Bioresource Technology 97 (2006) 2195–2210

Review Paper

A review on hydrolytic enzymes in the treatment of wastewater

with high oil and grease content
a,* b
M.C. Cammarota , D.M.G. Freire
a
School of Chemistry, Federal University of Rio de Janeiro, Rio de Janeiro, RJ 21945-970, Brazil
b
Institute of Chemistry, Federal University of Rio de Janeiro, Rio de Janeiro, RJ 21945-970, Brazil

Received 20 May 2005; received in revised form 7 February 2006; accepted 7 February 2006
Available online 18 April 2006

Abstract

Wastewater from dairies and slaughterhouses contains high levels of fats and proteins that present low biodegradability. A large num-
ber of pretreatment systems are employed to remove oil and grease (O&G) to prevent a host of problems that may otherwise arise in the
biological process, and reduce the efficiency of the treatment station. Problems caused by excessive O&G include a reduction in the cell-
aqueous phase transfer rates, a sedimentation hindrance due to the development of filamentous microorganisms, development and flo-
tation of sludge with poor activity, clogging and the emergence of unpleasant odors. Therefore the application of a pretreatment to
hydrolyze and dissolve lipids may improve the biological degradation of fatty wastewaters, accelerating the process and improving time
efficiency. However thus far, only a few studies describing the degradation of fats and oils by alkaline/acid/enzymatic hydrolysis have
been reported; the treatment of effluents from several origins is a new and promising application for lipases. Among the strains that pro-
duce the hydrolytic enzymes studied, the fungus Penicillium restrictum is a particularly promising one. When cultivated in low-cost solid
medium composed of agro-industrial waste, P. restrictum produces a pool of hydrolases capable of degrading the most complex organic
compounds. This degradation enables a considerable increase in organic matter removal efficiency to be realized, which results in the
attainment of a high-quality effluent in the subsequent biological treatment stage. Consequently, there is presently a wide variety of ongo-
ing scientific investigation in the field of developing enzymatic hydrolysis processes to precede traditional biological treatment.
 2006 Elsevier Ltd. All rights reserved.

Keywords: Fatty effluents; Hydrolases; Lipases; Enzymatic hydrolysis; Biological treatment; Solid-state fermentation; Babassu cake; Penicillium restrictum

1. Introduction
The steadily rising demand for food products has
required an increase in productivity from food industries,
and thus an increase in the number of dairies, slaughter-
houses and meat processing plants in many countries. In
Brazil, both the dairy and meat industry sectors have a sub-
stantial presence; they are responsible for an important
component of the economy, both in providing employment
and in the production of goods. In 2002, 21 · 109 L of milk
were produced in Brazil, which generated approximately
84 · 109 L of post-production and processing effluent, over
*
Corresponding author. Tel.: +55 21 2562 7568; fax: +55 21 2562 7567.
E-mail address: christe@eq.ufrj.br (M.C. Cammarota).
90% of which had not received any type of treatment (Jung
et al., 2002). In slaughterhouses, the consumption of water
per slaughtered animal varies according to the animal and
the industry-specific process employed, ranging from 1.0 to
8.3 m3. Most of this is discarded as wastewater, with 0.4–
3.1 m3 of water per slaughtered animal being reported in
the literature (Caixeta et al., 2002).
Wastewaters from dairies (Cammarota et al., 2001;
Danalewich et al., 1998; Jung et al., 2002; Marshall and
Harper, 1984; Omil et al., 2003) and slaughterhouses (Bat-
stone et al., 1997; Martı́nez et al., 1995; Massé et al., 2001,
2003) are rich in biodegradable organic molecules and
nutrients and usually contain high levels of fats and pro-
teins that have a low biodegradability coefficient. If not
treated, they cause gross pollution of land and water with

0960-8524/$ - see front matter  2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biortech.2006.02.030
2196 M.C. Cammarota, D.M.G. Freire / Bioresource Technology 97 (2006) 2195–2210
their high biochemical oxygen demand (BOD) and chemi-
cal oxygen demand (COD). The technology of lipid biodeg-
radation has not yet been fully exploited in the processing
of the tons of one of the major organic materials present in
wastewater streams; hence the restrictions on disposal. In
countries where food habits result in a large amount of
residual fat and oil in wastewater, it has become increas-
ingly difficult to fulfill the discard requirements (Tano-
Debrah et al., 1999).
Some conventional methods for wastewater treatment
include the reuse of certain fractions such as cheese whey
in dairies and blood in slaughterhouses. When this is not
possible, various treatment processes are used; these are
mainly aerobic processes, but in the last two decades anaer-
obic reactors have been increasingly applied (Omil et al.,
2003). However, it is necessary to reduce the concentration
of fat, oils and proteins or to eliminate these materials alto-
gether, in order to enable the biological treatment to pro-
ceed without any inhibition of the biological reduction of
organic matter in wastewater.
A large number of pretreatment systems (grease-trap,
tilted plate separators, dissolved air flotation systems and
physical–chemical treatment) are employed to remove oil
and grease (O&G) from these wastewaters prior to the
main treatment process itself, which is generally of a bio-
logical nature. However, the cost of such reagents is high,
the removal efficiency of dissolved and/or emulsified
O&G is low and extremely problematic sludge is produced.
(Kárpáti et al., 1995; Tano-Debrah et al., 1999; Willey,
2001).
The O&G not retained in the pretreatment systems enter
into the biological treatment system, becoming a consider-
able nuisance, especially in conventional mesophilic pro-
cesses (Massé et al., 2001). These O&G are associated
with several problems, including the reduction of the
cell-aqueous phase transfer rates (substrates, products
and oxygen) through the formation of a lipid coat around
the biological floc. In addition, filamentous microorganism
blooms (bulking) and floating sludge with undesirable
physical characteristics may develop. Furthermore, poor
activity associated with excessive amounts of O&G in the
wastewater hinders sedimentation and causes losses of
biomass through the reactor’s outflow. These detrimental
effects are further associated with clogging and the emer-
gence of unpleasant odors, and are frequently associated
with a reduction in the efficiency of treatment stations
(Grulois et al., 1993; Perle et al., 1995; Vidal et al., 2000).
Many systems act primarily as solid separators and operate
marginally as a biological treatment process. In addition,
fats in wastewaters produce glycerol and long-chain fatty
acids (LCFA) during the hydrolytic step, and the latter
are reported to inhibit the activity of various microorgan-
isms (Angelidaki and Ahring, 1992; Hanaki et al., 1981).
Fat and oil may become more accessible to microorgan-
isms and their lipolytic enzymes when the process occurs
under thermophilic conditions (Becker et al., 1999; Tho-
mas, 1987). Despite the increased efficiencies and decreased
residence times that can be achieved under thermophilic
conditions (in comparison with mesophilic processes),
large-scale application of this technology poses serious
challenges for treatment systems. Namely, expensive mod-
ifications involving the inclusion of heating systems, main-
tenance and temperature control are frequently required.
The application of a pretreatment process to hydrolyze
and dissolve fats may improve the biological degradation
of fatty wastewaters, accelerating the process and reducing
time. However, hydrolysis pretreatments have been mostly
tested on waste activated sludge or municipal waste (Dau-
ber and Boehnke, 1993; Knezevic et al., 1995; Lin et al.,
1997), which contain at least 10 times more suspended sol-
ids than slaughterhouse or dairy wastewaters. Moreover,
experiments have usually dealt with complex waste, and
the pretreatment effects on the different organic fractions
(protein, lipid, carbohydrate) have rarely been assessed.
There have been few studies describing the degradation
of fats and oils by alkaline/acid/enzymatic hydrolysis.
Especially lacking from the literature is the examination
of the biological treatment of wastewater with high fat con-
tent after an enzymatic hydrolysis step. The treatment of
such effluents from several origins is a new and promising
actuation area for lipases. Candida rugosa lipases were used
in the treatment of domestic wastewaters and in the clean-
ing of sewer systems, cesspools and sinkholes (Jaeger and
Reetz, 1998). The use of commercial lipases from several
sources (animal, vegetal and microbial) in the pretreatment
of slaughterhouses effluents (Massé et al., 2001) and the use
of lipases from Pseudomonas aeruginosa in the effluents of
restaurants (Dharmsthiti and Kuhasuntisuk, 1998) have
also been investigated. Lipases have also been used to
accelerate the biodegradation of polymers (Marten et al.,
2003; Sivalingam et al., 2003; Takamoto et al., 2001) and
of slurries from oil-well perforations containing synthetic
esters emulsified in water (Aliphat et al., 1998).
The employment of microbial enzymatic preparations
obtained by solid-state fermentation (SSF) in the pretreat-
ment of dairy industry and slaughterhouse effluents with
high fat content has been studied extensively by the Federal
University of Rio de Janeiro (UFRJ) research group (Cam-
marota et al., 2000, 2001, 2003; Jung et al., 2002; Leal et al.,
2002). Among the several strains that produce the hydro-
lytic enzymes isolated by the UFRJ research group, the
fungus Penicillium restrictum is the most promising. This
fungus, when cultivated in babassu cake solid medium
(waste from babassu oil industrial production), produces
lipases, proteases and amylases (Gombert et al., 1999;
Palma et al., 2000). The employment of SSF to produce
these enzymes provided lower production costs relative to
those obtained by submerged fermentation (SF) (Castilho
et al., 2000; Freire and Castilho, 2000). In the treatment
of effluents, the use of low-cost enzymatic preparations is
vital, since the use of high-cost commercial enzymatic prep-
arations would make the pretreatment procedure not feasi-
ble. In this context, it seems that the SSF process could be a
suitable technology once enzyme production can be per-
 M.C. Cammarota, D.M.G. Freire / Bioresource Technology 97 (2006) 2195–2210
formed in situ and industrial wastes can be employed as a
source of nutrients for the fermentative process. Indeed, an
enzymatic pre-hydrolysis stage with the enzymatic prepara-
tion obtained through SSF enabled a considerable increase
of the organic matter removal efficiency and the attainment
of a high-quality effluent, which was within discard stan-
dards required by the local environmental legislation, in a
subsequent stage of aerobic (Jung et al., 2002; Rosa,
2004) or anaerobic (Cammarota et al., 2001; Leal et al.,
2002, in press; Valladão, 2005) biological treatment.
2. Problems in the biological treatment of effluents
with high fat content
2.1. Operational problems
In aerobic bioreactors, O&G block the gas transfer
required for the biological degradation. Specifically they
reduce the oxygen transfer rates to the biological microbial
consortium by the formation of a lipid coat around the floc
(Chao and Yang, 1981; Grulois et al., 1993; Lemmer and
Baumann, 1988). O&G cause problems in the pumping
and aeration systems that arise due to the development
of filamentous microorganisms (Sphaerotilus natans, Thio-
thrix, Beggiatoa, Nocardia and Microthrix genuses). These
microorganisms are involved in the formation of scum and
stable foams on the surface of the aeration tank. The scum
and foams ultimately hinder the biomass flocculation and
sedimentation, a phenomenon known as bulking (Jenkins
et al., 1993). High O&G contents also generate agglo-
merates or pellets inside the secondary sludge flocs, which
hinders sedimentation, generating unpleasant odors and
reducing the efficiency of the treatment station (Ecken-
felder, 2000).
Several aerobic treatments have been used extensively
in the dairy industry. Such treatments include aerated
lagoons, activated sludge processes (Carta-Escobar et al.,
2005; Shack and Shandhu, 1989; Stephenson, 1989), a
membrane sequencing batch reactor (Bae et al., 2003),
trickling filters (Walsh et al., 1994) and rotating biological
contactors (Radick, 1992). However, the energy require-
ments for the aeration in these installations are high and
problems such as bulking and excessive biomass growth
frequently occur under these conditions (Timmermans
et al., 1993).
Carta-Escobar et al. (2005) studied the organic matter
removal kinetics of dairy wastewater in two reactor config-
urations. In this study, the authors made a brief description
of the general observations concerning the performance of
series reactor systems. At the volumetric organic loads
studied (0.24–0.70 kg COD/m3 d), frequent episodes of
foaming occurred with sludge floating on the surface of
the reactors. Clogging problems also occurred in the diffus-
ers due to the formation of very viscous films. In addition,
the samples exhibited pronounced resistance to filtration,
and the filter membranes became fouled by viscous films.
These foaming problems and difficulties in obtaining opti-
2197
mal filtration are frequent occurrences in the treatment of
dairy wastewaters (Bae et al., 2003; Danalewich et al.,
1998).
In anaerobic biological processes, O&G may solidify at
lower temperatures and thus cause operational damage
associated with clogging and unpleasant odors (Grulois
et al., 1993; Vidal et al., 2000). Meanwhile, the treatment
of dairy wastewaters by means of upflow anaerobic sludge
blanket (UASB) reactors (Hansen and Hwang, 1990;
Hawkes et al., 1995; Rico et al., 1991), hybrid UASB
reactors (Ozturk et al., 1993), expanded granular sludge
bed (EGSB) reactors (Petruy and Lettinga, 1997), as well
as others based on anaerobic filters (Méndez et al., 1989;
Veiga et al., 1994; Viraraghavan and Kikkeri, 1990), have
been reported. These studies demonstrated that anaerobic
treatment can be used effectively for these effluents, in
spite of the different operational problems described in
the literature, such as sludge flotation or toxicity/inhibi-
tion processes. However, in many of these studies the
reactors processed synthetic steady-composition wastes,
with dairy waste being simulated by powdered milk for
example. These synthetic wastes contain much less O&G
than real effluents. Indeed, real effluents also contain addi-
tives such as disinfectant and cleaning agents that may
jeopardize the biological treatment process. In the work
of Ramasamy et al. (2004), for example, the authors
established the feasibility of UASB reactors in treating
dairy wastewaters. They reported COD reduction rates
greater than 90% at hydraulic retention times (HRT) of
3 and 12 h, and COD loading rates ranging from 2.4 to
13.5 kg/m3 d. For these experiments, 1 g of powdered
milk per liter of water was used as effluent, providing a
COD of 1440 mg/L. From this stock solution, effluents
of a range of concentrations were prepared by appropri-
ate dilution.
Despite the results mentioned previously, the develop-
ment and flotation of sludges with different physical char-
acteristics or poor activity are known to cause biomass
loss through the reactor’s outflow, decreasing its quantity
inside the reactor and the treatment efficiency (Perle et al.,
1995; Rinzema et al., 1993). The O&G adsorbed on the
surface of the anaerobic sludge may limit the transport
of soluble substrates to the biomass and consequently
may reduce the substrate conversion rate (Rinzema
et al., 1994). Petruy and Lettinga (1997) showed that
70% of lipids were adsorbed by the granular sludge,
within approximately one day, and thereafter, the remain-
ing lipids were slowly converted into methane gas. Sayed
et al. (1988), studying the anaerobic degradation of vari-
ous fractions of slaughterhouse wastewater, verified that
the process-controlling factor was the liquefaction of col-
loidals adsorbed on the bacteria and the hydrolysis of
coarse suspended solids entrapped within the biomass
bed.
Martı́nez et al. (1995) evaluated the effluent treatment
plant of a slaughterhouse and detected some problems in
the operation of the anaerobic reactors caused by the pres-
2198 M.C. Cammarota, D.M.G. Freire / Bioresource Technology 97 (2006) 2195–2210
ence of fat and suspended solids in the effluent. A flotation
system using pressurized air injection was tested in the
plant, obtaining fat removal efficiencies of 63% and 37%
for two important streams (red water and green water,
respectively). In order to improve the hydrolysis of partic-
ulate matter, a system of two UASB reactors with recircu-
lation, connected in series, was tested on a laboratory scale.
The removal efficiency was 77% for soluble COD and 82%
for insoluble COD, at a volumetric load of 1.8 kg COD/
m3 d. Meanwhile, the first few cells of an anaerobic baffled
reactor treating meat packing plant wastewater eventually
became filled with solids, and as a result the system basi-
cally acted as a separator of solids and operated only mar-
ginally as a biological treatment process.
Saxena et al. (1986) treated slaughterhouse wastewater
containing approximately 1 g of suspended solids/L in a
packed-bed anaerobic reactor. Fat and suspended solids
deposition caused a rapid deterioration of the biomass at
organic loading rates above 2 kg COD/m3 d. These find-
ings together indicate that to improve wastewater treat-
ment processes researches need to develop techniques to
more efficiently reduce O&G levels in effluent.
2.2. Effect of long chain fatty acids on the biological
treatment
In addition to the necessity of longer residence times for
fat particles to be degraded and the design of a proper bio-
reactor to avoid undesirable fat accumulation, long chain
fatty acids (LCFA) produced from the hydrolysis of
O&G can exert toxic effects and inhibit anaerobic bacteria
(Angelidaki and Ahring, 1992; Hanaki et al., 1981). The
biodegradation of lipids is difficult due to their low bio-
availability (Petruy and Lettinga, 1997). Fats in waste-
waters produce glycerol and LCFA (saturated fatty acids
with 12–14 carbon atoms and unsaturated fatty acids with
18 carbon atoms) during the hydrolytic step. Glycerol was
found to be a non-inhibitory compound (Perle et al., 1995).
However, LCFA have been reported to inhibit the activity
of various microorganisms (Angelidaki and Ahring, 1992;
Hanaki et al., 1981; Ching-Shyung et al., 1996; Koster,
1987; Rinzema et al., 1994). They also decrease the avail-
ability of adenosine triphosphate (ATP) (Hanaki et al.,
1981; Perle et al., 1995). The inhibitory effect increases with
the number of double bonds and cis-isomers that are abun-
dant in natural lipids (Rinzema, 1988). As in the case of
volatile fatty acids (VFA), the toxicity of LCFA seems to
be related to the unionized form of these acids, namely
the long chain free fatty acids (LCFFA).
Studies carried out by Hanaki et al. (1981) showed that
LCFA content affects the amount of hydrogen produced
by acetogenic bacteria, responsible for the b-oxidation of
LCFA. The inhibition of acetogen and acetotrophic meth-
anogens causes a pronounced lag phase in batch experi-
ments. In addition, the inhibition of hydrogenotrophic
methanogens causes a decrease on the hydrogen conversion
rate (Rinzema et al., 1994).
Ching-Shyung et al. (1996) evaluated the effects of spe-
cific acetoclastic methanogenic activity and sludge adapta-
tion to lipids on toxicity degree. Fifty percent inhibition
concentration (IC50) values for sodium oleate obtained
from batch toxicity tests at 40 C ranged from 0.26 to
3.34 mM for the various sludges examined. Suspended
and flocculent sludges, which have a higher specific surface
area, displayed much greater inhibition than did granular
sludges. The authors suggested the use of granular sludges
as appropriate inocula for reactors treating lipids (fats, oils
and greases) wastewaters to decrease the toxic impact from
their hydrolysis products (LCFA). Indeed, an IC50 value 13
times higher for granular (1015 mg/L) than for suspended
sludge (79 mg/L) was obtained by these researchers. Koster
and Cramer (1987) found a higher IC50 value (1322 mg/L)
than did Ching-Shyung et al. (1996) (690 mg/L) for granu-
lar sludge taken from upflow anaerobic sludge blanket
(UASB) digesters treating effluents from potato processing
plants. This difference was attributed to the higher level of
calcium (35 mg/L) and the lower temperature (30 C) used
in Koster and Cramer’s study.
In accordance with research by Stoll and Gupta (1997),
Deckena et al. (1995) used both single and two-stage con-
tinuously operated anaerobic digesters for the digestion
of O&G residues and reported fat reduction rates in the
range of 88–94% with initial fat concentrations ranging
from 30 to 70 g/L in single-stage digesters operating with
hydraulic retention times (HRT) of 30–46 days. The spe-
cific biogas production obtained in the study was between
870 and 1320 L/kg fat. The authors attributed the high bio-
gas yield to the long HRT and the continuous mode of
digester operation.
Vidal et al. (2000) evaluated the influence of absolute
and relative concentrations of carbohydrates, fats and pro-
teins on the anaerobic biodegradability of dairy waste-
waters. They found that the anaerobic biodegradation rate
of wastewaters rich in fat was slower than that of waste-
waters with a low fat content, due to the slower rate of
the fat hydrolysis step. However, this reduced hydrolysis
rate prevented VFA accumulation and as a result the over-
all process was favored. Thus the presence of fats in the
wastewater prevented the periodic production of high
concentrations of VFA, which may adversely affect the
process. However, assays with wastewater with higher
COD levels presented problems of sludge flotation during
subsequent anaerobic biodegradation.
The effects of delivering increasing concentrations of
lipids on the characteristics of the sludge that develops in
anaerobic fixed-bed digesters was investigated by compari-
son with a control digester fed with a non-fat substrate
(Alves et al., 2001). The effect of gradually shifting the
delivered substrate from the original composition, to
sodium oleate as the sole carbon source, allowed the
researchers to evaluate whether prior contact with lipids
would increase the tolerance of acetoclastic bacteria to
oleic acid and the LCFA biodegradation capacity. Sludge
from a bioreactor that had been acclimated to lipids during
 M.C. Cammarota, D.M.G. Freire / Bioresource Technology 97 (2006) 2195–2210
the first operation period exhibited a higher tolerance to
oleic acid toxicity (IC50 = 137 mg/L) during sodium ole-
ate-based effluent treatment, than did sludge delivered with
a non-fat substrate (IC50 = 80 mg/L). This more tolerant
sludge also exhibited the highest biodegradation capacity
for oleic acid, achieving maximum methane production
rates between 33 and 46 mL CH4(STP)/g VS d and maxi-
mum percentages of methanization between 85% and
98% for oleate in the 500–900 mg/L range. When sodium
oleate was the sole carbon source delivered to both digest-
ers, the biomass became encapsulated with organic matter,
possibly oleate or an intermediate of its degradation, e.g.
stearate. These findings demonstrated that both tolerance
to toxicity and biodegradability of oleic acid were
improved by acclimatization to lipids or to sodium oleate
below a threshold concentration.
3. Conventional treatment systems for effluents
with high oil and grease contents
3.1. Physical–chemical pretreatment systems
for oil and grease removal of effluents
The main technique for separating fat and oil from
wastewater is the grease-trap method (gravity separation
of floatable fat/oil). In general terms, a fat trap can be con-
sidered as a rectangular or circular vessel through which
the wastewater passes under laminar-flow conditions at a
rate that allows the fat/oil particles to rise to the surface
by the time that they approach the outlet end of the trap.
The separation principle is based on Stokes’ law relating
rising velocity of a particle to its diameter, and on the the-
ory that the separation efficiency is independent of depth:
gðqs  qÞd 2
Vc ¼ ð1Þ
18l
where Vc is the terminal velocity of the particle, g is the
acceleration due to gravity, qs is the density of the particle,
q is the density of the fluid, d is the diameter of the particle,
and l is the absolute viscosity of the fluid.
In practice, fat traps have a depth of 1.5 m; though if the
accumulation of a bottom sludge is expected to occur, then
an additional 0.5 m should be added to the total liquid
depth (Willey, 2001). This method frequently becomes
unaesthetic and even causes air pollution in the vicinity
of the treatment facility. Studies conducted by Tano-
Debrah et al. (1999) with restaurant effluents in Japan
showed that some individual restaurants, which could pro-
duce approximately 100 tons of wastewater a day in full
operation, always possess too much residual fat/oil to rely
entirely on the gravity floatation method to meet
regulations.
Alternatively, oil/water separators (tilted plate separa-
tors – TPS), flotation systems and physical–chemical treat-
ments have been employed to reduce wastewater lipid
content. The TPS was developed in the petrochemical
industry and takes advantage of the fact that its surface
2199
area, rather than the depth, is the most important factor
in the oil separation. The introduction of inclined plates
into a vessel provides many parallel plate gravity separa-
tors with low liquid depth, but a high surface area. Typi-
cally, a TPS occupies less than 10% of the area of a
conventional fat trap. An additional advantage is that
these units are package plants, which can be moved to
accommodate site developments. There are, however, cer-
tain problems with edible oils and fats that must be consid-
ered, namely: (1) The narrow gaps (20 mm) between the
plates may be susceptible to fouling if solid or semisolid
fat is present in the effluent; (2) Removal of the plate pack
for cleaning is time consuming and requires the use of a
crane; (3) Careful selection of pumps and flow control
are required at the site where the wastewater is pumped
into the unit to avoid surging and undesirable fluctuations
in liquid depth (Willey, 2001).
In dissolved air flotation systems (DAF), air is dissolved
into wastewater under pressure (approximately 6 bar). The
air-saturated liquor is subsequently passed through nozzles
into a rectangular or circular vessel. Due to the pressure
drop, the dissolved air is released from the solution in the
form of microbubbles (60–70 lm diameter) clouds. The
microbubbles attach to the surface of the fat/oil particles
and increase their rise rate. As a result, typical surface load-
ing rates for DAF units are 3–6 m3/m2 Æ h. Generally, the
recycling of defatted effluent of up to 50% of the raw
wastewater flow is used as the air carrier to achieve an
air to solids ratio of 0.005–0.08 (w/w). As air solubility is
substantially reduced at elevated temperatures (Willey,
2001), wastewater temperature is an important consider-
ation in the design of DAF systems.
Physical–chemical treatments reduce the organic load
by protein and fat precipitation or flotation with different
chemical compounds. Aluminum sulfate, ferric chloride,
or more commonly, lime, may be used to break the fat
emulsion and coagulate fat particles, which can then be
readily separated by flotation or sedimentation. The settle-
ment or flotation rate can frequently be improved by a sec-
ond-stage flocculation. This involves the addition of low
levels of polyelectrolyte (0.5–5.0 mg/L) to the wastewater
once coagulation has taken place (Willey, 2001). The wide-
spread utility of physical–chemical treatments however is
questionable because the cost of reagents is high, the
removal efficiency of the dissolved and/or emulsified
O&G is low, and highly problematic sludge is produced
when flocculating agents such as polyelectrolytes and salts
are used (Kárpáti et al., 1995).
3.2. Chemical and enzymatic hydrolysis of oil and
grease as a pretreatment for biological systems
Chemical hydrolysis with NaOH increases the ratio of
soluble COD to total COD and reduces the volatile solid
content during anaerobic digestion (Knezevic et al., 1995;
Lefebvre et al., 1998; Lin et al., 1997; Rajan et al., 1989;
Ray et al., 1990). Massé et al. (2001) examined experiments
2200 M.C. Cammarota, D.M.G. Freire / Bioresource Technology 97 (2006) 2195–2210
reported by Karlsson (1990) in which the effects of various
chemical (HCl, NaOH and Ca(OH)2) and biological (acid
fermentation) hydrolysis pretreatments on waste activated
sludge were tested. In these experiments no biological pre-
treatment effect was seen for lipids, while both acid and
alkaline pretreatments reduced lipid in the sludge phase
by 28%. The author concluded, based in these experiments,
that the lipid portion of waste activated sludge is the most
difficult organic fraction to hydrolyze using pretreatments.
Omil et al. (2003) studied the anaerobic treatment of the
effluents generated during the quality control process for
raw milk (more complex than the ones commonly gener-
ated by dairies due to the presence of milk preservation
agents such as sodium azide or chloramphenicol). The
treatment of these effluents, carried out in a full-scale plant
comprising an anaerobic filter and an aerobic sequential
batch reactor, was investigated. After more than 2 years
of operation, a successful anaerobic treatment was
achieved without fat removal from the effluent prior to
its delivery to the anaerobic reactor (for organic loading
rates of 5–6 kg COD/m3 d, COD removal was greater than
90%). No biomass washout was observed and most of the
fat contained in the wastewater was successfully degraded.
However, the alkali added to neutralize acidic wastewaters
and to maintain an ideal alkalinity level, enhanced the
breakdown of fat in the feeding tank, thereby enhancing
fat biodegradation in the anaerobic filter. This addition
of alkali reduced the amount of fat in the effluent to a level
below 500 mg/L. Even with this alkaline hydrolysis, the
authors detected severe disturbances in the anaerobic pro-
cess as VFA accumulated and fatty floating matter was
observed in the upper part of the anaerobic filter, above
the liquid level. In many instances, in order to re-establish
as fast as possible the proper conditions, the industry was
forced to carry out a new inoculation of the anaerobic
reactor.
Massé et al. (2001) evaluated the effect of alkaline and
enzymatic pretreatments on the solubilization and size
reduction of fat particles in slaughterhouse wastewater.
Addition of 2–16 g NaOH/L did not increase soluble
COD in the wastewater, but the average particle size was
reduced to 73% of the initial average particle size at NaOH
concentrations ranging from 6 to 12 g/L. The authors con-
cluded that the high doses of NaOH required and the
resulting increase in pH rendered alkaline pretreatment a
poor choice for a hydrolysis pretreatment of fat particles.
Pretreatment with pancreatic lipase PL-250 reduced the
average particle size to a maximum of 60% of the initial
particle size (Massé et al., 2001). The bacterial lipase LG-
1000 was also efficient in reducing the average fat particle
size, but high doses (>1000 mg/L) were required to obtain
a substantial reduction after 4 h of pretreatment. No parti-
cle size reduction or changes in soluble COD were noted
after 4 h of pretreatment with the plant lipase EcoSystem
Plus. It was thus concluded that pancreatic lipase PL-250
was the best known pretreatment for hydrolyzing fat parti-
cles in slaughterhouse wastewater.
3.3. Biological treatment of oil and grease
under thermophilic conditions
The biological treatment of fats and oils under thermo-
philic conditions is advantageous due to the favorable
changes in most physical properties of hydrophobic com-
pounds at high temperatures. In the liquid state, these sub-
stances become more accessible to microorganisms and
their lipolytic enzymes. Both diffusion coefficients and the
solubility of fatty acids in aqueous media increase signifi-
cantly with rising temperatures, allowing a better mass
transfer (Thomas, 1987).
Thermophilic processes for the treatment of wastewaters
generated in various industries have been studied on a lab-
oratory scale. Becker et al. (1999) studied the biodegrada-
tion of olive oil and the treatment of the lipid-rich wool
scouring wastewater under aerobic thermophilic (65 C)
conditions using the newly isolated strain Bacillus thermo-
leovorans IHI-91. A severe growth inhibition was observed
when the delivered olive oil concentration was increased
to more than 4 g COD/L. Lipid removal efficiencies from
the highly loaded wool scouring wastewater (COD of
77,000 mg/L and lipid concentration of 17,000 mg/L) were
20–30% at a residence time of 10–20 h, while the COD
removal efficiencies were 15–20%. The lipid degradation
rates obtained under thermophilic conditions were mark-
edly greater than that obtained with mesophilic processes.
Lapara and Alleman (1999) attributed this difference to
the fact that the biology of thermophilic aerobic systems
differs from conventional activated sludge systems in that
nitrifying bacteria, floc-forming organisms and higher
organisms that aid flocculation are not present, which
affects sludge sedimentation.
3.4. Use of microorganisms, surfactants and/or
commercial enzymes for the treatment of effluents
with high oil and grease contents
There are research reports and patents that have
described the use of microorganisms and/or enzymes pools
developed in the laboratory for the biological treatment of
effluents with high fat and oil concentrations. For example,
the Japanese company Meito Sangyo Co. (41 Sasazuka-
Cho 2-ChomeNishi-Ku Nagoya, Aichi, Japan) produces
a lipase from Candida rugosa (Lipase-MY) for fat removal
in equipment of effluent treatment plants in the United
States (website: http://www.meito-sangyo.co.jp (28 Jan
2006)). In addition, Neozyme International Inc. (33
Journey, Aliso Viejo, CA, USA) manufactures and sells
patented bioorganic catalytic formulations that rapidly
break down organic contaminants including fats, oils,
and greases (FOG) due to the difficulty of successfully
treating these substances with conventional products and
equipment (website: http://www.daleco.com (18 Oct
2005)).
Mendes et al. (2005) cite various patent documents for
the application of hydrolytic enzymes, especially lipases,
 M.C. Cammarota, D.M.G. Freire / Bioresource Technology 97 (2006) 2195–2210
in the wastewater treatment. The patent developed by Tso-
chocke (FR pat.2,659,645 1990) describes a procedure for
lipid degradation in grease-traps that involves the addition
of lipase-producing bacteria. These bacteria are immobi-
lized on no-biodegradable support material and immersed
in the surface of grease-traps, and are reported to achieve
hydrolysis rates greater than 90%. Gardon and Lebesgue
(FR pat. 2,684,664 1991) proposed the use of a bioadditive
that consists of various microorganisms (Aerobacter aero-
genes, Bacillus subtilis, Cellulomonas biazotea, Nitrosomonas
sp., Nitrobacter winogradskyi, Pseudomonas denitrificans,
P. stutzeri and Rhodopseudomonas palustris) for the treat-
ment of wastewater with a high lipid content. Specifically,
a bacteria concentration of 107 to 109 cells/mL for a period
of 12–72 h under oxygen concentration of 4–8 mg/L was
recommended. Mendes et al. (2005) mentioned the use of
a mixture composed of emulsifiers, microorganisms and
enzymes (protease, amylase, lipase, cellulase and pectinase)
for lipid removal from wastewaters produced by the clean-
ing of industrial machines. The microorganisms included in
this enzymatic complex (Amerzyme-A-100, Applied Bio-
chemists – USA) are Bacillus subtilis, a protease and amy-
lase producing bacterium, and Aspergillus niger, a lipase,
cellulase and pectinase producing bacterium. In this mix-
ture, the enzymes are present in concentrations between 1
and 200 mg/L.
Various microorganisms have been used for the treat-
ment of grease-containing restaurant wastewater and other
grease-containing wastewaters. A method was proposed
for the treatment of waste by the direct cultivation of lipo-
philic yeasts into the waste (Anon, 1994). Wakelin and For-
ster (1997) investigated the microbial treatment of waste
from fast-food restaurants for the removal of fats, oils
and greases. They cultivated pure and mixed microbial
flora (known to produce lipases and other enzymes), and
found that Acinetobacter sp. was the most effective of the
pure cultures, typically degrading 60–65% of the fatty
material, whose initial concentration had been 8 g/L. P.
aeruginosa LP602 was tested for its ability to treat this type
of wastewater (Dharmsthiti and Kuhasuntisuk, 1998).
When these cells were added to the wastewater at approx-
imately 107 CFU (colony forming units)/mL, they domi-
nated the whole system, and no other microorganisms
were found in detectable numbers. Indeed, the BOD was
reduced by 94.1% after a 8-day incubation period. The lipid
content in the wastewater was also rapidly reduced, and all
the lipid content was removed within the first 5 days of
treatment. Crude lipase from P. aeruginosa LP602 (3.5 U/
mL) was added to this same lipid-rich restaurant waste-
water in a ratio of 1:1 and incubated with shaking at
37 C. The lipid content (approximately 200 mg/L) was
reduced by 70%, to less than 10 mg/L, during the first
24 h and was not detected after 48 h. Tano-Debrah et al.
(1999) developed an inoculum with high fat and oil degrad-
ing activities that consisted of a mixed culture of 15 bacte-
rial isolates from various fatty wastewater samples taken
from grease-traps of restaurants in Japan. All isolates were
2201
able to degrade various fats and oils to some degree. How-
ever, the extent of degradation varied for each fat/oil
tested, being lower for shea fat (fat obtained from nuts of
the tree Butyrospermum parkii). The variation in the com-
position of the fat/oil mixture caused variations in the
development of the inoculum bacteria. The authors sug-
gested that this variation highlights the need to identify
more microorganisms with degrading activities in order
to optimize and broaden the applications of lipid biodegra-
dation technology.
Mongkolthanaruk and Dharmsthiti (2002) evaluated a
mixed culture composed of P. aeruginosa LP602, Acineto-
bacter calcoaceticus LP009 (both lipase-producing bacteria)
and Bacillus sp. B304 (an amylase and protease producing
bacterium) to lower the biochemical oxygen demand
(BOD) value and lipid content of lipid-rich wastewater. It
was demonstrated that this consortium of 3 bacterial cul-
tures could be used successfully to treat lipid-rich wastewa-
ter. The BOD and the lipid content were reduced from
3600 mg/L and 21,000 mg/L, respectively, at day 0, to less
than 20 mg/L within 12 days under aerobic conditions.
Before these products can be widely applied however, it
needs to be examined whether there are hazardous species
present and whether they have any adverse environmental
effects, especially when used in places with conditions that
differ considerably from the conditions under which the
bacteria were isolated.
Another alternative method to deal with high fat con-
tent in wastewater is the use of surfactants to facilitate
the biodegradation by dissolving the fat and oils. Biosurf-
actants can easily be incorporated directly into the biolog-
ical process, thus eliminating the need for additional
processes and resulting in lesser operational costs. Nakhla
et al. (2003) demonstrated the effectiveness of a biosurfac-
tant derived from cactus (BOD-BalanceTM) in improving the
anaerobic digestion of high strength oil and grease laden
rendering wastewater within a mesophilic temperature
range. The biosurfactant applied at 500 mg/L to the raw
wastewater resulted in substantial improvement in the
overall anaerobic biodegradability with total and soluble
COD reduction of 62% and 74%, respectively. After
reduction of the O&G content by dissolved air flotation,
the biosurfactant appears not to offer any further lipid deg-
radation. However, the economic viability of using the bio-
surfactant with a cost of just 7–9 US dollars per liter (data
supplied by the authors) should be considered.
Enzymes and pure cultures have also been used to
increase hydrolysis during or prior to biological treatment
processes (Aoki and Kawase, 1991; Cail et al., 1986; De
Felice et al., 2004; Lagerkwist and Chen, 1993; Lanciotti
et al., 2005; Rintala and Ahring, 1994). Such pre- or co-
treatments methods generally consist of the cultivation of
lipase-producing microbial strains in the effluents. Cail
et al. (1986) tested an enzymatic mixture containing pro-
tease, amylase, cellulase and lipase and Bacillus subtilis
spores on wool scouring wastewater with high lipid con-
tent; this mixture increased the COD reduction from 59%
2202 M.C. Cammarota, D.M.G. Freire / Bioresource Technology 97 (2006) 2195–2210
in the control to 78%, increased grease removal from 47%
to over 70%, and improved solids reduction from 34% to
over 70%. Rintala and Ahring (1994) however reported
that the addition of an enzymatic supplement (lipase, cellu-
lase and protease) into an anaerobic reactor treating house-
hold solid waste did not improve reactor performance
relative to the control (without enzymes).
Lipases have also been used for the degradation of
wastewater contaminants from olive oil processing. De
Felice et al. (2004) cultivated the yeast Yarrowia lipolytica
ATCC 20255 on wastewater from an olive oil mill under
batch culture conditions. They found that the yeast was
capable of reducing the COD value (100–200 g/L) by
80% in 24 h and producing a useful biomass of 22.45 g/L
as single cell protein and enzyme lipases. Similar results
were described by Scioli and Vollaro (1997), who reported
lipolytic activity of 770 U/L in the fermentation medium
after 24 h. Lanciotti et al. (2005) evaluated the ability of
several Y. lipolytica strains to grow in olive mill wastewater
and to metabolize its lipidic fraction. All strains studied
were able to produce extracellular lipases. Some strains,
in addition to a high lipase activity, induced a COD reduc-
tion with respect to the uninoculated wastewater (control)
ranging from 20% to 40% after 72 h at 25 C.
As noted by Lanciotti et al. (2005), the biological char-
acteristics of a microorganism proposed for use in waste-
water treatment should be considered carefully. Indeed, it
is desirable to adopt species that are Generally Regarded
As Safe (GRAS). Furthermore it is preferable that the
species have limited nutritional requirements and have
the ability to antagonize the growth of pathogenic species
as well as the capacity to adapt to stringent environmental
conditions (i.e. low temperatures and water activity, high
concentrations of toxic substances).
The impact of the enzymatic hydrolysis of fat particles
on the efficiency of a downstream anaerobic digestion pro-
cess was evaluated by Massé et al. (2003). Slaughterhouse
wastewater containing fat particles was pretreated with
250 mg/L of pancreatic lipase PL-250 and delivered to an
anaerobic sequencing batch reactor operated at 25 C.
Approximately 35% of the neutral fat was hydrolyzed dur-
ing pretreatment. However, the pretreatment presented
only a small overall effect on the fat particle digestion,
marked by a decrease of about 5% (3 h) in the digestion
time to achieve 80% of reduction in the neutral fat and
LCFA concentrations.
4. Use of hydrolases obtained by solid-state fermentation
4.1. Lipase production systems
Among the most studied enzymes, the lipases (EC
3.1.1.3) constitute a group of enzymes that is becoming
increasingly attractive for industrial applications. Stability,
selectivity and versatility are the properties responsible for
their wide and diversified range of applications (Sharma
et al., 2001). Microbial lipases, like most industrial
enzymes, can be obtained either by submerged (SF) or by
solid-state fermentation (SSF). Although SF processes
have been dominant in the biotechnological industry in
recent decades (Elibol and Ozer, 2001; Ito et al., 2001),
SSF processes have been reassessed as of late. This recent
emergence of interest in SSF is mostly due to the possibility
that low-cost agro-industrial residues may used as raw
materials (Benjamin and Pandey, 1997; Bhusan et al.,
1994; Christen et al., 1995; Cordova et al., 1998; Gombert
et al., 1999; Haq-ul et al., 2002; Kamini et al., 1998; Maha-
dik et al., 2002; Ohnishi et al., 1994; Ortiz-Vazquez et al.,
1993; Palma et al., 2000; Pandey et al., 1999; Rao et al.,
1993a,b).
Fungi from the genera Mucor, Aspergillus, Rhizopus and
Penicillium have been used in lipase production studies.
Although the effects of media composition have been inves-
tigated (Dalmau et al., 2000; Gombert et al., 1999; Haq-ul
et al., 2002; Mahadik et al., 2002; Palma et al., 2000), there
is a general lack of understanding of the causes of the
physiological responses of the microorganisms to a given
change in culture conditions. For both cell growth and
lipase production, the use of triglycerides as a carbon
source is generally adequate, although the induction of
lipase biosynthesis by free fatty acids or triglycerides is con-
troversial (Freire et al., 1997; Mahadik et al., 2002; Ohnishi
et al., 1994). Although there are various commercial lipases
available, from various origins and with different selectiv-
ity, an intensive industrial application of these enzymes in
the future depends on the reduction of production costs
through selection of highly productive strains, high-level
expression of lipases in adequate hosts, development and
optimization of fermentation processes and a better under-
standing of the physiological mechanisms that govern the
microbial response to changes in the environmental condi-
tions (Bornscheuer et al., 2002; Pandey et al., 1999; Sharma
et al., 2001).
Lipase production by SSF may be suitable because this
type of fermentation process presents several advantages
in relation to conventional SF, in both laboratory and
industrial scales (Doelle et al., 1992; Freire and Castilho,
2000; Hesseltine, 1987; Mitchell et al., 2002; Nigam and
Singh, 1994; Pandey et al., 1999; Pandey, 2003; Robinson
et al., 2001; Viniegra-González et al., 2003). The small
amount of water employed in SSF processes yields several
advantages. Firstly, the space required for fermentators is
small in relation to the product’s yield, leading to high
‘‘volumetric productivity’’. At the end of the fermentation
process, the product is concentrated and, depending on
the application, the fermented material may be dried
and used directly. In addition smaller amounts of liquid
effluents are generated, thus resulting in smaller expenses
for waste treatment (Castilho et al., 2000; Ghildyal
et al., 1991; Kumar and Lonsane, 1987; Robinson et al.,
2001). Microorganisms, especially filamentous fungi, find
SSF conditions to be similar to their natural habitat,
enabling the excretion of large amounts of enzymes to
be obtained.
 M.C. Cammarota, D.M.G. Freire / Bioresource Technology 97 (2006) 2195–2210
In SSF, the culture medium is usually composed of bran,
cereal husk and other vegetal-origin complex materials. As
a result the fermentation medium needs few additional
nutrients. These materials are generally agro-industrial
wastes, which are inexpensive and plentiful in countries
such as Brazil.
The most commonly used culture media are waste prod-
ucts from vegetable oil production. Sources for the SSF
media include babassu cakes (Gombert et al., 1999; Palma
et al., 2000), coconut cakes (Benjamin and Pandey, 1997),
wastes from peanut pressing plants (Beuchat, 1982), olive
oil cakes (Cordova et al., 1998) and benne (sesame) cakes
(Kamini et al., 1998). Although the microorganism was
able to grow and excrete lipase when pure cake was
employed, its supplementation with additional carbon
and nitrogen sources considerably increased the process’
yield (Benjamin and Pandey, 1997; Gombert et al., 1999;
Palma et al., 2000). Several investigators have observed
that the C/N ratio has a substantial impact on the culture
medium formulation (Gombert et al., 1999; Palma et al.,
2000; Rao et al., 1993a,b). Other substrates such as wheat
bran and rice bran have also been employed successfully in
the production of lipase by SSF (Bhusan et al., 1994; Ortiz-
Vazquez et al., 1993; Rao et al., 1993a,b; Rivera-Muñoz
et al., 1991). Alternatively Bhusan et al. (1994) and Rao
et al. (1993a,b) demonstrated that the solid medium can
be supplemented with rice oil as an inductive agent.
Castilho et al. (2000) presented an economic evaluation
of lipase production by P. restrictum using both SF and
SSF. They demonstrated that for a production scale of
100 m3 of lipase concentrate per year, the total capital
investment required for the SF process was 78% greater
than that required for the SSF process. Therefore SF
proved to be economically unfeasible, as the unitary prod-
uct cost was 68% greater than the product-selling price. By
contrast, the SSF process was quite economically sound.
The SSF unitary product cost was 47% lower than the sell-
ing price; thus the return investment time was just 1.5
years, the return on investment was 68% and the internal
return rate was 62% for a 5-year-project life. Furthermore,
the profitability of this process remained high even with
eventual increases of 40% in product concentration or total
capital investment, or decreases of 20% in product price.
The major advantage of the SSF process is the low cost
of the raw material used as main substrate.
4.2. Use of enzymatic preparations obtained
by solid-state fermentation in the pretreatment
of effluents with high oil and grease contents
Leal et al. (2002) used an enzymatic extract with a high
level of lipase activity for the treatment of dairy wastewater
with high fat contents. The wastewater was taken from a
dairy cooperative, after fat separation in a flotation unit,
and had the following composition: pH = 3.8; COD =
11,300 mg/L; BOD = 8000 mg/L; O&G = 250 mg/L; Total
Kjeldalhl Nitrogen = 340 mg/L. The O&G separated in the
2203
flotation unit were used to adjust the levels of O&G in the
wastewater under the conditions studied. The enzymatic
preparation was produced by the P. restrictum fungus
strain, isolated from the solid waste of a Brazilian babassu
oil industry (Freire et al., 1997), under SSF conditions
using the same solid waste as substrate (Gombert et al.,
1999). After fermentation, the cake containing lipase activ-
ity of 17.2 U/g of initial dry weight, on average, was mixed
with phosphate buffer and the liquid fraction was
extracted, and labeled as the liquid enzymatic preparation.
The lipase activity of the fermented cake was determined as
described by Freire et al. (1997). One unit of lipase activity
(UL) was defined as the amount of enzyme that produced
1 lmol of fatty acid equivalent per minute under the assay
conditions. The wastewater was treated with 10% (v/v)
enzymatic extract of 2.1 U/mL of lipase activity for 12 h
at 35 C, without agitation. Both raw and hydrolyzed efflu-
ents were subsequently submitted to an anaerobic biologi-
cal treatment in sealed 120-mL glass flasks. The effects of
the enzymatic pretreatment on the COD removal kinetics
are summarized in Fig. 1, which shows the COD variation
with time at 35 C for an effluent with an initial O&G con-
tent of 1200 mg/L. The initial COD removal rate was much
higher for the hydrolyzed wastewater than for the crude
wastewater. That is, during the first 24 h, the rate was
0.18 kg COD/m3 d for the crude wastewater, whereas for
the hydrolyzed wastewater it was 1.87 kg COD/m3 d, a
value ten times higher than that observed for the pretreated
effluent.
The results obtained by Leal et al. (2002) for dairy efflu-
ents with different initial O&G contents are summarized in
Table 1. The COD removal efficiencies for hydrolyzed ef-
fluents (80–95%) were higher than those obtained for raw
effluents (without enzymatic pre-hydrolysis; 19–55%) at
different O&G concentrations. In general, the difference
of COD removal efficiency obtained between the reactors
fed with raw or hydrolyzed effluent increased with initial
O&G content. The combination of enzymatic hydrolysis
and anaerobic digestion for the treatment of dairy waste-
1.6
COD/initial COD
1.2
0.8
0.4
0.0
0 20 40 60 80 100
Time (hours)
Fig. 1. Effect of enzymatic pretreatment on COD removal kinetics during
anaerobic treatment (35 C) of a dairy effluent containing 1200 mg/L of oil
and grease: (s) effluent pretreated with 10% v/v enzymatic extract (2.1 U/
mL of lipase activity) for 12 h at 35 C, (d) raw effluent (without
enzymatic treatment). The COD values are normalized in relation to initial
COD (from Leal et al., 2002).
2204 M.C. Cammarota, D.M.G. Freire / Bioresource Technology 97 (2006) 2195–2210

Table 1 100
COD removal efficiency in raw versus enzymatically hydrolyzed dairy
effluents 90

COD removal (%)

Initial O&G contents COD removal efficiency (%)
(mg/L) 80
Raw Hydrolyzed
180 55 95 70
450 70 91
900 37 80 60
1200 19 80
From Leal et al. (2002): For hydrolysis, effluents were treated with 10% (v/ 50
v) liquid enzymatic extract (2.1 U/mL lipase activity) for 12 h at 35 C, 0 25 50 75 100 125 150 175 200 225 250 275 300
without agitation. Raw and hydrolyzed effluents were submitted to a 96-h A Time of operation (days)
anaerobic biological treatment in sealed 120 mL glass flasks. O&G – oil
and grease. 1000

800
water yielded promising results that justify its investigation

O&G (mg/L)
in a pilot study or even on an industrial scale. The SSF pro- 600

cess examined enabled an enzymatic extract with high

lipase activity to be produced at a low cost. This achieve-
ment is a critical prerequisite if the enzymes are to be
applied to industrial effluent treatment. Furthermore, the
findings indicate that the integrated process may be applied
to treat wastewater with fat content as high as 1200 mg/L
through anaerobic processes with no operating problems
and with high COD removal efficiencies and relatively
low treatment times, which contribute to a reduction in
the reactor volume.
Cammarota et al. (2001) evaluated the efficacy of an
enzymatic hydrolysis stage in dairy industry wastewater
prior to the anaerobic biological treatment. In that study,
the authors employed a solid enzymatic preparation
(SEP) with high lipase activity (21 U/g of fermented cake)
obtained by drying fermented medium for 4 h at 45 C.
This preparation had a relatively low production cost, once
the recovery costs of the fermented medium enzymatic pool
were reduced. The process was tested in an upflow anaero-
bic sludge blanket reactor (UASB) operated for a period
of 182 days at 33 C with an average organic load of
4.0 kg COD/m3 d. During this period, the O&G content
of the wastewater was gradually raised (from 203 to
868 mg/L) until it reached the limit the reactor in the con-
ditions under study. The reactor limit was defined as the
point when operational problems (formation of scum, bio-
mass flotation and loss of COD removal efficiency) became
so immense that had the reactor continued running under
the same conditions, it would have collapsed. The evolu-
tion of total COD removal efficiency and the O&G affluent
and effluent concentration data throughout the operational
period from this study are presented in Fig. 2. The results
provide a clear demonstration of the effects of O&G accu-
mulation in the reactor. Initially, the reactor operated with
low loads of O&G (0.25 kg O&G/m3 d or 203 mg O&G/L
on average) in the feeding mixture and presented total
COD and O&G removal efficiencies of 91% and 82%,
respectively. However, the removal efficiencies diminished
drastically when the reactor received wastewater containing
high levels of O&G (868 mg/L on average), reaching values
400
200
0
0 25 50 75 100 125 150 175 200 225 250 275 300
B Time of operation (days)
Fig. 2. Evolution of total COD removal efficiency (A) and oil and grease
affluent and effluent concentrations (B) observed while monitoring the
upflow anaerobic sludge blanket (UASB) reactor treating crude dairy
wastewater (data before vertical line) and dairy wastewater pre-hydrolyzed
enzymatically (data after vertical line), with a hydraulic retention time of
20 h at 33 C. The following raw wastewater O&G concentrations were
tested: 203 (0–90 days), 404 (91–125 days) and 868 mg/L (126–182 days)
on average. Pre-hydrolyzed wastewater (with 0.1% fermented babassu
cake with 21 U/g lipase activity) contained an initial oil and grease
concentration of 800 mg/L (183–301 days). (s) influent, (d) effluent (from
Cammarota et al., 2001).
on the order of 50% for COD and 40% for O&G on the
182nd day (data before vertical line in Fig. 2). In addition,
the crude wastewater (without enzymatic hydrolysis pre-
treatment) resulted in effluents of high turbidity (757
FTU) and volatile suspended solids (VSS, up to 944 mg/
L). When wastewater pre-treated with 0.1% (w/v) of
fermented babassu cake containing P. restrictum lipases
began to be included in the feed mixture, the reactor’s per-
formance improved rapidly; within approximately 15 days,
the average COD and O&G removal efficiencies had risen
to their initial values of 92% and 89%, respectively (data
after vertical line in Fig. 2). Effluent turbidity (189 FTU)
and VSS (97 mg/L) also exhibited notable improvement,
being reduced by 75% and 90%, respectively, in relation
to the raw wastewater.
Leal et al. (2006) studied the optimization of the culti-
vation conditions of fungus P. restrictum through SSF sup-
plemented with various carbon and nitrogen sources (olive
oil and agro-industrial liquid wastes such as a waste from
butter production and molasses), pH, buffer, temperature
and moisture content; a statistical design was adopted for
these experiments. The fungus was capable of growing
 M.C. Cammarota, D.M.G. Freire / Bioresource Technology 97 (2006) 2195–2210
and producing lipases with high activity in SSF with
babassu cake as the basal medium and molasses as the sup-
plementation source, both industrial wastes obtained at
low cost. Lipase activity values (approximately 22 U/g)
were obtained after 24 h of fermentation at 35 C, with
70% moisture content. There were distinct advantages
gained by using molasses supplementation rather than
olive oil. First of all, a decrease in the O&G content added
to the effluent in the pre-hydrolysis stage was achieved. Sec-
ondly, molasses is less expensive than olive oil. The kinetic
production profile of this solid enzymatic preparation is
described in Fig. 3. With SSF, the peak lipase activity level
was obtained within 24 h of fermentation and decreased
thereafter. By contrast this same strain did not peak until
after 64 h of fermentation with SF (Freire et al., 1997).
These findings suggest that SSF is a promising alternative
to SF, as it provided high lipase productivity and uses
waste materials as culture media and supplementation
(babassu cake and molasses, respectively). The accumula-
tion of proteases, which begins at approximately 20 h of
fermentation, results in deamination of amino acids and
ammonia liberation, which ultimately alkalinizes the
media. Protease activities were determined as described
by Freire et al. (1997), where one protease activity unit
(UP) was defined as the amount of enzyme that produces
a unitary difference in absorbance (428 nm) between the
reaction blank and the sample, per minute, under the assay
conditions. The decrease in lipase activity after 24 h of fer-
mentation could be due to pH inactivation, proteolysis (of
the lipase itself) or both (Gombert et al., 1999). Therefore,
the production of the enzymatic preparation was per-
formed within 24 h due to the fact that the protease and
lipase concentration balance favors the biological treat-
ment process under these conditions (Leal et al., 2006).
Leal et al. (in press) also evaluated the effect of an enzy-
matic pre-hydrolysis stage (0.1% (w/v) of fermented cake
for 14 h at 35 C) on the anaerobic treatment of dairy
wastewaters with different fat contents (200, 600 and
25
20
Activity (U/g)
15
10
5 (800 mg/L), the O&G content in the sludge of the bioreac-
0
16 20 24 28 32
Time (hours)
Fig. 3. Kinetics of lipase and protease production by the fungus
Penicillium restrictum using babassu cake as the basal medium, supple-
mented with molasses. Fermentation conditions: T = 35 C, moisture
content 70%. (s) protease activity (UP/g), (d) lipase activity (UL/g) (from
Leal et al., 2006).
2205
1000 mg of O&G/L) in bench UASB reactor. The UASB
reactor fed with the enzymatically pre-treated effluent was
able to support increments in the O&G contents in the
effluent up to 1000 mg/L. When the reactor was fed with
crude effluent with O&G concentration at 600 mg/L, prob-
lems manifested, such as loss of efficiency, biomass wash-
out and fat accumulation in the sludge.
Jung et al. (2002) evaluated the treatment of dairy
wastewater containing different O&G contents in batch
activated sludge systems with and without (control) an
enzymatic pre-hydrolysis stage, which entailed a 0.2% (w/
v) of fermented babassu cake containing P. restrictum lip-
ases (11 U/g) for 8 h at 30 C. When the O&G concentra-
tion in the control bioreactor was increased (400, 600 and
800 mg/L), the COD removal efficiency decreased (86%,
75% and 0%, respectively). However, in the reactor fed
with pre-hydrolyzed wastewater, the COD removal effi-
ciency was maintained (93%, 92% and 82%, respectively).
At an O&G concentration of 800 mg/L, the control biore-
actor yielded final VSS values in the supernatant ten times
greater (2225 mg/L) than those obtained with the bioreac-
tor treating the pre-hydrolyzed wastewater (201 mg/L).
The initial and final COD levels for the bioreactors treating
crude (A) versus pre-hydrolyzed wastewater (B) are pre-
sented in Fig. 4. After enzymatic hydrolysis the COD
values in the wastewater are initially greater than that in
the control because the enzymatic cake possesses a series
of organic compounds in addition to the microorganisms
(P. restrictum) and their proteins and enzymes. Neverthe-
less, the bioreactor treating the pre-hydrolyzed wastewater
maintained COD removal efficiencies within the range
from 82% to 93% (Fig. 4B). This sustained removal
capability is likely due to a facilitated ability of the micro-
bial consortium present in the activated sludge to meta-
bolize the products of enzymatic hydrolysis. However, at
increased O&G concentrations, the COD removal capacity
of the control bioreactor was challenged, such that the final
COD levels in the last stage of the experiment (800 mg
O&G/L), at approximately 170 days, were higher than
the initial COD (Fig. 4A).
Jung et al. (2002) reported that in the control bioreactor,
a 1.7-fold increase in the sludge O&G content was observed
when the O&G concentration in the feed was brought to
800 mg/L. However, in the bioreactor fed with pre-hydro-
lyzed wastewater the O&G values in the sludge (4–
5 mg O&G/g of sludge wet weight) always came out below
those of the control bioreactor under all conditions
studied. Indeed at this high level of O&G in wastewater
tor fed with pre-hydrolyzed wastewater (4 mg/g) was just 1/
5 of that in the control bioreactor sludge (20 mg/g). There-
fore these findings indicate that 800 mg/L represents a crit-
ical O&G concentration level in wastewater for the
operation of aerobic biological reactors. Sludge in the bio-
reactor operated with previously hydrolyzed wastewater
also presented better sedimentability, a characteristic that
is extremely important in activated sludge processes
2206 M.C. Cammarota, D.M.G. Freire / Bioresource Technology 97 (2006) 2195–2210

2500 600 mg/L mended for optimal sedimentation (Eckenfelder, 2000;

400 mg/L 800 mg/L Tchobanoglous, 1991).
2000 Rosa (2004) investigated the efficiency of the solid
COD (mg/L)

enzymatic preparation (SEP) produced by P. restrictum

1500 in the pre-hydrolysis of dairy wastewater with high fat
content (400–800 mg O&G/L) before biological treatment
1000
in activated sludge reactors continuously operated at room
500
temperature (25 C). Lipase and protease production was
monitored under a range of molasses concentrations,
0 because molasses can be used as an alternative carbon
0 30 60 90 120 150 180 210 source in SSF; molasses is a plentiful sugar industry by-
A Time of operation (days) product in Brazil that has the benefits that it is inexpensive
and can be stored at room temperature. Under the most
3000 optimal fermentation conditions (0.75% (w/w) of molasses,
400 mg/L 600 mg/L 800 mg/L 35 C, 70% of moisture content and 20 h) a lipase and pro-
2500 tease average activity of 29 U/g and 3.8 U/g of fermented
cake was obtained, respectively. The hydrolysis of O&G
2000
COD (mg/L)

present in the effluent was conducted at 30 C, for 24 h

1500
1000
500
0 similar COD removal efficiencies (80–90%); however, the
0 30 60 90 120 150 180
B Time of operation (days)
Fig. 4. Concentration of initial (d) and final (s) COD levels in
bioreactors (batch activated sludge) fed with crude dairy wastewater (A)
and dairy wastewater pre-hydrolyzed enzymatically (B), both operating in
sequential batches of 24 h at 25 C. The bioreactors were operated with
the following wastewater O&G content: 400, 600 and 800 mg/L (dashed
lines). Enzymatic pre-hydrolysis stage was carried out with 0.2% (w/v) of
fermented babassu cake containing P. restrictum lipases (11 U/g) for 8 h at
30 C. COD = soluble chemical oxygen demand (from Jung et al., 2002).
(Tchobanoglous, 1991). At 800 mg O&G/L of wastewater,
an accentuated increase of sludge volumetric index (SVI) in
the control bioreactor (569 mL/g) becomes evident, provid-
ing further evidence that operational problems are emerg-
ing. Meanwhile, in the hydrolyzed bioreactor, the sludge
maintained SVI values (117 mL/g) in the range recom-
and with 0.1% (w/v) of fermented cake. Two identical acti-
vated sludge systems operated continuously with an HRT
of 20 h and 3 kg COD/m3 d, where one system was fed with
crude effluent (control) and the other system was fed with
enzymatically hydrolyzed effluent. Both systems produced
210 O&G accumulation rate in the control bioreactor flocs
was 1.7 times higher than that in the bioreactor fed with
the hydrolyzed effluent.
The degradation constants (k) for a first-order substrate
consumption model obtained from the control and hydro-
lyzed bioreactors in each regime studied, considering versus
not considering the biomass concentration, are presented in
Table 2 (Rosa, 2004). This analysis verified that for both
models, the degradation constant is always higher for the
bioreactor fed with effluent that had previously been
hydrolyzed. The specific oxygen uptake rates (SOUR) in
the different regimes studied are also presented in this
Table. As the organic matter of the effluent previously
hydrolyzed is presented as less complex molecules, its
assimilation by the microorganisms is facilitated, which
explains the higher SOUR values obtained in the hydro-
lyzed bioreactor in all regimes. This result is certainly
related to the lesser O&G content accumulation on the sur-

Table 2
Degradation constants (k) for first-order substrate consumption model and specific oxygen uptake rate (SOUR) in activated sludge systems operated with
(hydrolyzed) and without enzymatic pre-hydrolysis (control) in the treatment of dairy wastewaters containing different fat contents
Regime (mg O&G/L) k (d1)a k (mL/mg VSS d)b SOUR (mg DO/g VSS d)
Control Hydrolyzed Control Hydrolyzed Control Hydrolyzed
Without fat 4.0 3.9 2.5 6.1 10.2 18.7
400 5.1 7.8 1.8 3.3 31.2 53.7
600 0.7 1.6 8.3 9.1 17.9 45.6
800 0.3 1.2 3.0 6.5 28.0 36.9
From Rosa (2004): Activated sludge systems were operated at 25 C, HRT = 20 h, average volumetric organic load = 3 kg COD/m3 d.
DO – dissolved oxygen.
a
Substrate consumption described according to dS
dt ¼ kS.
b
Substrate consumption described according to dS dt ¼ kXS. S = substrate concentration (COD), k = degradation constant, t = reaction time and
X = biomass concentration (VSS).
 M.C. Cammarota, D.M.G. Freire / Bioresource Technology 97 (2006) 2195–2210
face and inside of the flocs in this bioreactor, which facili-
tates the transport and absorption of substrate and oxygen.
Valladão (2005) evaluated the anaerobic biodegradabil-
ity of poultry industry effluents with and without enzymatic
hydrolysis. The wastewater was taken after flotation unit
and had the following composition: pH = 5.7; COD =
1180 mg/L; BOD = 646 mg/L; O&G = 341 mg/L; total
Kjeldahl nitrogen = 194 mg/L. The O&G separated in the
flotation unit were used to adjust the levels of O&G in
the wastewater under the conditions studied. Three
P. restrictum-produced SEP levels (0.1%, 0.5% and 1.0%
(w/v)) were tested. The average lipase and protease activi-
ties of the SEP were 29 U/g and 3 U/g of fermented cake,
respectively. The total COD removal obtained for effluent
hydrolyzed or unhydrolyzed by SEP after anaerobic treat-
ment with the different O&G concentrations tested (150,
300, 750 and 1200 mg O&G/L) is shown in Fig. 5. The lit-
erature reports that optimal removal efficiencies in full-
scale bioreactors are attained when 150 mg O&G/L is used
(Campos et al., 2000; Perle et al., 1995). At this value, COD
removal efficiencies between the hydrolyzed and unhydro-
lyzed effluents did not differ substantially (61–76%), dem-
onstrating that this low O&G concentration did not
cause assimilation problems by the microbial consortium.
However, in the absence of enzymatic hydrolysis, the
increase of the O&G concentration reduced COD removal
efficiency almost linearly, such that it reached only 21%
with 1200 mg O&G/L. However, in tests with effluent pre-
viously hydrolyzed with 0.1% (w/v) of SEP, O&G concen-
tration increases did not seem to affect COD removal
efficiency, which ranged from 64% to 73%. At higher SEP
levels (0.5% and 1.0% w/v) the COD removal efficiencies
stabilized at approximately 40% and 20%, respectively,
suggesting that microbial activity may be inhibited with
higher SEP levels. The poultry effluent’s anaerobic degra-
100
COD removal (%)
80
60
40
20
0
150 300 750 1200
O&G (mg/L)
Raw effluent Hydro 0.1% Hydro 0.5% Hydro 1.0%
Fig. 5. Total COD removal efficiency across a range of O&G content in
experiments conducted over 6 days at 35 C with raw (without SEP
treatment) and hydrolyzed (with SEP treatment) poultry industry efflu-
ents. Crude effluent (effluent without pre-hydrolysis), Hydro 0.1% (effluent
pre-hydrolyzed with 0.1% (w/v) SEP), Hydro 0.5% (effluent pre-hydro-
lyzed with 0.5% (w/v) SEP), Hydro 1.0% (effluent pre-hydrolyzed with
1.0% (w/v) SEP). Solid enzymatic preparation (SEP) produced by the
fungus P. restrictum, containing 29 U/g lipase activity, was utilized (from
Valladão, 2005).
2207
dation kinetics at the highest O&G level evaluated
(1200 mg O&G/L) are summarized in Fig. 6 (Valladão,
2005). The effect of the pre-hydrolysis stage of O&G pres-
ent in the effluent is clear. The substantially higher COD
removal efficiencies (85% versus 53%) and biogas produc-
tion levels (175 mL versus 37 mL) obtained after 4 days
with pre-hydrolyzed effluent, relative to that obtained with
crude effluent containing the same initial O&G content,
even after 6 days of treatment should be noted.
Based on the results reported thus far, the present enzy-
matic/biological process verified that the optimum hydro-
lysis conditions depend upon effluent type and upon the
kind of further biological treatment incorporated (aerobic
or anaerobic, batch or continuous). However, based on
our analysis we can make the following three general rec-
ommendations: (1) 0.1% (w/v) of crude SEP, with lipase
and protease activities of 20–30 U/L effluent and 2–3 U/L
effluent, respectively; (2) hydrolysis times of 8–12 h for
dairy effluents, and approximately 22 h for poultry efflu-
ents; and (3) hydrolysis temperatures in the range of 30–
35 C. These recommendations are based on experiments
that tested O&G contents up to 1200 mg/L for dairy efflu-
ents and up to 1000 mg/L for poultry effluents. However
our research group will continue to investigate the condi-
tions that affect these processes.
In summary, consideration of the findings thus far in-
dicates that the employment of enzymatic preparations
produced through SSF (known as SEP) from low-cost
industrial wastes may represent an important contribution
as an adjuvant in the treatment of effluents with high O&G
contents. These enzymes catalyze the hydrolysis of complex
organic compounds, transforming them into substances
that can be readily biodegraded by the microbial consor-
2000
COD (mg/L)
1600
1200
800
400
0
0 1 2 3 4 5 6 7
A time (days)
250
Biogas volume (mL)
200
150
100
50
0
0 1 2 3 4 5 6 7
B time (days)
Fig. 6. Evolution of COD reduction (A) and biogas production (B) in
experiments with poultry industry effluents containing 1200 mg O&G/L.
Crude effluent (d) and effluent hydrolyzed with 0.1% (s) (w/v) of the solid
enzymatic preparation (SEP) produced by the fungus P. restrictum (from
Valladão, 2005).
2208 M.C. Cammarota, D.M.G. Freire / Bioresource Technology 97 (2006) 2195–2210
tium present in the subsequent biological treatment. In this
context, there is a wide range of scientific studies investigat-
ing enzymatic hydrolysis processes to precede traditional
biological treatments with the objectives of improving the
characteristics of the treated effluent and optimizing
the performance of the biological treatment of interest.
The development of improved techniques in this regard is
especially important for the treatment of wastewater with
high O&G concentrations.
Acknowledgement
This work was supported by project funds from the Fun-
dação Carlos Chagas Filho de Amparo à Pesquisa no Estado
do Rio de Janeiro (FAPERJ), Conselho Nacional de Pesqu-
isa e Desenvolvimento (CNPq) and Fundação José Bonifácio
(FUJB). The authors also wish to thank Global Ciência e
Tecnologia Co. (GCT).
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