ability to decompose proteins. Growth enhancing amino acids and peptides
include glycine, glutamic acid, and aspartic acid, whereas leucine can re-
sult in poor growth (Carlile & Watkinson, 1994). A balanced fungal medium
should contain about 10 times more carbon than nitrogen (i.e., carbon to
295
nitrogen [C/N] ratio =10:1). A C/N ratio of 10:1 or less ensures biomass high
in protein content, whereas a C/N ratio in excess of 50:1 favors the accumu-
lation of alcohols, secondary metabolites, lipids, or extracellular polysaccha-
rides (Carlile & Watkinson, 1994).
A major concern in fungal wastewater treatment is nutrient requirements.
300
There have been very few studies that examined the effect of micronutri-
ent supplementation on organic removal and fungal biomass production
during wastewater treatment. Costa et al. (2004) investigated the effects of
different nitrogen sources (sodium nitrate, phenylalanine, and tryptophan)
on mycelial production and glucose uptake by Fonsecaea pedrosoi in the
305
culture media with and without tricyclazole. Tricyclazole is an inhibitor of
1.8-dihydroxynaphthalene (DHN) melanin synthesis. The main characteristic
of F. pedrosoi is the production of DHN melanin. These pigments are not
essential to the growth and development of the fungus, but, on the other
hand, they increase its lifespan and competitive capacity for food in certain
310
environments. Stevens and Gregory (1987) studied the growth of Cephalospo-
rium eichhorniae in potato-processing wastewater to produce MBP. It was
found that 0.61 g (dry weight) of biomass and 0.3 g of crude protein per
gram of carbohydrate could be produced at pH 3.75, with the addition of
monoammonium phosphate, ferric iron, and nitrogen in the form of ammo-
315
nium hydroxide. The pH was maintained using sulfuric acid. The authors
reported that C. eichhorniae utilized organic protein in absence of inorganic
nitrogen. When inorganic nitrogen was added, the fungi preferred inorganic
nitrogen rather than organic nitrogen.
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10 S. Sankaran et al.
Wu et al. (2005) found that the addition of glucose and ammonium 320
tartrate was beneficial for degradation of lignin by white-rot fungi. Cing and
Yesilada (2004) indicated that glucose and cheese whey supplementation
improved the stability and dye decolorization performance by the fungal
pellets. Jasti et al. (2006) operated a Rhizopus oligosporus attached growth
bioreactor with plastic composite support (PCS) tubes, with and without 325
the addition of nutrients (ammonium bicarbonate and potassium hydro-
gen phosphate) for treating corn processing wastewater. The results indi-
cated that although the addition of nutrients improved COD removal rates
and observed yields, it did not affect fungal protein production to a great
extent. 330
Nutrient removal using fungal species has also been studied by vari-
ous researchers. Recently, it has been established that a few fungal species
have the ability to use urea and ammonia as a source of oxidizable ni-
trogen energy as well as a nutrient source (Guest & Smith, 2002). Earlier,
Eylar and Schmidt (1959) conducted a comprehensive study on the ability 335
of fungi to transform ammonia into nitrate and nitrite that undeniably con-
firmed fungal nitrification. Falih and Wainwright (1995) tested the ability of
a wide range of filamentous fungi and yeasts to oxidize urea or ammonium.
Kurakov and Popov (1996) reported that fungi had one to four orders of
magnitude greater resistance to inhibitory compounds such as herbicides 340
and germicides (e.g. atrazine, butylate, metolachlor, trifluralin, nitrapyrin),
as well as greater nitrate and nitrite formation ability than autotrophic nitri-
fying bacteria. Fusarium solani has been recognized independently to show
nitrification and denitrification (Guest & Smith, 2002). Fungal denitrification
lacks nitrous oxide reductase and consequently yields nitrous oxide as the 345
final end product. Other studies on fungal nitrification and denitrification
include those of Shoun and Tanimoto (1991), Shoun et al. (1992), Kobayashi
et al. (1996), and others.
Wheat starch-processing wastewater was treated with R. oligosporus in
a laboratory-scale airlift reactor with a working volume of 3.5 L (Jin et al., 350
1999c). Production of fungal proteins and glucoamylase was also evaluated
during this study. COD removal efficiency exceeding 95% was achieved with
fungal biomass production of 4.5–5.2 g/L (dry wt.) within 14 hr of cultivation.
The effect of nutrient supplementation on the biomass yield and COD re-
moval in a few studies is shown in Table 3. Jin et al. (1999c) and Guimar˜
aes 355
et al. (2005) did not observe any notable improvement in biomass yield or
COD removal with nutrient addition, whereas other studies found a signifi-
cant improvement in the performance during fungal wastewater treatment.
The effect of nutrient addition in wastewater treatment is case-specific, even
depending on the specific type of food processing wastewater used. It is 360
necessary to evaluate the effect of nutrients for a particular type of wastew-
ater prior to fungal treatment. Many studies have not assessed the effect of
combined nutrient supplementation.
BEST_A_328061 bestxml-als-v1.cls March 26, 2010 14:4
TABLE 3. Effect of Nutrient Supplementation on Fungal Biomass Yield and Chemical Oxygen Demand (COD) Reduction in the Treatment of
Different Types of Wastewater
Change in Change in COD
Type of wastewater Nutrients Fungal species biomass yield reduction (%) Source
Food processing
wastewater
Ammonium bicarbonate
and dipotassium
hydrogen phosphate
Rhizopus oligosporus 0.32–0.56 gVSS/
gCODremoved
53–85 Jasti et al., 2005
Sugar refinery wastewater Tween 80, thiamine and
nitrogen
Phanerochaete
chrysosporium
— No change (54–59
decolorization)
Guimar˜
aes et al.,
2005
Olive mill high-strength
wastewater
Sucrose and yeast extract Panus tigrinus 2.2–3.4 g/L 39.5–53.2 D’Annibale et al.,
2004
Cassava-starch-processing
wastewater
Ammonium sulfate Aspergillus oryzae ∼0.65–0.74 g/g COD 90 with nutrient Tung et al., 2004
Wheat milling wastewater Dipotassium hydrogen
phosphate
Rhizopus oligosporus 4.66–5.21 g/L 95.8–97.8 Jin et al., 1999c
Purified potato starch
waste
Ammonium sulfate Cephalosporium
eichhorniae
0.19–7.17 g/L - Stevens &
Gregory, 1987
11
BEST_A_328061 bestxml-als-v1.cls March 26, 2010 14:4
12 S. Sankaran et al.
Essential micronutrients for fungal growth are iron, zinc, copper, man-
ganese, molybdenum, and either calcium or strontium. Different fungal 365
species can have their own specific nutrient needs. For example, Aspergillus
niger requires gallium and scandium, whereas other fungi require cobalt
(Moore-Landecker, 1990). Certain fungi require vitamins in trace quantities,
whereas others synthesize their own vitamins. Many fungi are deficient in
thiamin (e.g., Phycomyces), biotin (e.g., Neurospora), riboflavin, pyridoxine, 370
nicotinic acid, and others (Carlile & Watkinson, 1994; Kendrick, 2000). Vi-
tamin deficiency can be temporary. Some fungal species require additional
growth factors such as inositols and heme (e.g., Pilobolus,Physarum poly-
cephalus; Carlile & Watkinson, 1994).
TEMPERATURE 375
Temperature is an extremely important operational parameter in the growth
rate of fungi. The vast majority of fungi employed in wastewater treatment
are mesophiles, thriving at moderate temperatures of about 20–40◦C. Psy-
chrophilic fungi with an optimum temperature below 10◦C occur commonly
in regions covered with snow and ice during the major part of the year. 380
There are also thermophilic fungi that grow above 45◦C. Maheshwari et al.
(2000) described the physiology and enzymes derived from thermophilic
fungi. Temperature controls the specific growth rate, metabolism, total yield,
and lag period of the fungi.
Temperature affects growth rate, metabolism, nutritional requirements, 385
regulation mechanisms of enzymatic reactions, and cell permeability in a
microorganism. The structure and composition of cytoplasmic membranes in
cells are also altered by temperatures that determine the substrate utilization
rate of fungi (Madigan et al., 2000).
Each species of microorganism prefers a narrow range of temperatures, 390
optimum for its growth and development. Sclerotium rolfsii has a maximum
growth at a temperature of 25◦C with reduced growth at temperatures be-
low 20◦C and above 35◦C (Hussain et al., 2003). In addition to affecting
metabolism, temperature also plays a major role in determining fungal spore
survival. Hong et al. (1997) developed a model to predict the effect of tem- 395
perature on fungal spore longevity and found a negative semi-logarithmic
relationship between longevity and temperature. The authors also indicated
that the sensitivity of spore longevity to both temperature and equilibrium
relative humidity varied considerably among different types of fungi, among
species within each group, and among different strains within certain species. 400
Thus, temperature effects need to be considered in fungal wastewater treat-
ment. In nonaseptic operation, the effects of temperature on bacterial growth
are also critical in fungal wastewater treatment.
Thermophilic fungi require a minimum temperature of growth at or
above 20◦C and a maximum temperature of growth extending up to 60 405
or 62◦C. The hypothesis behind using thermophilic fungi is that a higher
BEST_A_328061 bestxml-als-v1.cls March 26, 2010 14:4
Fungal Wastewater Treatment 13
growth rate achieves a better degradation rate (Maheshwari et al., 2000).
The thermophilic fungi break down the soluble-protein fractions at a rate
twice that of mesophilic fungi.
PH410
The effects of pH on fungal metabolism include availability of metal ions, cell
permeability, and enzymatic activity. The optimum pH for most fungi is in
the acidic range of pH <5.0. Lower pH increases iron availability, whereas
higher pH increases enzymatic activity. It is possible to have two optimum
values in a pH growth curve for fungi (Moore-Landecker, 1990).
415
The biochemical and enzymatic reactions in any biological system vary
with pH. Numerous studies were conducted to determine the optimum pH
for different fungal species (Jasti et al., 2005; Jin et al., 2002; Mishra & Arora,
2004; Riscaldati et al., 2000; Stevens & Gregory, 1987; van Leeuwen et al.,
2002). In these studies, pH was optimized based on their research objectives
420
(e.g., organic removal or fungal biomass and byproduct production). Van
der Westhuizen and Pretorius (1998) reported that the yield coefficients of
Aspergillus fumigatus were 0.44, 0.39, and 0.38 g/g COD at pH 5.0, 5.5,
and 6.1, respectively. As pH increased, the number of opportunistic bacteria
under nonaseptic conditions increased from 1 ×106to 30 ×106colony-425
forming units (CFU)/ml. Similar results were also reported by Jasti et al.
(2006) while using R. oligosporus to treat wet corn-milling wastewater. The
bacterial biomass concentration was found to be greater at a pH of 4.5 than
at pH 4.0. The fungal and bacterial biomass concentration is given in Table 4.
Thus, it could be concluded that the pH affects growth rate as well as the
430
microbial competition in a mixed culture.
The morphology of fungi is also affected by the pH. The morphological
changes attributed to pH variation were from fluffy to clumpy and compact
fungal pellets (Jin et al., 1999d; Metz & Kossen, 1977). Jin et al. (1999b)
investigated the morphological characteristics of three different strains of A.
435
oryzae (e.g., DAR 1679, 3699, 3863) and their yields at varying pH (3.0–6.0)
conditions while treating wheat starch-processing wastewater. The fungal
morphology as well as the yield coefficients differed to a great extent at
different pH conditions as summarized in Table 5. The morphology of fungi,
TABLE 4. Effect of pH on Fungal and Bacterial Biomass Con-
centration in Attached Growth Systems (Jasti et al., 2005)
Fungal biomass Bacterial biomass COD removal
pH (mg VSS/L) (mg VSS/L) (%)
3.5 80 30 34
4.0 625 50 80
4.5 370 70 68
Note. COD =chemical oxygen demand.
BEST_A_328061 bestxml-als-v1.cls March 26, 2010 14:4
14 S. Sankaran et al.
TABLE 5. Morphology and Biomass Yield (g dry weight/L culture media) with Varying pH
for Aspergillus oryzae Strains Grown in Starch-Processing Wastewater (Jin et al., 1999b)
Growth
medium DAR 1679 DAR 3699 DAR 3863
pH Morphology Yield Morphology Yield Morphology Yield
3.0 Dispersed mycelia 2.67 Clumpy mycelia 4.69 Dispersed mycelia 2.83
3.5 Dispersed mycelia 3.21 Compact pellets 5.03 Fluffy mycelia 3.45
4.0 Fluffy mycelia 4.32 Compact pellets 5.18 Clumpy pellets 4.26
4.5 Fluffy mycelia 5.22 Compact pellets 4.94 Compact pellets 5.17
5.0 Clumpy mycelia 5.37 Clumpy pellets 4.12 Compact pellets 5.47
5.5 Clumpy mycelia 5.25 Clumpy mycelia 3.25 Compact pellets 5.42
6.0 Clumpy mycelia 4.54 Fluffy mycelia 3.05 Compact pellets 5.18
in turn, affects the viscosity of the medium, thereby affecting the oxygen 440
transfer efficiency and mixing in the biological system. Van Suijdam and
Metz (1981) described the influence of various parameters (chemical factors:
carbon dioxide, substrate, pH; physical factors: shear, temperature, pressure)
on the morphology of filamentous fungi.
OXYGEN 445
Most fungi are obligate aerobes, requiring molecular oxygen for their growth.
Therefore, fungi are usually found growing on or near the surface of the
substrate. Some fungi are facultative anaerobes, which survive in oxygen-
limited environments, including sewage sludge and polluted waters (Tabak &
Cooke, 1968). Utilization of carbon and nitrogen may be affected by oxygen 450
availability. Insufficient oxygen supply increases the nutritional demand and
thereby decreases fungal growth (Moore-Landecker, 1990).
ADVANTAGES OF FUNGI IN WASTEWATER TREATMENT
A traditional biological wastewater treatment system generates large quanti-
ties of sludge (mainly bacterial biomass), which is of low value and expensive 455
to treat before disposal. Meanwhile, fungi are often cultivated in industry
as a source of a variety of valuable biochemicals. Integrating wastewater
remediation with recovery of valuable resources may possibly lead to an
economically viable solution for sustainable waste management. From these
perspectives, the fungal wastewater treatment process can be an attractive 460
alternative that utilizes a low-cost organic substrate as a feed to generate
high value fungal byproducts with concomitant wastewater remediation.
The fungal wastewater treatment process offers several inherent mer-
its, including (a) higher degradation rates of complex organic compounds
present in wastewater due to the presence of specific fungal enzymes, 465
BEST_A_328061 bestxml-als-v1.cls March 26, 2010 14:4
Fungal Wastewater Treatment 15
(b) efficient solid separation of the fungal biomass from the mixed liquor,
and (c) the possibility to recover valuable fungal byproducts.
Pollutant Removal
Removal and detoxification of pollutants can be achieved by physical, chem-
ical, or biological means. However, a biotechnological approach is widely
470
adopted due to its cost-effectiveness, higher efficiency, and generation of
nontoxic value-added products. Thus, the fungal process not only offers a
solution to wastewater remediation, but also provides an opportunity for
byproducts recovery. One of several advantages of the fungal process is the
enzyme-mediated activity that provides solution to the treatment of waste
475
streams containing hazardous or xenobiotic organic pollutants. The enzymes
are produced during all phases of the fungal life cycle and are present even
at low pollutant concentrations (Ryan et al., 2005). Fungal biomass secretes
specific and nonspecific extracellular enzymes that have attracted the atten-
tion of researchers working on degradation of complex high-molecular-mass
480
organic pollutants. For instance, the extracellular enzymes secreted by white-
rot fungi catalyze PAH degradation through nonspecific oxidation reactions
leading to the formation of varieties of quinones and hydroxylated aromatic
compounds (Giraud et al., 2001). White-rot fungi produce highly oxidative
enzymes, such as ligninase, phenol-oxidase, and manganese-peroxidase, that
485
are capable of degrading lignin, phenol, dyes, and various other xenobiotic
pollutants (Boyle et al., 1992; Elisa et al., 1991; Libra et al., 2003; Yesilada
et al., 1999). Examples of a few fungal species used for treating specific types
of waste and wastewater include Myrothecium verrucaria and Trametes hir-
suta that degrade cellulose-rich waste; A. niger that degrades apple distillery
490
waste; P. chrysosporium that degrades lignin; Pleurotus ostreatus that de-
grades lignocellulosic biomass; Alternaria tenuis, A. niger, and Trichoderma
viride that degrade plastic; Humicola grisea that degrades raffinose; and
P. chrysosporium that degrades veratryl alcohol (National Collection of In-
dustrial Microorganisms, 2005). This is a short list of information compiled
495
by the National Collection of Industrial Microorganisms (NCIM), National
Chemical Laboratory, New Delhi, from WFCC-MIRCEN World Data Centre
for Microorganisms (WDCM) listing for fungal species degrading specific
types of wastes.
In addition to extracellular enzyme production, fungal cell walls and
500
their components play a major role in biosorption of toxic compounds during
wastewater treatment. A detailed review of heavy metal biosorption by fungi
can be found elsewhere (Kapoor & Viraraghavan, 1995), and is not discussed
in the present review. Aksu (2005) reported biosorption of organic pollutants
by various types of fungal species. Denizli et al. (2004) found that fungal
505
biomass removes considerable amounts of organic pollutants from aqueous
BEST_A_328061 bestxml-als-v1.cls March 26, 2010 14:4
16 S. Sankaran et al.
solution through adsorption. Fungal biomass has been identified as a most
effective biosorbent for removal of toxic metals, such as chromium (Park
et al., 2005; Say et al., 2004), copper (Ozsoy et al., 2008), mercury, nickel,
cadmium, and lead (Ozsoy, in preparation) from wastewaters.
Q3 510
Excess Wastewater Sludge Treatment
Separation and treatment of excess bacterial sludge produced during con-
ventional biological wastewater treatment is one of the most important and
expensive steps. Sludge dewatering has been cited as one of the most ex-
pensive and least understood processes (Alam & Fakhru’l-Razi, 2003). The 515
enhanced separability of fungal biomass offers an advantage over bacterial
biomass in various facets. Fungi can grow in submerged culture in different
forms ranging from dispersed mycelia (filamentous growth) to filamentous
pellets. Fungal pellets also have a potential application as immobilized cell
systems because they do not require cross-linking or entrapment (Truong 520
et al., 2004). The fungal pellets autoimmobilize the secreted enzymes in-
volved in hydrolysis within its fungal matrix.
The easier separation of fungal biomass is based on its filamentous na-
ture, facilitating better screening or filtration and therefore easier separation
of the biomass from wastewater, leading to reduced treatment costs (Truong 525
et al., 2004). The pellet is still easier to separate by either settling or screening
(Jin et al., 1999b).
Fungal systems are also employed in wastewater treatment, in a bacterial
sludge treatment called the liquid state bioconversion process. Filamentous
fungi are used to entrap and immobilize solid particles of sludge to form 530
larger pellets or flocs in the liquid state bioconversion process, which en-
hance the subsequent separation and biodegradation (Alam & Razi, 2003).
The fungal filamentous mycelia modify the structure of biosolids, thereby
enhancing their bioseparation, dewaterability, and filterability (Mannan
et al., 2005). 535
Byproduct Recovery
The potential for utilizing various fungal byproducts produced from the
wastewater treatment process has not been explored in large-scale wastew-
ater treatment systems. Fungi are normally cultured under aseptic condi-
tions in monocultures on relatively expensive substrates, such as starch, corn 540
syrup, or molasses. Fungal processes, however, could also utilize a low-cost
substrate such as corn processing waste streams for simultaneous recovery
of fungal byproducts and concomitant wastewater remediation (Jasti et al.,
2005, 2006; van Leeuwen et al., 2003). Researchers evaluated different fun-
gal species as a potential source of byproducts, as given in Table 6. Table 7 545
BEST_A_328061 bestxml-als-v1.cls March 26, 2010 14:4
TABLE 6. Fungal-Derived Byproducts and Their Economic Importance
Product Fungal species Source Application
Amylase Aspergillus niger, Aspergillus oryzae,
Lentinula edodes, Neurospora crassa
El-Zalaki & Hamza, 1979; Jin
et al., 1998; NCIM, 2005
Used in brewing and fermentation
industries, the laundry industry, the
paper and food industry
Asteriquinone Aspergillus terreus NCIM, 2005 Used for biomedical applications
Cellulase Aspergillus fumigatus, Aspergillus niger,
Cladosporium sp., Fusarium sp., Myrothecium
verrucaria, Phanerochaete chrysosporium,
Gloeophyllum trabeum, Trichoderma reesei
NCIM, 2005; Rasmussen, 2007;
Shrestha, 2007
Used as digestive aids, for the management
of flatulence, ethanol production from
cellulose
Cell-wall lytic
enzyme
Trichoderma reesei, Trichoderma viride NCIM, 2005 Used in hydrolysis
Collagenase Arthrobotrys conoides NCIM, 2005 Used in medicines and ointments
Chitin Phycomyces blakesleeanus, Aabsidiarepens,
Absidia blakesleeanus, Cunninghamella
elegans
Allan et al., 1978; Knorr & Klein,
1986; Davoust & Hansson,
1991; NCIM, 2005
Used to reduce serum cholesterol levels,
for treating drinking water, used in
medicine, cosmetics, and other
biomedical applications
Chitinase Aspergillus flavus, Aspergillus niger, Aspergillus
oryzae, Beauveria bassiana, Penicillium
chrysogenum
Allan et al., 1978; Andrade et al.,
2000
Used in ointments, medicines, and other
clinical applications
Glucoamylase Mucor javanicus, Neurospora crassa, Aspergillus
sp., Rhizopus sp.
Manjunath et al., 1983; Stone
et al., 1993; Tan et al., 1996;
Norouzian et al., 2006; NCIM,
2005
Used in saccharification for starch
conversion and alcohol production
Lactic acid Rhizopus oryzae
Rhizopus arrhizus
Rosenberg & Kriof´
ıkova, 1995;
Mirdamadi et al., 2002; Jin et
al., 2003; Huang et al., 2005;
NCIM, 2005
Used to ferment milk products, as a
preservative in foods
Protease Acremonium chrysogenum, Penicillium
roqueforti, Beauveria feline
NCIM, 2005 Used in food fermentation, medicine
Note. NCIM =National Collection of Industrial Microorganisms. COD =chemical oxygen demand.
Q4
17
BEST_A_328061 bestxml-als-v1.cls March 26, 2010 14:4
TABLE 7. Studies on Fungal Wastewater Treatment and Byproduct Recovery
Substrate Fungal species Byproduct recovered Growth condition Source
Apple distillery wastewater Aspergillus awamori,
Trichoderma reesei
Fungal protein Nonaseptic Friedrich, 1987
Starch wastewater from wheat
milling
Aspergillus oryzae α-amylase, fungal protein Nonaseptic Jin et al., 1998
Soybean and mung bean residues
(food-processing byproducts)
Aspergillus niger, Rhizopus
oryzae, Candida albicans
Chitosan Nonaseptic Suntornsuk et al., 2002
Potato-processing wastewater Rhizopus arrhizus Lactic acid Nonaseptic Huang et al., 2003
Food-processing wastewater Rhizopus oligosporus Fungal protein Nonaseptic van Leeuwen et al., 2003
Cassava-starch-processing
wastewater
Aspergillus oryzae Fungal protein Aseptic Truong et al., 2004
Potato-processing wastewater Aspergillus foetidus Aspergillus
niger
Fungal protein Nonaseptic Mishra & Arora, 2004
Corn-processing wastewater Rhizopus oligosporus Chitin Nonaseptic Jasti et al., 2006
xxx
Q5
18
BEST_A_328061 bestxml-als-v1.cls March 26, 2010 14:4
Fungal Wastewater Treatment 19
summarizes some of the wastewater treatment studies carried out using fun-
gal species to recover specific byproducts.
FUNGAL PROCESSES
Application in Wastewater Treatment
Studies conducted on fungal wastewater treatment have different objec-550
tives varying from organic removal to fungal biomass production. Broadly,
wastewater treatment can be classified as (a) wastewater treatment for recal-
citrant pollutant degradation, and (b) wastewater treatment for concomitant
pollutant degradation and byproduct recovery. The focus of this review is
on fungal wastewater treatment with concomitant recovery of value-added
555
products.
RECALCITRANT POLLUTANT DEGRADATION
In view of the excellent recalcitrant compound degradability of certain fungi,
researchers have been more inclined to explore fungal degradation of toxic
industrial wastewater. These include wastewaters from textile, olive milling,
560
and food-processing industries, and others. The objective of using fungi for
wastewater purification in textile and olive mills is mainly to remove aromatic
compounds present in the wastewater. Coulibaly et al. (2003) reviewed some
of the fungal wastewater treatment processes treating domestic wastewater,
agro-based industrial wastewater, dyes, and metal-containing effluents. The
565
operating conditions and the results from various studies conducted on dif-
ferent types of wastewater are presented in Table 8.
The textile industry could use fungi for wastewater treatment to degrade
organic dyes. Several studies have been conducted on the ability of fungi
to decolorize specific dyes using fungi (Cing & Yesilada, 2004; Libra et al.,
570
2003; Santos et al., 2004). Dye degradation by fungi is possibly due to the
production of the lignin-modifying enzymes laccase, manganese peroxidase
(MnP), and lignin peroxidase (LiP) that mineralize lignin or dyes (Chander
and Arora, 2007; Harazono & Nakamura, 2005; Michel et al., 1991; Mohorcic
et al., 2006; Raghukumar, 2000; Raghukumar et al., 1996). Additional decol-
575
orization is also obtained through adsorption even by dead cells. Fu and
Viraraghavan (2001) exclusively reviewed the fungal decolorization of dye
wastewaters, and summarized various studies on decolorization using dead
and live cells. Various additional factors that affect fungal decolorization were
also described. More than 35 fungal species were identified for their ability
580
to decolorize various dyes. However, white-rot fungi are most commonly
used for dye decolorization.
Cing and Yesilada (2004) studied the decolorization of Astrazon Red dye
by Funalia trogii. The decolorization rate obtained by two different methods,
namely the growing cell method (growth under agitated culture conditions)
585
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TABLE 8. Review of Operational Parameters in Fungal Wastewater Treatment Processes
Wastewater
source
Treatment
process Operational parameters Fungal species Objectives Significant findings Source
Recalcitrant pollutant degradation
Textile
wastewater
Sequencing
batch
reactor
(4 L)
Temperature: 18–24◦C
pH:5;COD:n/a
Initial inoculum: 100 mL
Aeration: 200 rpm
HRT: 5–14 days
Trametes versicolor Decolorization and
bacterial
contamination
COD reduction: n/a
Optimum pH: n/a
Optimum temperature: n/a
Color removal: 91–99.5%
Unstable performance with
white rot under nonsterile
condition
Borchert &
Libra, 2001
Textile
wastewater
(diazo dye)
Sequencing
batch
reactor
(4 L)
Temperature: 18–24◦C
pH:5.5;COD:n/a(RB5
dye—100 mg/L)
Initial inoculum: 10–20 g
Aeration rate: 300 rpm
HRT: 5–10 d
Trametes versicolor Optimization of
treatment process
(dye degradation)
99% decolorization under
sterile conditions, 80–95%
decolorization under
non-sterile conditions after
5–10 days
Libra et al.,
2003
Cork-boiling
wastewater
Batch studies
(200 mL)
Temperature: 25◦C
pH: 4.7
COD: 7.5 ±0.5 g/L
Initial inoculum: 2 g/L
Aeration rate: 150 rpm
Incubation time: 48 hr to 5
days
Sporothrix, Trichoderma
koningii, Penicillium
glabrum, Fusarium
flocciferum,
Phanerochaete
chrysosporium
Detoxification and
degradation of cork
boiling wastewater
COD reduction: 54.7% ±
4.7% Optimum pH: n/a
Optimum temperature: n/a
Other findings: 50% of the
COD was nondegradable
Mendonca
et al., 2004
Phenolic
wastewater
Airlift
bioreactor
(3.5 L)
Temperature: n/a
pH: 5
COD: n/a
(Phenol: 3.45 mM p-cresol:
1.04 mM m-cresol: 1.08
mM o-cresol: 3.20 mM)
Initial inoculum: 10% v/v
Aeration rate: 2.2 L/min
HRT: n/a
Trametes pubescens Bioremediation COD reduction: n/a
Optimum pH: n/a
Optimum temperature: n/a
Other findings: Optimum air
supplywas3.5L/min
Removal rates of phenol,
p-cresol, m-cresol, and
o-cresol was 0.033, 0.011,
0.012 and 0.035 g
phenol/g biomass/d,
respectively
Ryan et al.,
2005
20
BEST_A_328061 bestxml-als-v1.cls March 26, 2010 14:4
Olive oil mill
wastewater
Batch studies
(200 mL)
Temperature: 28◦C
pH: 4
COD: n/a
Initial inoculum: 13 U
laccase
Aeration rate: 160–180 rpm
Incubation time: 8 days
Mediators used
Pycnoporus
coccineus(laccase)
Optimization of
treatment process
(aromatic
compounds
degradation)
COD reduction: 71.55% ±
4.65%
Optimum pH: 3.5–4.0
Optimum temperature: 60◦C
Other findings: Cu2+and
ethanol increased fungal
activity; Mediator
enhanced degradation.
Jaouani et al.,
2005
Sugar refinery
wastewater
Rotating
biological
contactor
(1.5 L)
Temperature: 38◦C
pH: 4.5
COD: n/a
Initial inoculum: 10% v/v for
attached growth
Aeration: 4 rpm disk rotation
HRT: 3 days
Phanerochaete
chrysosporium
Decolorization and
phenol removal
COD reduction: 48%
Optimum pH: n/a
Optimum temperature: n/a
Other findings:
Color removal: 55%
Phenol removal: 63%
Guimar˜
aes
et al., 2005
Pulp mill
wastewater
Batch studies
(100 mL)
Temperature: 28◦C
pH: 3–6
COD: 4.6 g/L
Initial inoculum: n/a
Aeration rate: No agitation
Incubation time: 16 days
Phanerochaete
chrysosporium,
Trametes versicolor
Optimization of
treatment process
(lignin degradation)
COD reduction: 48%
Optimum pH: 8–11
Optimum temperature: n/a
Other findings:
Lignin removal: 71%
Addition of 1 g/L glucose
and 0.2 g/L ammonium
tartrate was beneficial for
the lignin degradation
Wu et al.,
2005
Olive mill
wastewater
Stainless
steel airlift
reactor
(100 L)
Temperature: 20–35◦C
pH: 4.9
COD: 70–130 g/L
Initial inoculum: n/a
Aeration rate: 0.25 v/v per
minute
HRT: 3–5 days
Phanerochaete
chrysosporium
Wastewater
pretreatment for
reuse
COD removal: 20–50%
Optimum pH: n/a
Optimum temperature: n/a
Other findings:
Reduction in relative toxicity
was 70%
Dhouib et al.,
2006
(Continued on next page)
21
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TABLE 8. Review of Operational Parameters in Fungal Wastewater Treatment Processes (Continued)
Wastewater
source
Treatment
process Operational parameters Fungal species Objectives Significant findings Source
Textile
wastewater
Continuous
biological
rotating
contactor
reactor
(1 L)
Temperature: n/a
pH: 3–4
COD: n/a
Initial inoculum: Attached
growth
Aeration rate: DO—7 mg/L
HRT: 3 days
Trametes versicolor,
Pleurotus flabellatus
Decolorization of
Reactive Blue 4
(anthraquinone dye)
COD reduction: n/a
Optimum pH: n/a
Optimum temperature: n/a
Other findings:
Variation in C source did
not affect decolorization
Decolorization efficiency:
70%
Nilsson et al.,
2006
Concomitant pollutant degradation and byproduct recovery
Starch-
processing
wastewater
Airlift
bioreactor
(3.5 L)
Temperature: 35◦C
pH: 5.5
COD: 5.5–10 g/L
Initial inoculum: 5% v/v
spore suspension
Aeration rate: 1–1.7 v/v per
minute
HRT: 24–30 hr
Aspergillus oryzae Fungal protein and
α-amylase
production
COD reduction: 47–96%
Optimum pH: 5.0
Optimum temperature: 35◦C
Other findings: α-amylase
had stable activity at pH
5–9 and temperature
25–35◦C
Jin et al., 1998
Starch-
processing
wastewater
External
airlift
bioreactor
(160 L)
Temperature: 35–38◦C
pH: 4.5–6.5
COD: 16–22 g/L
Initial inoculum: 8% v/v
Aeration rate: 0.5–1.2 v/v
per minute
HRT: 8–10 hr
Aspergillus
oryzae,Rhizopus
oligosporus
Fungal protein
production;
wastewater
reclamation
COD reduction: 71.55% ±
4.65%
Optimum pH: 5–6
Optimum temperature:
35–38◦C
Other findings: 65.65% ±
3.05% nitrogen removal
Jin et al., 2002
Corn-
processing
wastewater
Completely
stirred tank
reactor
(1.5 L)
Temperature: 20–50◦C
pH: 2.5–7
COD: 0.8–1.2 g/L
Initial inoculum: n/a
Aeration rate: n/a
HRT: 2–8 hr
Rhizopus oligosporus Selective cultivation
of fungal biomass
COD reduction: max. 82%
Optimum pH: 4.0
Optimum temperature: 38◦C
Other findings:
Microscreen was effective in
reducing bacterial
contamination.
van Leeuwen
et al., 2003
22
BEST_A_328061 bestxml-als-v1.cls March 26, 2010 14:4
Potato-chip-
industry
wastewater
Batch studies
(25 mL)
Temperature: 28 ±1◦C
pH: 4–6
COD: 8.122 g/L
Initial inoculum: 5% v/v
Aeration rate: n/a
Incubation time: 96 hr
Aspergillus foetidus,
Aspergillus niger
Fungal protein
production;
optimization of
treatment process
COD reduction: max. 90%
Optimum pH: 6
Optimum temperature: n/a
Other findings:
Max. accumulation of
reducing sugars at pH 4
Mishra &
Arora, 2004
Cassava-
starch-
processing
wastewater
Batch studies
(100 mL)
Temperature: 28◦C
pH: 3–6
COD: 8.8–8.92 g/L
Initial inoculum: 1–10% v/v
Aeration rate: 120 rpm
Incubation time: 120 hr
Aspergillus oryzae SCP production COD reduction: max. 92%
Optimum pH: 4.0
Optimum temperature: n/a
Other findings:
N-enhanced biomass
production
Truong et al.,
2004
Potato-
processing
wastewater
Batch studies
(100 mL)
Temperature: 22–38◦C
pH: 3–7
COD: 20–30 g/L
Starch: 12.8–62.8 g/L
Initial inoculum: 5% v/v
Aeration rate: 150 rpm
Incubation time: 48 hr
Rhizopus arrhizus Starch treatment and
lactic acid
production
COD reduction: 96%
Optimum pH: 5–6
Optimum temperature: 30◦C
Other findings:
Max. lactic acid produced
was 21 g/L, with
conversion efficiency of
95% in 52 hr
Huang et al.,
2003
Note. n/a =not available; COD =chemical oxygen demand; HRT =hydraulic retention time.
23
BEST_A_328061 bestxml-als-v1.cls March 26, 2010 14:4
24 S. Sankaran et al.
and the pellet method (free and immobilized fungal pellets), were compared.
The pellet method decolorized Astrazon Red dye, a monoazo textile dye,
rapidly, to 96% in 24 hr without any visual sorption of any dye to the pellets,
while growing cell method could only decolorize up to 16–24%. Glucose and
cheese whey supplementation improved the dye decolorization performance 590
of the pellets, which remained high and stable for 10 days. Santos et al.
(2004) screened several fungal species to identify their ability to degrade a
reactive dye (Blue BF). Among the 13 different fungal species, it was found
that P. chrysosporium exhibited the highest growth rate with decolorization
efficiency of 39–51%. 595
Kim et al. (2004) employed a membrane bioreactor (MBR) for decol-
orization of reactive dyes using Trametes versicolor. The MBR was employed
in treating synthetic textile wastewater as it can handle high microbial load,
long retention times and is easy to operate and control. A white-rot fungus
was selected for its ability to produce laccase, LiP, and MnP. The fungal 600
MBR with reverse osmosis (RO) removed >98% total organic carbon (TOC)
and dyes (reactive black 5 and reactive blue 49). However, for reactive blue
19, though the treatment scheme was able to remove 98% TOC, the decol-
orization was lower (76.8%) than that of the other two dyes. One of the
drawbacks of a MBR process is frequent fouling of membranes. Hai et al. 605
(2006) optimized MBR design and operation to obtain maximum TOC re-
moval and decolorization with minimal membrane fouling while treating
synthetic textile wastewater under nonaseptic conditions with the white-rot
fungus Coriolus versicolor. The optimum design comprised a cylindrical hol-
low fiber bundle with a nonwoven coarse-pore (50–200 µm) mesh cage. 610
High-pressure back-washing (100 ml/min−1) for 3 s after every 10 min fil-
tration along with chemical (NaOCl) back-washing (100 ml/m−2) every third
day was recommended to minimize fouling. The TOC (Influent TOC =2
g/l−1) and color removal (Influent Dye =100 mg.l−1) efficiency was 97%
and 99%, respectively, at pH ∼4.5, temperature ∼29◦C, hydraulic retention 615
time (HRT) 15 hr and average flux 0.021 m/d−1. Fujita et al. (2000) experi-
mentally found that the contamination of MBR (external membrane) with air-
and water-borne microorganisms decreased the decolorization efficiency of
immobilized Coriolus hirsutus under nonaseptic condition, during the treat-
ment of the liquor from the heat treatment of waste sludge. 620
Fungi are effective in degrading complex aromatic organic compounds
present in wastewater. For instance, phenolic compounds present in olive
mill wastewater are similar to those derived from lignin degradation (Jaouani
et al., 2005). Olive mill wastewater also consists of additional nutrients and is
usually acidic, which favors fungal growth. Sassi et al. (2006) evaluated the 625
toxicity of olive mill wastewater with barley seed germination and found that
olive mill wastewater was highly toxic. The study determined that the yeast
was predominant in the wastewater in comparison to molds and bacteria.
The authors recommended that olive mill wastewater should be utilized
BEST_A_328061 bestxml-als-v1.cls March 26, 2010 14:4
Fungal Wastewater Treatment 25
to grow yeast that yield SCP for animal feed as well as promote ethanol630
production.
Fungi use nonspecific oxidative systems, including extracellular oxidore-
ductases, low-molecular-mass metabolites, and activated oxygen species to
degrade recalcitrant organic compounds (Jaouani et al., 2005). Khiyami et al.
(2005) demonstrated the ability of P. chrysosporium to grow and detoxify
635
different concentrations of diluted corn stover and diluted cornstarch pyrol-
ysis liquors in a shaker flask test with addition of 3 mM veratryl alcohol.
Pyrolysis is a physical and chemical process that liquefies a natural car-
bon reservoir, such as plant biomass, into a dark brown liquor that contains
anhydro-sugars, such as levoglucosan (1.6-anhydro β-D-glucopyranose), and
640
a wide range of oxygenated compounds. Unfortunately, the liquor contains
toxic compounds (phenolic and furfuryl derivative compounds) that inhibit
microbial growth unless removed. Diebold and Bridgwater (1997) illustrated
that the ligninolytic enzymes produced by P. chrysosporium were capable of
detoxifying these liquors in situ or when exogenous enzymes were added.
645
D’Annibale et al. (2004) conducted a similar study on the degradation
of phenolic compounds by Panus tigrinus in a shaker flask test. They found
that phenolic compounds were effectively degraded, especially hydroxy-
substituted phenolics such as 2-hydroxyphenylethanol, 4-hydroxybenzoic
acid, 4-hydroxyphenylacetic acid, and 4-hydroxy-3-methoxyphenylacetic
650
acid. These compounds were completely degraded in 13 days. The authors
indicated that the polymeric aromatic fraction underwent simultaneous poly-
merization and depolymerization. While studying the role of fungal species in
PAH (anthracene and fluoranthene were used as model compounds) removal
in constructed wetlands, Giraud et al. (2001) found that more fungal species
655
were effective in degrading fluoranthene than anthracene. Among the 40 fun-
gal species isolated from wetlands contaminated by PAH, 33 species showed
an ability to degrade fluoranthene (60–99%), whereas only two species were
able to degrade anthracene over 70%. The probable reason for these results
is that the wetland contaminated with PAH particularly contained fluoran-
660
thene that might have resulted in adaptation of the fungal species to the
compound.
Enzymatic treatment of wastewaters to accelerate biodegradation of spe-
cific recalcitrant components by fungal species has been a recent advance-
ment in wastewater remediation (Karam & Nicell, 1997). Among various
665
types of microorganisms, fungi constituted a major proportion of the micro-
bial population that produces these enzymes. The examples of recalcitrant
waste streams degraded by fungal enzymes are summarized in Table 9.
CONCOMITANT POLLUTANT DEGRADATION AND BYPRODUCT RECOVERY
Concomitant wastewater treatment studies with byproduct recovery have670
been conducted both under aseptic and nonaseptic conditions (see Table 7).
Early fungal studies on wastewater treatment to derive fungal byproducts
BEST_A_328061 bestxml-als-v1.cls March 26, 2010 14:4
26 S. Sankaran et al.
TABLE 9. Enzymatic Degradation of Different Wastewater Types (Karam & Nicell, 1997)
Type of wastewater
Enzymes used for
degradation Enzymes producing fungi
Phenolic-laden
wastewater
Lignin peroxidase,
chloroperoxidase,
manganese peroxidase,
laccase
Phanerochaete chrysosporium,
Caldariomyces fumago,
Rhizoctonia praticola
Cyanide-rich wastewater Cyanide hydratase Gloeocercospora sorghi,
Stemphylium loti
Food-processing
wastewater
L-galactonolactone oxidase
Chitinase
Candida norvegensis, Serratia
marcescens
were conducted under aseptic conditions. Recently, the focus has been on
nonaseptic cultivation of fungal biomass, as it would be more cost effective
for full-scale applications. Fungal wastewater treatment is a low-cost, techni- 675
cally simple process for the production of protein-rich animal feed (Stevens
& Gregory, 1987). Most of the studies listed in Table 7, except Jin et al.
(1998), Suntornsuk et al. (2002), and Huang et al. (2003), focused on fungal
Q6
biomass production using wastewater treatment to be used for animal feed.
Jin et al. (1998), Suntornsuk et al. (2002), and Huang et al. (2003) treated 680
starch-processing wastewater, soybean and mung-bean residues, and potato-
processing wastewater for the production of α-amylase, chitosan, and lactic
acid, respectively.
Fungal wastewater treatment with concomitant byproduct recovery has
various advantages, as indicated in a previous section. One additional advan- 685
tage of the concomitant fungal treatment process is that a single species could
be used for deriving multiple valuable biochemical substances. For example,
Aspergillus oryzae was used to produce proteinase (Christensen et al., 1988),
lipase (Huge-Jensen et al., 1989), lactoferrin (Ward et al., 1992), lysozyme
(Tsuchiya et al., 1992), α-amylase (Jin et al., 1998), protein (Jin et al., 2002; 690
Truong et al., 2004; van Leeuwen et al., 2003), and chitin/chitosan (Jasti
et al., 2006).
Typically, food-processing wastewaters have a high carbohydrate con-
tent, which favors fungal biomass production for fungal protein and
byproduct recovery. Many studies on concomitant fungal treatment process 695
have used food-processing wastewater as their substrate medium. Food-
processing wastewater streams are relatively free of toxic substances and
pathogenic organisms, making for a suitably safe substrate for fungal biomass
production for animal feed. Moreover, potentially high proportions of
carbohydrates, proteins, and lipids are desirable for fungal cultivation. 700
Wastewater containing starch is commonly used as substrate for fungal cul-
tivation. Fungal treatment of food-processing wastewater was studied by
several researchers (Huang et al., 2003; Mishra & Arora, 2004; Truong et al.,
2004). One of the prevailing problems in nonaseptic wastewater treatment,
BEST_A_328061 bestxml-als-v1.cls March 26, 2010 14:4
Fungal Wastewater Treatment 27
however, is the proliferation of bacteria, which not only compete with fungi705
for organic substrates, but also deteriorate the dewaterability and quality
of the fungal biomass. Mikami et al. (1982) used a thermophilic fungus,
C. eichhorniae, to produce protein from cassava at 45–47◦C. The temperature
and pH were optimized to 45◦C and 3.8, respectively, in batch fermentors.
Optimum conditions were used in a pilot-scale fermentor with a working
710
volume 50 L. In the pilot-scale fermentor, nutrient sources, (NH4)2SO4and
KH2PO4were required to convert starch into microbial biomass (0.45 g of
biomass per 1 g of cassava meal). Although thermophilic fungi were used for
treatment in order to minimize the effect of contamination under nonaseptic
conditions, the study did not enumerate the bacterial count. However, the
715
toxicity of the spores was tested with mice and chickens. The animals did
not exhibit any symptoms of infection.
Another important technique that can be appropriate for degrading re-
calcitrant compounds in the wastewater is by utilizing a reactor design, which
provides an optimum condition under which fungi can attain slight domi-
720
nance, while bacteria can also remain functional. Many fungal species can
degrade complex structures difficult for bacteria to degrade (Nilsson et al.,
2006). Thus fungi can initiate the biodegradation process by secreting target-
specific enzymes, whereas bacteria can utilize the degradation byproducts
(intermediates) produced by fungi. A few comparative studies on bacterial
725
and fungal degradation of polyaromatic hydrocarbons have been performed
(Ch´
avez-G´
omez et al., 2003; Grant et al., 2004) that suggest that a coculture
of bacteria and fungi can perform better than individual fungal or bacteria
species. However, further investigation on combined (bacteria and fungi)
wastewater treatment needs to be investigated.
730
Bioreactor Design
Bioreactor design plays a significant role in determining the microbial domi-
nance during nonaseptic fungal treatment. Microscreens are used in bioreac-
tors to aid fungal predominance as well as eliminate bacterial contamination
(Pretorius & Lempert, 1993; van Leeuwen et al., 2003). Microscreens consti-
735
tute a part of bioreactor design, which is discussed in subsequent sections
of this article. Other designs used to obtain the predominance of a fungal
monoculture include application of an attached or biofilm growth system
and application of specific bioreactor configurations (e.g., airlift bioreactor).
Airlift bioreactors have received considerable attention particularly from
740
the bioprocessing industry and are well suited for aerobic waste treatment.
The term airlift bioreactor refers to a bioreactor in which the reaction
medium is kept under completely mixed conditions by introduction of air
or another gas (mixture) at the base of a column-like reactor. The reactor is
equipped with either a draft tube or another device (e.g., an external recycle
745
tube) by which the reactor volume is separated into gassed and ungassed
BEST_A_328061 bestxml-als-v1.cls March 26, 2010 14:4
28 S. Sankaran et al.
regions, thus generating a vertically circulating flow up in the gassed re-
gion and down flow in the ungassed region. When compared with stirred
tank reactors, airlift bioreactors provide advantages such as simple construc-
tion, low shear rates, low energy consumption, high biomass concentrations, 750
high momentum, and heat transfer rates (Jin et al., 2002; Trager et al., 1989).
Trager et al. (1989) compared the stirred and airlift bioreactors using A. niger
and found that the desired morphology of the fungal culture (i.e., pellet
growth) could be obtained under low stress conditions in both laboratory
and pilot-scale airlift fermentors (260 L) compared with stirred tanks, which 755
required high agitation to achieve similar results. Armillaria mellea produced
loose fluffy pellets in an airlift fermentor, whereas compact sclerotic pellets
appeared in a stirred tank reactor (Hansson & Seifert, 1987). Under the same
agitation conditions, Aspergillus awamori produced 20–30% larger pellets in
a 100-L reactor than in a 2-L vessel when pellet size was measured by means 760
of image analysis (Cui et al., 1998).
Airlift bioreactors are also suitable for cultivation of filamentous fungi
with high oxygen demand and sensitivity to shear, which would be suitable
for processes involving MBP production (Jin et al., 2002). It is possible to
carry out fermentation processes in large-scale airlift bioreactors at relatively 765
low aeration rates, as aeration efficiency increases with vessel size. The ex-
ternal airlift bioreactor is important for large-scale aerobic fermentation and
wastewater treatment, particularly for growth media with high viscosity, in
which high mass and heat transfer rates are required (Gavrilescu & Roman,
1995). The authors evaluated the ability of a pilot-scale external-loop air- 770
lift bioreactor to overcome the problems arising from the morphology and
rheology of the broth for cephalosporin C production from Cephalosporium
acremonium. The specific power consumption of the airlift bioreactor was
two-thirds to that of a stirred tank reactor, whereas cephalosporin C pro-
duction was comparable between the two reactor configurations. Contrarily, 775
Bayer et al. (1989) found lower yield of cephalosporin C in a 60-L loop air-
lift reactor due to oxygen limiting condition resulting from increased broth
viscosity. Jin et al. (1998) developed a simple, nonaseptic, low-cost airlift
bioreactor for producing protein and α-amylase while treating wheat starch-
processing wastewater. The bioreactor was constructed with cylindrical glass 780
of 9.5 cm in diameter and 70 cm in length and had a working volume of 3.5 L.
The aeration was regulated at about 1.0–1.7 volumes per reactor volume per
minute (vvm) to keep the dissolved oxygen tension >50%. Jin et al. (1999d)
developed an external airlift reactor with double spargers in another study.
The reactor was found to be suitable for cultivating selective filamentous 785
microfungi in continuous mode. An airflow rate up to 1.5 vvm was required
to maintain the dissolved oxygen level >50% saturation because of the high
BOD of the wastewater. Such high aeration rates could be achieved in an
airlift reactor with external circulation of mixed liquor and some additional
sparged air in the external loop. Compact pellets and clumpy-coalesced 790
BEST_A_328061 bestxml-als-v1.cls March 26, 2010 14:4
Fungal Wastewater Treatment 29
mycelia of A. oryzae and R. arrhizus cultures could be produced at high
velocity gradients resulting from high agitation rates or high airflow rates.
Separation, effective oxygen transfer, and biomass harvesting were simpli-
fied greatly by this morphology. Jin et al. (2002) further developed the airlift
reactor with external loop for pilot-scale study. Several other researchers also
795
studied airlift bioreactors for treating wastewater (Dhouib et al., 2006; Ryan
et al, 2005).
The geometric configuration, mycelial broth rheology, and superficial
gas velocity affect the hydrodynamic parameters (e.g., gas holdup, oxygen
transfer coefficient and mixing time), and thus the fungal production in
800
a bioreactor (Jin et al., 1999a). The authors found that an external airlift
reactor (with double sparger) having a downriser-to-riser diameter ratio of
0.71 (√1
2) and a height just shorter than the main airlift reactor column was
best suited for fast mycelium growth and high biomass production.
Attached or biofilm reactors are also a good option for fungal cultiva-
805
tion. Gibbs et al. (2000) suggested in their review that the application of
various forms of immobilization technology with filamentous fungi could be
an innovative option for overcoming various drawbacks of other bioreac-
tor types. The first known application of biofilm technology was industrial
wastewater treatment by trickling filters in the early 1880s in Wales, Great
810
Britain (Lazarova & Manem, 2000). Biofilm is a natural form of cell immobi-
lization that results from microbial attachment to solid supports in submerged
environments and cell immobilization is one way to enhance cell density in
a system (Ho et al., 1997a, 1997b, 1997c). The higher cell density of specific
microorganisms can suppress the growth of competitive microorganisms un-
815
der mixed culture conditions, leading to better yields. The higher affinity of
filamentous fungi to attach on organic or inorganic surfaces can be used as a
factor for fungal selection. However, when grown simultaneously, the faster
growing bacteria could become the dominant species in a mixed culture
of fungi and bacteria (Banks & Byers, 1991; Elvers et al., 1998). Therefore, Q7
820
an aseptic operation condition for initial cultivation of pure fungi may be
required to achieve fungal domination during nonaseptic attached growth
wastewater treatment. Alternatively, high fungal inoculums to biological re-
actors may also lead to high fungal cell density. Additionally, the capability
of attached growth systems to efficiently operate at smaller HRTs could result
825
in bacterial washout from the reactor.
The use of PCS tubes, composed of 50% (w/w) polypropylene and 50%
(w/w) agricultural products, as attached growth media in a bioreactor helped
to selectively cultivate fungi through cell immobilization in the presence of
unwanted bacterial competition (Jasti et al., 2006). Jirku et al. (2001) com-
830
pared the inhibitory effects of xenobiotics on Candida maltosa and Fusarium
proliferatum in suspended culture, and in artificial or natural biofilms. The
results indicated that the attached cell population showed increased resis-
tance to xenobiotic compounds. Moreover, only the attached fungal cells
BEST_A_328061 bestxml-als-v1.cls March 26, 2010 14:4
30 S. Sankaran et al.
had the ability to degrade acetone and phenol. Jasti et al. (2006) cultivated 835
R. oligosporus in a PCS attached growth system using wet corn-processing
wastewater and achieved a high fungal yield of 0.56 gVSS/g COD removed.
Wu et al. (2005) used porous plastic support media while growing white-rot
fungi, and 71% lignin degradation was achieved while treating black liquor
from the paper and pulp industry. Khiyami et al. (2006) demonstrated pro- 840
duction of LiP and MnP by P. chrysosporium in a defined medium using
PCS biofilm stirred tank reactors. Although LiP and MnP were produced by
the fungi, laccase was not produced in the reactor. The formation of the
P. chrysosporium biofilm on PCS was essential for MnP and LiP production.
The bioreactor was operated as a repeat batch, and no reinoculation was 845
required between the batches. Addition of veratryl alcohol and MnSO4on
Day 0 with 300-rpm agitation and continuous aeration at 0.005 vvm resulted
in the fastest MnP production with a final yield of 63.0 U/L after 3 days in
P. chrysosporium peroxidase culture medium. The potentials and limitations
of reactor designs are summarized in Table 10. 850
Hydraulic and Solids Retention Time
Physical attributes of microorganisms such as mass and size can be dynamic
tools for selection of a desired species from two or more microbial species
in a bioreactor. For instance, the filamentous bacterial population can be
favored against nonfilamentous bacterial forms by controlling operational 855
parameters, such as HRT and solid retention time (SRT; Cenens et al., 2000;
Chudoba et al., 1973; Contreras et al., 2002) or with ozone or chlorine (Saay-
man et al., 1996; van Leeuwen, 1988). Different researchers have evaluated
the various factors influencing microbial domination in a nonsterile wastewa-
ter. Harder and Kuenen (1977) suggested that the dominance of a particular 860
type of microorganism in a continuous system is primarily determined by
the specific growth rate and initial microbial inventory in a bioreactor. Con-
treras et al. determined that the competitive growth between Sphaerotilus
natans and Acinetobacter anitratus was dictated by dilution rate (reciprocal
of HRT), which correlated directly to specific growth rates. 865
Spontaneous mutations can occur in the microorganisms during long-
term continuous bioreactor operation (Lindegren, 1963). The growth-limiting
nutrient in the wastewater exerts a selection pressure on the microorgan-
isms that can selectively favor the mutated organism. This selection process
could result in improved yields. Pretorius (1987) introduced the concept 870
of size-based selection of microorganisms with desirable characteristics in
wastewater treatment. Pretorius and Lempert (1993) developed continuous
systems using a cross-flow microscreen for MBP production during fun-
gal wastewater treatment. A microscreen system is useful in maintaining
fungal predominance by allowing the bacterial cells to wash out while 875
BEST_A_328061 bestxml-als-v1.cls March 26, 2010 14:4
TABLE 10. Merits and Demerits of Reactor Design Summarized from Different Studies
Reactor design Potentials of reactor design Limitations of reactor design Source
Attached growth
or biofilm
reactor
•Biofilms reactors enhance cell density in a system by promoting
microbial attachment and cell immobilization and eliminating
wash out.
•Attached growth system can act as selection mechanism to
maintain a dominant fungal culture under nonaseptic conditions.
•No batch inoculation is required.
•Support biofilm reactor posses increased end-product
production rate, minimal lag phase, tolerance to high
concentration of nutrient, reduced requirement of
micronutrients, and increased cell density for ethanol, lactic
acid, and succinic acid production.
•Biofilm thickness was controlled by agitation speed.
•The biofilm increases the cell interfacial contact with culture
fluid, decreases the shear force, and permits medium to circulate
through the tubes.
•Harvesting the attached film may
be of concern.
•SRT may be difficult to maintain.
Jasti et al., 2005;
Khiyami et al., 2006
Stirred-tank
reactors •Simple design and easily controllable parameters.
•Pellet morphology (size and structure) can be controlled by
controlling agitation speeds.
•Tensile strength of the pellets is stronger than the exerted
mechanical forces.
•It is easier to harvest the fungal pellets for byproduct recovery.
•A negative effect of agitation on
enzyme production may be
found due to the shear stress
effect in a stirred tank reactor
•Pellets may break after a critical
size is reached
Hansson & Seifert, 1987;
Cui et al., 1998;
Khiyami et al., 2006
Airlift reactors •Provide simple construction, low shear rates, low energy
consumption, high biomass concentrations, high momentum,
and heat transfer rates.
•Desired configuration can be obtained under low stress
conditions.
•Promotes cultivation of filamentous fungi with high oxygen
demand and sensitivity to shear.
•Aeration efficiency is high.
•Mass transfer rates are high and power consumption is low.
•Higher initial capital investments
due to large-scale processes.
•Foaming may occur due to
excessive aeration, which may
result in loss of certain amount of
biomass.
Trager et al., 1989; Jin
et al., 2002
Bioreactors with
microscreens •Aid in fungal predominance as well as eliminate bacterial
contamination
•Fungal and bacterial sludge age can be controlled separately.
•Fouling of microscreens is an
issue of concern.
•Fungi may act as media for
bacterial growth
Pretorius, 1989;
Pretorius & Lempert,
1993; van Leeuwen
et al., 2003
Note. SRT =solid retention time.
31
BEST_A_328061 bestxml-als-v1.cls March 26, 2010 14:4
32 S. Sankaran et al.
retaining fungal filaments in the bioreactor (van Leeuwen et al., 2003). Van
der Westhuizen and Pretorius (1998) successfully limited bacterial contami-
nation without autoclaving the wastewater by controlling fungal and bacte-
rial retention time separately by using a microscreen. Fungal and bacterial
biomass ages were controlled separately by SRT (12 or 9 hr) and HRT (3.3 880
hr or less), respectively. Bacteria were effectively washed out regardless of
fungal biomass concentration at an HRT of less than 4 hr. The authors found
that fungal dominance in the bioreactor can be achieved by controlling SRT.
Van Leeuwen et al. introduced a cylindrical microscreen (100 µm) in a by-
pass recirculation system while working under nonaseptic conditions. Fungal 885
mycelia with a diameter >5µm and lengths of several hundred micrometers
were retained in the bioreactor, whereas the bacterial cells (0.5–2 µm) passed
through the microscreen and were washed out of the bioreactor. Thus, the
bacterial retention time in the bioreactor was limited by the HRT, whereas
the SRT of the fungal biomass could be controlled by the rate of harvesting. 890
This technique was successful in maintaining predominance of R. oligosporus
during nonaseptic fungal treatment of corn-processing wastewater.
Jasti et al. (2006) limited the bacterial fraction to <10% of the total
biomass concentration at an HRT of 2.5 hr while treating of corn wet- milling
wastewater with R. oligosporus in a PCS biofilm continuous reactor. The 895
system demonstrated an ability to withstand smaller HRTs without fungal
biomass washout. In comparison to the suspended growth bioreactors used
in precedent studies, the PCS biofilm continuous reactor demonstrated an
obvious advantage of achieving optimal fungal bioconversion and bacterial
elimination at relatively lower HRTs. The higher treatment rates obviously 900
offer an economical advantage on industrial scale with lower capital and
operational costs.
Biochemical Kinetics
Table 11 summarizes the biokinetic parameters of few studies described in
Table 8. Except for Truong et al. (2004), the studies reported in the Table 11 905
were conducted under nonsterile conditions. The biokinetic parameters were
compared to the biokinetics of activated sludge processes (bacteria) treating
domestic wastewater. The biokinetic constants of fungal process treating
cassava wastewater (Truong et al., 2004) and phenolic wastewater (Ryan
et al., 2005) were compared with those of bacterial treatment, and the yield 910
coefficients were found to be similar. However, it should be noted that the
former study was under aseptic condition, whereas the latter was under
nonaseptic condition. The specific growth rate (0.14–0.18 hr−1) of a fungal
treatment system treating wheat starch-processing wastewater from starch
and gluten production process (Jin et al., 1998, 2002) was higher than that of 915
bacterial treatment system (0.104 hr−1). The growth rate, however, was much
smaller for a fungal treatment system treating phenolic wastewater from a
BEST_A_328061 bestxml-als-v1.cls March 26, 2010 14:4
Fungal Wastewater Treatment 33
TABLE 11. Biokinetic Parameters of Fungi Treating Different Types of Wastewater and
Bacteria Treating Domestic Wastewater
Wastewater source Biokinetic parameters Source
Wheat starch processing
wastewater
Specific growth rate (µ): 0.14 hr−1Jin et al., 1998
Wheat starch processing
wastewater
Yield (Y): 8.80 ±0.40 g/L for Aspergillus
oryzae, 8.00 ±0.40 g/L for Rhizopus
oligosporus µ: 0.18 ±0.35 hr−1for Aspergillus
oryzae, 0.15 ±0.03 hr−1for Rhizopus
oligosporus
Jin et al., 2002
Corn-processing
wastewater
Endogenous respiration rate (kd): 0.001 hr−1van Leeuwen
et al., 2002
Cassava-starch-processing
wastewater
Y: 0.67 g/g COD Truong et al.,
2004
Phenolic wastewater Y: 0.87 g biomass/g glucose µ: 0.0104 hr−1Ryan et al.,
2005
Olive oil mill wastewater Half saturation constant (Km) with
2.6-dimethoxyphenol: 27 ±2.1 µM
Jaouani et al.,
2005
Kmwith 2.2-azino-bis(3)-
ethylbenzothiazoline-6-sulphonic acid: 36 ±
1.2 µM
Domestic wastewater
treatment using bacteria
Y: 0.50 mg VSS/mg BOD Kd: 0.002 hr−1µm:
0.104 hr−1
Metcalf & Eddy
et al., 2003
Note. COD =chemical oxygen demand; BOD =biochemical oxygen demand.
coal gasification plant (0.0104 hr−1) (Ryan et al.). The endogenous decay
rate of fungal process applied to wet corn-milling wastewater (van Leeuwen
et al., 2003) was half that of a bacterial treatment process (Metcalf & Eddy
920
et al., 2003). By comparing with the biokinetic constants, it is apparent that
the performance of a fungal wastewater treatment system was better than
that of a bacterial treatment system, but the former is often slower, depending
on the types of substrate.
Fungal Harvesting Technologies925
Fungal harvesting technologies involve various methods employed for sep-
aration of large quantities of fungal biomass cultivated in different wastew-
aters. Separation methods such as settling, centrifugation, screening, filtra-
tion, and drying can be used for fungal biomass separation. Though there
are a few research papers (Nigam, 1994; Jin et al., 1999d) indicating the
930
high settleability and easier separation of fungal biomass, the true settleabil-
ity characteristics of fungal biomass have not yet been determined. A few
laboratory studies describe the separation of fungal biomass from the sub-
strate or mixed liquor.
Barros J´
unior et al. (2003) recovered the fungal biomass through a vac-
935
uum filtration method to recycle fungi for biosorption studies. O’Brien and
BEST_A_328061 bestxml-als-v1.cls March 26, 2010 14:4
34 S. Sankaran et al.
Heiland (1993) developed a patented attached growth process for growing
and harvesting filamentous fungi on a large scale. The reactor consisted of
a rigid cylinder, partially submerged and rotating in a biological medium
containing a fungal medium with nutrients. The filamentous fungi grew in 940
the rotating drum, wherein it was removed with a doctoring blade (i.e., a
thin metal plate or scraper that is in contact with a rolling cylinder along its
entire length to keep the cylinder clean; Reid, 1981). Two other technologies
used for immobilization of microbial biomass are adsorption and entrap-
ment. In the adsorption process, cells are directly linked to water-insoluble 945
carriers such as ion-exchange resins or fritted glass. Entrapment involves
the use of inert gels, such as polyacrylamide or calcium alginate, in which
cells are trapped within the polymeric matrix. O’Brien and Heiland (1993),
however, specified that adsorption and entrapment technologies have only
limited application in fungal treatment. Fungal colonization on the surfaces 950
of the bioreactor is a nuisance contributing to difficulties for harvesting fungi
in conventional fermentors. Unkles et al. (2004) harvested filamentous fungi
through filtration and yeast through centrifugation on a micro-scale, while
studying the relationship between nitrate reductase activity and nitrate trans-
port into fungal cells. Many laboratory techniques involving fungal cultivation 955
use filtration or centrifugation for biomass recovery (Barton, 2000). Jasti et al.
(2006) developed a process to grow R. oligosporus attached to the surface
of an extruded material consisting of 50% polypropylene and 50% agricul-
tural products attached to an impellor shaft. Removal of excess biomass was
achieved by continuous sloughing off and subsequent settling of the readily 960
settleable fungal mass.
Microscreening is yet another possible technique for fungal separation.
In addition to fungal separation, microscreening helps in size-based selec-
tion of microorganisms, thereby establishing the predominance of a fungal
monoculture under nonaseptic conditions. A bioreactor with a microscreen 965
can be considered as a completely mixed reactor with fungal cell recycling
(see Figure 1). It is possible to control fungal retention in the bioreactor by
using a microscreen at the effluent port. The fungi with a size larger than
the screen pore size remains in the reactor, whereas the bacteria or other
particles with a size smaller than the screen pore size are expected to pass 970
through the screen and leave the system along with the effluent. Thus, the
fungal retention can be controlled independently by the rate of harvesting.
HRT then governs the bacterial biomass retention time, and thereby limit
the population size, whereas the SRT allows optimal growth of the fungal
biomass. 975
Solids retention time (tSF; for microfungi):
tSF =XFV/XFq=V/q(1)
BEST_A_328061 bestxml-als-v1.cls March 26, 2010 14:4
Fungal Wastewater Treatment 35
pH
controller
Pump
Effluent
pump
Influent
pump
Micros
creen
Harvesting, q
Air diffuser
Q
Q
FIGURE 1 Microscreen cell recycle reactor (van Leeuwen et al., 2003).
where
tSF =Biomass in the reactor/biomass harvested per day [d]
V=Reactor working volume [L]
q=Flow rate of harvesting stream [L/d]
980
XF=Biomass concentrations in the reactor and harvested stream [mg dry
biomass/L]
Therefore, the SRT of the fungal biomass can be controlled by withdraw-
ing mixed liquor (at a flow rate, q) from the reactor. A high fungal biomass
concentration facilitates fungal predominance in the bioreactor. Several in-
985
vestigators successfully demonstrated the use of microscreens for selective
fungal biomass cultivation and recovery during wastewater treatment (K¨
uhn
& Pretorius, 1989; Pretorius, 1987; Pretorius & Lempert, 1993; van Leeuwen
et al., 2003). However, one of issues concerned with the application of mi-
croscreens is that fouling may occur during continuous operation of these
990
reactors. Mitigation techniques to overcome this problem can be air-scouring
or effluent backwash similar to MBR operation but at longer intervals with
sufficient back-pressure to eliminate the fouling in the screens. Techniques
similar to manual or mechanical grit removal (primary wastewater treatment)
can be applied to clean the screens.
995
BEST_A_328061 bestxml-als-v1.cls March 26, 2010 14:4
36 S. Sankaran et al.
BACTERIAL CONTAMINATION IN FUNGAL TREATMENT SYSTEMS
The goal of many nonsterile operations in fungal wastewater treatment is
the biodegradation of organic matter and cultivation of fungal biomass as
a value-added byproduct. One of the most prevalent problems in fungal
wastewater treatment under nonaseptic conditions is the control of bacterial 1000
contamination. Bacterial proliferation during fungal wastewater treatment
results in intense competition for available organic substrate, and it affects
the fungal metabolism since the bacterial growth takes place on the fungal
filaments as support media. Thus, restraining bacterial growth by creating
an environment in which fungi can dominate would make fungal cultivation 1005
much easier in wastewater bioremediation.
Sterilization of wastewater is extremely expensive in industrial applica-
tions. Hence, maintaining dominance of fungal biomass in nonsterile wastew-
ater requires development of technologies to increase their competitive abil-
ity over the bacterial microflora and suppressing bacterial growth. Selective 1010
mechanisms for preventing bacterial growth without inhibiting fungal growth
as discussed in previous sections include modification of the physical and
operational (such as HRT and SRT modification, and application of micro-
screens), environmental (such as pH and temperature modification), and
physiological conditions during bioreactor operation. A higher operating 1015
temperature and pH is expected to reduce bacterial contamination in fungal
treatment systems (Fujita et al., 2000; Libra et al., 2003). Jin et al. (1999c)
carried out batch shake-flask experiments under nonaseptic conditions on
starch processing wastewater using R. oligosporus (now classified as R. mi-
crospores). The study found that at pH 4 and 35◦C, bacterial contamination 1020
was not a problem and occurred only after 18 hr. Further increasing the
temperature >40◦C and decreasing pH <3.5 or both were successful in
eliminating the bacterial contamination. The increase in temperature was fa-
vorable, exhibiting enhanced rate of starch hydrolysis and biomass growth,
but the decrease in pH had an adverse effect on protein synthesis and en- 1025
zyme production.
Bacterial contamination may occur even after sterilization of media and
wastewater as it is difficult to maintain aseptic conditions throughout the
bioreactor operation. Nilsson et al. (2006) treated textile wastewater using the
white-rot fungus T. versicolor. Continuous experiments were conducted in 1030
cylindrical reactors and rotating biological contactors to treat textile wastew-
ater containing Reactive Red 2 and Reactive Blue 4 under sterile conditions.
The air was led into and out of the reactors through sterile filters (0.2 µm) in
order to avoid contamination of the reactors. Researchers found that it was
difficult to maintain sterile conditions. After 15 days of continuous operation, 1035
the absorbance of the dye increases and no decolorization occurred after
34 days. The authors hypothesized that the absence of decolorization was
due to disturbance in enzyme production or bacterial contamination. Further
BEST_A_328061 bestxml-als-v1.cls March 26, 2010 14:4
Fungal Wastewater Treatment 37
microscopic examination of the effluent revealed the presence of bacteria
and protozoans.
1040
Fountoulakis et al. (2002) treated olive mill wastewater using the white-
rot fungus Pleurotus ostreatus under different conditions such as using steril-
ized, thermally treated (100◦C), diluted and undiluted olive mill wastewater.
The degradation of phenols was up to 78.3% for sterilized wastewater with
50% dilution. Phenol degradation rate was 66.7 and 64.7% for thermally
1045
treated diluted and undiluted wastewater, respectively. The study indicated
that thermal treatment by raising the wastewater temperature (40◦C) to a
higher temperature could not prevent bacterial contamination, unless the
temperature was maintained above 100◦C. One important observation in
the study was that though the fungi growing on thermally treated wastewa-
1050
ter displayed a lag phase of about 6 days unlike fungi growing in sterilized
wastewater; the thermally treated wastewater yielded a higher mycelium con-
tent than sterilized wastewater. The reason stated in the study was a probable
change in the composition of the wastewater, reducing the nutrient availabil-
ity for fungal growth. Thus, this signifies another possible concern dealing
1055
with sterilization of wastewater for fungal growth.
Libra et al. (2003) stated that though dye-house wastewater often has a
high temperature or pH and is relatively free from bacteria, treatment with
fungi requires temperature and pH adjustments of the wastewater that are
also conducive for fungal growth. The ability of T. versicolor to produce oxi-
1060
dizing enzymes is used for decolorization of dye in flask experiments as well
as sequencing batch reactors (Borchert & Libra, 2001). By shearing of sus-
pended pellets of T. versicolor in each cycle via high agitation speeds, rapid
decolorization of reactive textile dyes (91–99%) was repeatedly attained with-
out decrease in enzymatic activity over time in pure culture. However, under
1065
nonaseptic conditions, bacterial contamination occurred frequently, causing
a decrease in decolorization efficiency (72%) in the fifth cycle (Borchert &
Libra). Fujita et al. (2000) had similar results, with a decrease in decolorization
efficiency under nonaseptic conditions during treatment of liquor from the
heat treatment of waste sludge using an MBR process. The paper suggested
1070
that this problem could be solved by heating the returned concentrate at 50◦C
for 10 min. When Nilsson et al. (2006) faced a decrease in decolorization
due to bacterial contamination, the authors suggested that certain strategies
that may decrease the effective of bacterial contamination are the following:
(a) a larger amount of fungal biomass can decrease the negative effect of
1075
bacteria depending on the higher concentration of enzymes to counteract
the enzymes destroyed by bacteria; (b) a solid lignocellulosic support mate-
rials for fungi can give an advantage to fungi over bacteria; and (c) without
the addition of an easily degradable carbon source, it could be possible to
suppress bacterial contamination as fungi are capable of degrading com-
1080
plex compounds. The authors found that though decolorization efficiency
of T. versicolor treating textile wastewater decreased due to bacterial and
BEST_A_328061 bestxml-als-v1.cls March 26, 2010 14:4
38 S. Sankaran et al.
protozoan contamination, decolorization efficiency of Pleurotus flabellatus
was not affected by the presence of bacteria and protozoa. Thus, using a
fungal species resistant to bacterial presence may be of importance in further 1085
studies. Though only few studies have been conducted to quantify the effect
of bacterial competition on fungal wastewater treatment under nonaseptic
conditions, bacteria are expected to hinder fungal activity significantly.
In biological wastewater treatment, different microbial communities are
selected for use in the purification process based on plant design and op- 1090
erating conditions. A technical solution to bacterial contamination in fungal
treatment system is to design a process that helps to obtain an enriched
culture of a desired microbial community and to effectively suppress bac-
terial growth under nonsterile conditions. Thus, in order to use the fungal
byproducts as an animal feed or for deriving other valuable byproducts, it is 1095
necessary to identify and quantify the microbial composition present in the
derived byproducts.
Bacterial proliferation can be prevented by a suitable mechanism for
inactivation of bacteria. Cenens et al. (2000) developed a model to describe
the competition between filamentous and floc-forming bacteria in an acti- 1100
vated sludge process. The model was based on both kinetic selection and
filamentous backbone theory, and they were successful in describing the
coexistence of floc-forming and filamentous bacteria. Similar models should
be developed for describing the growth kinetics of bacteria and fungi in
nonaseptic conditions, as it would help in developing a mechanism to con- 1105
trol bacterial proliferation.
One of the methods for preventing bacterial propagation in a fungal
treatment system is the use of selective disinfection, wherein a mild disin-
fectant dose could inhibit bacterial growth without suppressing fungal cells.
Selective disinfection has also been employed in activated sludge bulking 1110
control. Chlorine (Lakay et al., 1988) and ozone (van Leeuwen, 1988) have
both been used to selectively control the growth of filamentous bacteria with-
out significant disruption of floc-building bacteria. Van Leeuwen (1989) and
Camel and Bermond (1998) found that fungi are more resistant to ozonation
than bacteria. Van Leeuwen (1989) investigated the possibility of activated 1115
sludge bulking control in fuel synthesis wastewater treatment with ozona-
tion. Although ozone could effectively reduce the growth of filamentous
bacteria, it had little or no effect on fungal mycelia. Traditional disinfection
methods, such as ozonation or UV treatment, hydrogen peroxide, chlorine,
and others, could also be used as selective disinfectants. 1120
RESEARCH NEEDS
The operation of fungal wastewater treatment on large scale is limited. Fur-
ther technical and feasibility details are required for better understanding and
BEST_A_328061 bestxml-als-v1.cls March 26, 2010 14:4
Fungal Wastewater Treatment 39
operation of fungal process for wastewater treatment. Descriptive models of
fungal degradation kinetics and byproduct recovery are needed to serve as a
1125
basis for designing wastewater treatment systems. Furthermore, techniques
for preventing bacterial contamination under nonaseptic operation are
critical.
Advances in biotechnological applications in recent years have made it
possible to obtain desired characteristic in various microorganisms through
1130
genetic engineering. It is also possible to increase fungal byproduct produc-
tion by genetically engineered fungal cultures to enhance a specific product
excretion (Timberlake & Marshall, 1989). This enhanced byproduct produc-
tion could be obtained through classical mutagenesis, which can help in
both MBP production and microbial screening. It also may be possible to
1135
use recombinant DNA technology for developing fungi to obtain specific
desirable byproducts at available growth conditions (pH, temperature, spe-
cific substrate, and others). Microbial growth parameters, mutagenesis, strain
selection, and genetic engineering of filamentous fungi are well established
and could be used to improve competitiveness, organic conversion, and
1140
separability.
There have been a few studies on genetic modification of fungal species
(Christensen et al., 1988; Ward et al., 1992). For example, when chitin syn-
thase genes isolated from R. oligosporus were probed into Saccharomyces
cerevisiae, elevation in synthase activity was achieved (Motoyama et al.,
1145
1994). Genetic modification of fungal species for organic compound re-
moval and biomass production during wastewater treatment has not been
investigated, but could be expected to significantly enhance the concept of
fungal wastewater treatment with production of valuable biochemicals.
SUMMARY1150
Filamentous fungi can be used for treatment of a variety of wastewaters,
ranging from readily degradable food-processing wastewater to highly pol-
luting recalcitrant milled olive oil processing wastewater. Fungal treatment
technology is well suited for resource recovery and new byproduct devel-
opment based on the ability of fungi to produce various enzymes, amino
1155
acids, and other valuable biochemicals. This comprehensive review has dis-
cussed various studies conducted on different types of wastewater and fun-
gal species suited for organic removal and fungal biomass cultivation with
valuable byproducts. Particular emphasis has been given to the operational
conditions and fungal byproduct recovery during wastewater treatment. The
1160
occurrence of bacterial contamination in fungal wastewater treatment sys-
tems under nonaseptic conditions has also been discussed in detail. There
are very few studies on fungal harvesting and the nutrient requirements for
fungal treatment. Moreover, the growth and development of specific fungal
BEST_A_328061 bestxml-als-v1.cls March 26, 2010 14:4
40 S. Sankaran et al.
species for particular types of wastewater is also limited. Thus, an inventory 1165
based on economics and regulatory requirements needs to be developed
to arrive at a viable solution for constructing a full-scale fungal wastewater
treatment process for resource recovery.
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