A. Vergara-Fernández et al.

Biotechnology Advances 36 (2018) 1079–1093

Fig. 2. Conceptual model of an aerial biofilm (a) and a monolayer biofilm (b) (Vergara-Fernández et al., 2016).

Fig. 3. Model for the formation of aerial hyphae aided by hydrophobins. The model was adapted from Wösten et al. (1999). A very similar model was proposed to explain the aerial
growth of the bacteria Streptomyces coelicolor (Claessen, 2003).

tension from 72 mJ m−2 to values as low as 30 mJ m−2 (Wösten et al.,
1999). To overcome the barrier imposed by the surface tension of
water, the submerged hyphal cells secrete hydrophobins, see Fig. 3.
Hydrophobins are thus surfactant proteins (with molecular weight of
≈10 kDa), capable to self-assembly at hydrophilic-hydrophobic inter-
faces and which protect the fungal aerial structures (Vigueras et al.,
2009). These proteins, containing 100 or more amino acids (Schor
et al., 2016; Vigueras et al., 2008), also form a hydrophobic coating on
the aerial hyphae, fruiting bodies and spores, allowing hyphae to co-
lonize hydrophobic materials (Wösten et al., 1994), possibly playing a
role in preventing desiccation (Linder et al., 2005) and facilitating the
dispersion of spores into the air (Bayry et al., 2012). Hydrophobins

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A. Vergara-Fernández et al.
promote the solubilization of hydrophobic VOCs in biofilters by acting
as a coating of the aerial mycelium. The solubilizing of hydrophobic
VOCs can be quantified using partition coefficients.
In general, models reported in the available literature are based on
mass transfer principles, with the main objective of determining the
elimination capacity of fungal biofilters. In order to do this, the gas
phase and biofilm transport equations are mainly considered, as well as
their relation to diffusion conditions of the contaminants and nutrients,
dispersion (described in the previous section), partition coefficient, and
mass transfer coefficient, among others (Kraakman et al., 2011).
2.1.1. Partition coefficient
In general, fungal biofilters are modeled considering a contaminant
partition coefficient in the gas phase/biomass as an air/water Henry's
constant. Systems were this assumption was made include α-pinene
degradation using an Ophiostoma specie (Jin et al., 2006), xylene and
mixed bacteria and fungi (Li and Liu, 2006), hexane and Fusarium solani
(Arriaga and Revah, 2009; Hernández-Meléndez et al., 2008; Vergara-
Fernández et al., 2006), toluene and a fungi consortium (Dorado et al.,
2008). Nevertheless, the partition is modified when fungal biomass is
present in the aqueous biofilm. This situation was observed by Vergara-
Fernández et al. (2006), by determining that n-hexane solubility can
increase up to 200 times with respect to the solubility of water in
presence of filamentous fungi.
The partition coefficient (Kbiomass) of fungal biomass may be ex-
pressed as proposed by Vergara-Fernández et al. (2006) and Davison
et al. (Davison and Barton, 2000):
Cheadspace
KBiomass =
Cbiomass (9)
where, Cheadspace and Cbiomass are the concentration of the contaminant
in the headspace and biomass, respectively.
The partition coefficient of the dried biomass can be expressed as a
function of its composition and the individual partition coefficients of
the lipid-free biomass and the lipid component by Eq. (10). Similarly,
the partition coefficient for the biomass on a wet basis can be calculated
by Eq. (11) that includes the water fraction and the partition coefficient
in water (which correspond to the Henry Coefficient).
lipid − free
1 xlipid xbiomass
= +
dry
Kbiomass Klipid lipid − free
Kbiomass (10)
1
dry
xbiomass x water
wet
= dry
+ at the bottom of the packing. Then, the following equations represent
Kbiomass Kbiomass Kwater (11)
where, x represents the mass fractions of water, lipids or biomass; and K
represents the partition coefficient in biomass, water and lipids.
Vergara-Fernández et al. (2011b; 2006), obtained biomass partition
coefficient values with Fusarium solani in the range of 0.033–0.04 for n-
hexane and 0.015–0.032 for n-pentane, respectively, which are around
three orders of magnitude lower as compared to water. Furthermore,
Vergara-Fernandez et al. (2011b), determined the n-pentane/dry-bio-
mass partition coefficient in microcosms for the same fungus when
grown in four packing materials (compost, peat, perlite and vermicu-
lite) at different temperatures (15 °C, 25 °C and 35 °C). Results indicated
that the n-pentane/wet-biomass partition coefficients in organic
packing material were 160-fold lower (0.21 ± 0.09) than those in
water (33.2 ± 9.4), while for inorganic packing material they were
700-fold lower (0.05 ± 0.04). The authors attributed this result to the
fact that, when grown over an inorganic packing material, the growth
of F. solani is supported only by n-pentane in the gas phase and not from
the carbonaceous material present in the organic packing material.
Thus, an increased hydrophobicity of the surface of the mycelium,
caused by the presence of hydrophobins, would result in a more effi-
cient mass transfer of n-pentane from the gas phase. The role of hy-
drophobins in the biofiltration of slightly soluble contaminants is
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Biotechnology Advances 36 (2018) 1079–1093
supported by the fact that when F. solani is grown in glucose, or other
soluble carbon sources such as 1-hexanol and glycerol, the partition
coefficients are higher than those obtained when grown in n-hexane (in
the gas phase).
In another approach, Salehahmadi et al. (2012), in their mathe-
matical model for fungal biofiltration, applied the method described by
Mackay (Mackay, 2001), in order to determine the n-hexane/biomass
(K) partition coefficient. The model is based on that the organic content
of the biomass is responsible for the hydrophobic VOCs absorption,
which in turn can be related to the octane/air (Koa) (Eq. 12) coefficient.
1
= 4.1·10−4 [K oa yρb ]
Kbiomass (12)
where, ρb is the biofilm density and y represents the organic content.
2.1.2. Axial dispersion
Axial dispersion is the deviation from the ideal plug-flow regime in
a pipe, column or reactor. It can be caused by multiple factors, in-
cluding the packing geometry and size, a non-uniform distribution of
the feed stream, channeling or short-circuiting creating dead zones, and
specifically for fungal biofilters, the growth of aerial hyphae occupying
the void space (Prenafeta-Boldú et al., 2008).
The correlation proposed by Edwards and Richardson (1968) was
used by Vergara-Fernández et al. (2008) in the estimation of the axial
dispersion coefficient in the phenomenological modeling of a fungal
biofilter treating n-hexane. Helfferich (Helfferich, 1985) proposed an
empirical correlation for the axial dispersion coefficient in a packed-bed
as a function of the bed porosity (ε), gas velocity (ug), packing diameter
(dp) and the specie diffusion coefficient (DA) (Eq. 13). Although this
expression does not account for the presence of bacterial or fungal
biofilms, it was nevertheless used by Salehahmadi et al. (2012) in
fungal biofilters.
D = (0.45 + 0.55ϵ) DA + 0.5dP ug /ϵ (13)
In a different approach, Prenafeta-Boldú et al. (2008) showed that
the gas residence time distribution (RTD) can be determined using an
air dispersion model. The model was experimentally validated in a
fungal biofilter inoculated with Cladophialophora sp. using methane as
tracer-gas. The dispersion study was carried out in two laboratory scale
biofilters packed with granular perlite and polyurethane foam. The
recorded pulse RTD curves were modeled as being equivalent to a series
of CSTR (Eq.15). Two additional CSTR compartments of constant vo-
lume were added to account for the void spaces present at the top and
the proposed CSTR system for the estimation of the RTD:
dM0 Q
= − M0
dt Vin (14)
dMi n
= (Mi − 1 − Mi ), i = 1, 2, …, n with Mi − 1 = 0 if i = 1
dt τ (15)
dMn + 1 Q
= (Mn − Mn + 1)
dt Vout (16)
where Mi is the contaminant gas concentration in tank i and τ is the
mean residence time of the tracer gas equivalent to that of the flowing
air, the effective bed void volume can be calculated as:
Ve = τ ·Q (17)
Spigno and De Marco Faveri (2005) analyzed the RTD in a biofilter
model of a fungal biofilter inoculated with Aspergillus niger for n-hexane
abatement. They found a small axial dispersion coefficient of
1.22·10−4 m2 s−1.
2.1.3. Diffusion coefficient in the biofilm
The main parameters affecting the diffusion of a contaminant in the
fungal biofilm are the temperature and the biomass density, where the
A. Vergara-Fernández et al.
diffusion coefficient for biofilms is found to be slightly lower than the
one for water. However, this parameter should be considered only for
the cases in which the biomass attached to the support is modeled (not
the aerial biomass), since it is customary to consider that the con-
taminant reacts instantaneously once it is in contact with the surface of
the aerial hyphae (Vergara-Fernández et al., 2016, 2008).
The diffusion coefficient for fungal biofilters, might be approxi-
mated by applying the empirical correlation of Wilke and Chang
(1955). This method was used by Spigno and De Marco Faveri (2005)
by assuming the value of the diffusion coefficient to be 40% of its value
in water, with a resulting estimated diffusion coefficient of 0.74
×10−9 m2 s−1. Instead, Arriaga and Revah (2009) applied the ex-
pression for impermeable sphere-packed porous media by Neale and
Nader (1973) in a mathematical model of hexane degradation in fungal
biofilters, obtaining the following effective diffusivity for hexane (Eq.
18):
Def 2ϵ
= 2.3. Drying, metabolic activity and heat generation
DHwater 3−ϵ (18)
where, Def is the effective diffusivity in the biofilm, ε is the bed void
fraction, and DHwater is the diffusivity in water. The bed void fraction
can be obtained according to:
Vwater
ϵ=
Vtotal (19)
where, Vwater is the volume occupied by the water in a total volume
(Vtotal) packed with a support material. The authors reported a Def/
DHwater of 0.35 for bacterial biofilms, and 0.19 for fungal biofilms, with
effective diffusivity in fungi of 4.1×10−7 m2 h−1.
2.2. Pressure drop
The pressure drop in fungal biofilters is one of the most important
operational parameters to be considered. The increase in the pressure
drop can be caused by several factors including entrapment of particles
transported in the air entering to the reactor, bacterial growth and by
the aerial growth of filamentous fungi, diminishing the void fraction,
and therefore reducing the permeability of the porous media of the
bioreactor. Since air entering to biofilters is commonly prefiltered and
humidified, the load of particles that can cause cloging of the reactor is
reduced, along with the concentration of highly water soluble VOCs.
Estrada et al. (2013) found pressure drop increments for fungal (Pae-
cilomyces variotti) and bacterial biofilters of 91 to 912 Pa m−1
bed and 91 to
372 respectively, for the treatment of a VOC mixture, showing how
fungi can introduce significant resistance to the moving flow compared
to bacteria. Similar values, between 98 and 350 Pa m−1 bed, were obtained
Prenafeta-Boldú et al. (2008) in a fungal biofilter inoculated with Cla-
dophialophora sp. strain CBS 110553 for the removal of toluene using
perlite granules as packing material.
The pressure drop evolution over time in a fungal biofilter was
evaluated by Vergara-Fernández et al. (2008) by applying the Darcy
equation (Eq. 20).
ΔP 72ω (1 − ϵ)2 ⎞ ⎛ μf ⎞
⎛ ⎞ = ⎛⎜
⎝H ⎠ ⎝ ϵ 2dp2
⎟⎜ ⎟ vg
⎠ ⎝ ρg g ⎠ (20)
where, ΔP is the pressure drop, H is the biofilters height, ϖ is the tor-
tuosity factor, ε is the bed porosity in the biofilters, dp is the particle
diameter, μf dynamic viscosity of the fluid, ρg is the gas density, g is the
acceleration of gravity, and vg is the gas superficial velocity.
Where the void fraction can be evaluated as a function of fungal
growth by application of Eq. (21):
ϵ(t ) =
Vr − ⎡VE +
⎣ ( )d
π
4
2
hyphae Lhyphae, Total (t ) ⎤
⎦
Vr (21)
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Biotechnology Advances 36 (2018) 1079–1093
Above, Vr is the reactor total volume, VE is the total volume of the
support, dhyphae is the hyphae diameter obtained from morphological
observations and Lhyphae,Total is the total length of the hyphae which can
be calculated as shown in Eq. (26).
An important aspect to be considered when simulating the pressure
drop in a fungal biofilter at the initial stages of operation, is the amount
of static liquid present in the bed. This behavior can generate a de-
viation of up to 11% from experimental measurements when it is not
accounted for in the mathematical model (Vergara-Fernández et al.,
2008). The maximum pressure drop obtained in the latter study goes up
to about 45 mmH2O m−1 bed. On the other hand, Zamir et al. (2011a,
2011b) obtained a pressure drop approximately four times smaller
(11 mmH2O m−1 bed), than those obtained by Vergara-Fernández et al.
(2008) using a gas flow three times lower and half the residence time in
the toluene elimination in a fungal biofilter inoculated with the fungus
Phanerochaete chrysosporium.
Packing and biofilm moisture content, metabolic activity and heat
generation are intimately intertwined. Moisture balance is affected by
thermodynamic phenomena, the equilibrium between the content of
water in the biofilm and the moisture in the gas stream, the water
transfer rate from the solid support to the biofilm (in biofilters).
Humidification of the contaminated air stream entering the biofilter is a
common practice to avoid biofilm drying, incomplete humidification of
the air stream (relative humidity < 100%) evaporates moisture from
the solid support and biofilm at a rate that depends on inlet tempera-
ture, initial relative humidity and empty bed residence time. The heat
liberated by the oxidation of gaseous pollutants, especially those with
high upper heating values such as alcohols, alkanes and alkenes, in-
creases the evaporation rate. van Lith et al. (1997) estimated that a
biofilter operated with inlet contaminant concentration of 0.5 g m−3
and volumetric load of 50 g m−3 h−1 loses 30 L of water per cubic
meter of packing in a period of 3 to 6 days. This drying mechanism is
especially relevant near the biofilter influent where the highest VOCs
concentrations are found. In this regard, Gostomski et al. (1997) pro-
vide experimental results showing that the degradation caused by heat
generation and water evaporation started in a narrow zone at the inlet
of the bed, however, as the loss of water inhibited the microbial activity
of this area, the degradation region progressed through the bed.
Insufficient moisture prevents the formation of either bacterial or
fungal wet biofilms, that can support microbial growth and respiration,
promotes the contraction of the support and the consequent medium
cracking reducing retention times (Swanson and Loehr, 1997). On the
other hand, excessive moisture reduces mass transfer rates of hydro-
phobic substances, reduces biofilm surface area by clogging available
pore space, increases pressure drop (Ferdowsi et al., 2017; van Lith
et al., 1997) and creates anaerobic zones that promote odor formation
and low degradation rates (Ferdowsi et al., 2017; Swanson and Loehr,
1997).
The moisture content of the biofilter support, both for fungal and
bacterial biofilters, is one of the main parameters affecting reactor
performance. The control of the moisture content requires under-
standing the dynamics of drying of the biologically active support due
to the air temperature changes at the inlet, the air relative humidity,
and the metabolic heat produced by the oxidation of VOCs. Morales
et al. (2003) developed a mathematical model to study the drying effect
on a biofilter inoculated with five different types of bacteria and two
types of yeast used for the elimination of toluene. The model considers
the effect of temperature, water content, and contaminant concentra-
tion in the biological reaction. The latter model allows the prediction of
the biofilter performance and water evaporation from the support
media because of the metabolic heat generation and the variation of the
inlet air current relative humidity. The water vapor in the gaseous
phase (air absolute humidity) can be estimated from Eq. (22):
A. Vergara-Fernández et al.
∂XH F ∂XH −RV ρC
= +
∂t ρa ηg ∂Z ηg (22)
where XH is the absolute humidity of air, F is the air mass flow, ηg is the
ratio of gas-phase volume to elementary representative volume, ρa is the
real density of air, ρc is the apparent density of the packing material,
and RV is the water evaporation rate based on dry support.
When analyzing the energy balance in the biofilter, the convective
heat must be considered due to its importance in: the biofilter drying,
the water evaporation and the reaction heat, as stated in Eq. (23):
∂T
β = Qconv + Qevap − Qrx
∂t (23)
where, β is a parameter associated to the accumulation of heat and it
represents the average heat capacity of the medium, Qconv represents
the convective volumetric heat, Qevap is the evaporative volumetric
heat, and Qrx depicts the volumetric heat associated to VOCs degrada-
tion and T is the operation temperature. The combustion heat from the
reaction can be estimated from each VOCs combustion enthalpy. The
CO2 production rate because of the VOC mineralization can be used as a
correction factor of the combustion enthalpy.
Water content can affect also other important aspects of fungal
biofilters such as spore production, emission and retention. The effect of
biofilter water content on spores generation was studied by Vergara-
Fernández et al. (2012b) and Saucedo-Lucero et al. (2014) operating
fungal biofilters for the elimination of n-pentane and n-hexane, re-
spectively. The number of colony forming units (CFU m−3 air ) in the PDA
Petri dish according to Eq. (24), was used for the determination of the
spore concentration (SC):
CFU
SC =
Q·tm (24)
Where, tm is the elapsed time for spore collection and Q the filtered gas
flow rate.
The results show that a low water content in the biofilter promote
sporulation, while a higher water content decreases sporulation and
favors spore retention. Vergara-Fernandez et al. (2012), concluded that
the periodic addition of mineral medium in biofilters based on in-
organic materials promotes rapid recovery of the biodegradation per-
formance and reduces spore emission. However, an excess of irrigation
can increase the pressure drop across the biofilter bed and hinder hy-
drophobic VOCs transfer from the gas phase, with a subsequent de-
crease in EC. Interestingly, the increase in VOCs inlet load in a non-N-
limited scenario triggered spore emission and increased the EC, with
average spore counts of 1.8 ×104 CFU m−3 −3
air and ECmax of 110 g mreactor
−1
h . Finally, lower empty bed residence times (EBRTs) can support an
increase in the emission of spores and a deterioration in the EC. Similar
results, between 2.4×103 to 9.0×104 CFU m−3, were reported by
Saucedo-Lucero et al. (2014) in the hexane abatement in a fungal
biofilter inoculated with a fungal consortium, obtaining a EC of
35 g m−3 h−1.
2.4. Biomass growth and biodegradation kinetics
Though the main problem in biofilters relates to the description of
the mass and momentum transfer phenomena between the gas, the li-
quid and the biofilm; the knowledge of the intrinsic microbial growth
and biodegradation rates is a key issue in the design and optimization of
this type of bioreactors. Coupling both phenomena can be done con-
sidering a micro approximation in order to determine the intrinsic
biodegradation kinetics (Iranmanesh et al., 2015).
2.4.1. Fungal biofilm
Fungal biofilm modeling in biofilters has been scarcely studied. In
general it is considered as a flat biofilm with constant thickness
(Fig. 2b), within the range of several μm to > 100 μm (Dorado et al.,
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Biotechnology Advances 36 (2018) 1079–1093
2008; Spigno and De Marco Faveri, 2005). In contrast, studies carried
out by Vergara-Fernández et al. (2016, 2008) show that the aerial
growth of filamentous fungi plays an important role in the VOCs bio-
degradation. This is mostly due to the increase of transport area, which
can reach values up to 107 m2 m−3, in comparison to 102 m2 m−3 for
non-aerial biofilm (Arriaga and Revah, 2009).
Vergara-Fernández et al. (2016) developed a simple mathematical
model with the objective of describing the contribution of aerial and
non-aerial biodegradation of n-pentane using Fusarium solani. They
found that, although the aerial mycelium represents only 23% of the
biomass on a dry weight basis, is responsible for 71.6% of n-pentane
degradation. This result highlights the key role of the aerial hyphae on
hydrophobic VOCs degradation. In this study, together with mass bal-
ances in the gas phase and the bioreactor biofilm, the mass transfer
surface area (av) was determined, according to Eq. (25), where the
biofilm area (aa) can be determined as reported by Arriaga and Revah
(2009), considering a total hyphal length, Lhyphae (Eq. 25).
a v = am + aa ·α (25)
where, α denotes the fraction of the aerial biomass. Hence, the super-
ficial area available for VOCs transfer can be defined as:
Vbiomass
Lhyphae =
dh yphae 2
π ( 2 ) (26)
where, Lhyphae is the hyphae length, Vbiomass is the biomass volume and
dhyphae is the hyphae diameter.
The total fungal interfacial area per reactor volume, aa, is:
dhyphae Lhyphae π
aa =
Vr (27)
where, Vr is the reactor volume.
A similar approach was applied by Spigno and De Marco Faveri
(2005), to establish the amount of aerial biofilm in the mass balance of
the gaseous phase of a fungal biofilter. This was carried by sensibility
analysis of the variation of the contaminants concentration throughout
the biofilter for values of α between 0.25 and 1.0.
The specific transport area obtained from several simulations reach
values around 2×105 m2 m−3, considering hyphae diameters between
2.1 μm and 2.9 μm (Arriaga and Revah, 2005; Vergara-Fernández et al.,
2008). These values are between 50 and 130 times larger than those
obtained for bacterial biofilters (Iliuta and Larachi, 2004; Zhu et al.,
2004).
2.4.2. Kinetics
Biofilter performance is ultimately determined by the catabolic ac-
tivity of the microbial biomass which, in turn, depends both on the
microbial mass present and the intrinsic activity of this biomass. The
amount of biomass results from growth and decay rates and the in-
trinsic activity on the identity of the microbial population (genotype),
how it is expressed (phenotype) and the reactor conditions (pH, tem-
perature, water content, presence of inhibitors, etc.). Substrate uptake
is utilized for biomass growth when conditions are appropriate using
the chemical energy generated by the catabolic reactions and producing
CO2.
Modeling fungi growth decay has received less interest due to the
complex processes involved in the growth of filamentous fungi. The
nature in which fungi grow, makes it troublesome to define cellular
death when compared to unicellular organisms. Szewczyk and Myszka
(1994) applied an Arrhenius type of equation to represent specific
growth rate as a function of temperature, where there is a term de-
scribing growth and another describing cellular death. Smits et al.
(1999) did not develop an explicit model for cellular death, yet they
proposed an equation describing specific respiration activity of the
microbial mass decay over time.
In the other hand, hyphae elongation of the first hyphae, during the