Biochemical Engineering Journal 128 (2017) 8392

Contents lists available at ScienceDirect

Biochemical Engineering Journal

journal homepage: www.elsevier.com/locate/bej

Review

A mini review on bioreactor congurations and gas transfer

enhancements for biochemical methane conversion
Kyle A. Stone, Matthew V. Hilliard, Q. Peter He, Jin Wang
Department of Chemical Engineering, Auburn University, Auburn, AL, 36849, USA

a r t i c l e i n f o a b s t r a c t

Article history: Methane is an essential component of the global carbon cycle and one of the most powerful greenhouse
Received 6 December 2016 gases (GHGs), yet it is also a rich source of carbon and energy. Methanotrophs that use methane as
Received in revised form 30 August 2017 their sole carbon and energy source have drawn renewed interest due to their capability of converting
Accepted 5 September 2017
methane under ambient conditions in an environmentally benign fashion. In this work, we provide a mini
Available online 8 September 2017
review on recent progress in process development, particularly on bioreactor design and gas transfer
enhancements for biological methane conversion. Bioreactor congurations reported in recent research
Keywords:
papers and patents are tabulated, together with their key characteristics, performances, pros and cons.
Methane
Bioconversion
Bioreactor congurations for gas feed with high concentration of methane (e.g., natural gas) and that
Methanotrophs with low methane concentrations (e.g., anthropogenic emission) are reviewed. For gas transfer promoting
Gas-liquid mass transfer agents, recent results on using vectors, polymers, nanoparticles, electrolytes, and non-ionic surfactants to
Bioreactor enhance mass transfer of methane are reviewed and summarized with a table. Process safety and future
research directions are also briey discussed.
2017 Elsevier B.V. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83
2. Mass transfer of methane and transfer rate measurement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84
3. Bioreactor congurations and operations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85
3.1. Bioreactor congurations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85
3.1.1. For high concentration methane feed . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85
3.1.2. For low concentration methane feed . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85
3.2. Gas transfer enhancements in STRs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87
3.3. Operation safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87
4. Promoting agents of gas transfer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87
4.1. Vectors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87
4.2. Polymers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87
4.3. Nanoparticles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89
4.4. Electrolytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89
4.5. Non-ionic surfactants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89
5. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92
Web References [WR]: . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92

1. Introduction

Methane is a rich source of carbon and energy and is the most

Corresponding author at: Auburn University, 212 Ross Hall, Auburn, AL 36849, abundant organic gas in the atmosphere. At the same time, methane
USA. is a powerful greenhouse gas (GHG) with a global warming poten-
E-mail address: wang@auburn.edu (J. Wang). tial over 20 times that of CO2 . There are two main sources of

http://dx.doi.org/10.1016/j.bej.2017.09.003
1369-703X/ 2017 Elsevier B.V. All rights reserved.
84 K.A. Stone et al. / Biochemical Engineering Journal 128 (2017) 8392
methane: non-renewable natural gas and renewable biogas. For
natural gas, which contains 80%95% CH4 , there is more than 6800
trillion cubic feet of proven reserves globally and the production
of natural gas is expected to continue increasing based on the U.S.
Energy Information Administration (EIA) projection [WR1]. On the
other hand, biogas, which contains 50%70% CH4 and 30%40%
CO2 , can be produced within a short period of time from anaerobic
digestion of organic matter. The potential of biogas production is
enormous, and a 2014 U.S. government report estimated that 654
billion cubic feet of biogas per year can be produced in the country
[1].
Methane is mainly used for heating, cooking and electricity
generation. In its compressed form (e.g., compressed or liqueed
natural gas), methane can be used also as a transportation fuel.
However, such usage is constrained owing to methanes inher-
ently low volumetric energy density and the lack of infrastructure
required for its broader adoption. An alternative means to use
methane as a transportation fuel involves thermochemical gas-to-
liquid (GTL) conversion technologies and subsequent conversion
via the Fischer-Tropsch (FT) process. However, the technical com-
plexity of the GTL-FT process results in exceptionally large-scale
facilities (>$20 billion per facility) that cannot be economically
scaled down [2], and therefore are not suitable for smaller, dis-
tributed biogas sites and natural gas wells [3].
In contrast to thermochemical processes, nature has its own way
of converting methane through methanotrophs, which are largely
aerobic bacteria that consume methane as their sole carbon and
energy source. With the use of the particulate and/or soluble form
of methane monooxygenase (MMO), methanotrophs can catalyze
the oxidation of methane to methanol under ambient conditions
in an environmentally benign fashion [46]. Driven by the need for
renewable energy, plus recent increases in unconventional natural
gas production in the United States, as well as the record breaking
development of biogas plants in Europe in recent years [WR2], there
is a renewed interest in biological methane conversion. As a result,
methanotrophs are starting to gain a foothold in bioindustries with
great progress seen in aquaculture and for energy production [7].
However, their industrial applications and developments are lim-
ited compared to the conversion of biomass to ethanol through
industrial microorganisms [8]. To fully industrialize biological con-
version of methane, several technical hurdles have to be overcome
which are discussed in [9]. Among the identied technical hurdles,
a major one is bioprocess development. This includes biocatalyst
development, such as strain selection and modication, and pro-
cess development, such as bioreactor design, and optimization of
operation conditions. Several excellent reviews have been pub-
lished in the last few years to capture recent advances [7,1022].
Together, these recent reviews provide comprehensive coverage of
various biological conversion routes of methane to potential prod-
ucts [16,22], especially on methanol and biodiesel [10,12,17] and
associated challenges and opportunities [7,10,12,16,17]. All of these
recent reviews focus on the biochemistry background and biolog-
ical considerations; while only three of them lightly touched on
process considerations [7,12,20].
In this work, we aim to provide a brief but comprehensive
review on two aspects of bioprocess development, i.e., bioreactor
design and gas transfer enhancement, as they are critical enablers of
commercializing methane bioconversion into various products. By
doing so, we hope to promote the research in the process devel-
opment for methane bioconversion, and to help accelerate the
process of translating the recent progress in biocatalyst discov-
ery and development (such as methanotroph mutant design) into
real applications that can convert natural gas and biogas into value
added products. This paper is organized as follows: rst, we briey
discuss the challenges and solutions in measuring mass transfer
rate of methane from the gas phase to the liquid; then, we provide
a comprehensive review on different bioreactor congurations that
have been used for methane conversion; next, we examine differ-
ent approaches to enhance mass transfer of methane, and nally
summarize the review with conclusion and discussion. It should be
noted that this review focuses on lab scale set up as that is what
most research was published on.
2. Mass transfer of methane and transfer rate measurement
A common challenge associated with gas phase fermentation is
the low mass transfer rates of gaseous substrates into the liquid cul-
ture medium. Detailed reviews on gas (predominantly oxygen) to
liquid mass transfer can be found in [2331]. The mass transfer rate
is usually characterized by the volumetric mass transfer coefcient,
kL a, of the substrate. In this work, kL a is used as one of the major
metrics to evaluate the performance of different bioreactor cong-
urations and mass transfer enhancement approaches. However, it
is worth noting that kL a is not a direct measure of a process perfor-
mance. In addition, it is highly sensitive to cell density and broth
properties. As a result, it can vary signicantly during the course of
any experimental run [32]. Therefore, more direct metrics, such as
biomass productivity and methane consumption or removal rate,
are also used wherever they are available.
Several methods to measure kL a have been reported and
reviewed [3335]. Among them, a few commonly applied
approaches (such as dynamic method and its variations) require the
measurement of dissolved gas concentration. For methanotrophs
this would require the measurement of both dissolved oxygen
and dissolved methane. Dissolved oxygen probes have long been
used and are commercially available; however, very few probes
for measuring dissolved methane are available. In fact, much of
the dissolved methane sensor technology has been developed for
geochemical studies and can be prohibitively expensive [36].
To address this challenge, a couple of in-house developed
methane probes have been reported [37,38], which usually con-
sists of a permeable membrane and a fast response methane sensor
[38]. Another approach to measure dissolved methane is to strip the
dissolved gas in the liquid into the gas phase, and then convert this
gas phase concentration under equilibrium back to liquid phase
concentration using Henrys Law. The gas phase methane concen-
tration can be measured through methane detectors or standard
analytical techniques such as gas chromatography [39].
Besides these approaches, the kL a of methane can also be esti-
mated through the kL a of oxygen. For the applications of methane
bioconversion, since the gas phase consists of mixed methane and
oxygen, both gases share the same specic interfacial area and their
volumetric mass transfer coefcients are only differentiated by
their corresponding mass transfer coefcient kL . Both the penetra-
tion theory and the surface renewal theory of mass transfer suggest
a linear relationship between the gas components mass trans-
fer coefcient, kL , and the square root of its diffusion coefcient,
D [40]. For methane and oxygen, their diffusion coefcients are
1.49 105 cm2 /s and 2.1 105 cm2 /s [40], respectively, which
suggests that kL aCH4 = 0.842kL aO2 . This relationship is similar to
what Yu et al. [41] have reported: kL aCH4 = 0.855kL aO2 , which is
used in this work. Because dissolved oxygen probes are widely
available, using the measured kL a of oxygen to estimate the kL a of
methane seems to be one of the easiest ways to quantify the mass
transfer rate of methane from gas to liquid for methane bioconver-
sion applications.
In the following sections of this review paper, kL a of methane
and/or oxygen is used as one of the major criteria to compare differ-
ent reactor congurations for mass transfer performance. Although
all cited work in this paper focuses on methane utilization, not all
sources reported the kL a of methane, mostly due to the lack of dis-
 K.A. Stone et al. / Biochemical Engineering Journal 128 (2017) 8392
solved methane probes. For those cases, we computed the kL a for
methane based on the measured kL a for oxygen using the above
mentioned linear relationship of kL aCH4 = 0.855kL aO2 .
Finally, as discussed earlier, kL a is not a very reliable and direct
way of measuring process performance. Besides kL a measurement,
it is important to measure how much methane is actually consumed
or removed. For continuous operations, this can be accomplished
through mass balance of methane from the inlet and off gases [42].
3. Bioreactor congurations and operations
Among different factors that affect gas-liquid mass transfer,
bioreactor conguration and operation arguably have the most sig-
nicant impact. In this section, we review major research ndings
in these areas over the past few decades.
3.1. Bioreactor congurations
Research and applications in biological methane conversion can
be categorized into two major groups based on the gas feed: gas
feed with high concentration of methane such as natural gas, and
gas feed with low methane concentration such as anthropogenic
emissions, which are reviewed separately in the following subsec-
tions.
3.1.1. For high concentration methane feed
The upper part of Table 1 summarizes different bioreactor
congurations commonly used for converting gas feed with high
methane concentration. The key characteristics, reported kL a val-
ues, and pros and cons are summarized and compared.
FCLBs have been utilized for production of poly-3-
hyroxybutyrate (PHB) [43], single cell protein (SCP) [4447],
and biomass [4850] using methanotrophs. It has been demon-
strated that productivity of 4 kg/(m3 h) SCP was achieved in a 15 m
long FCLB at a kL a of 180/h [46]. It is worth noting that Dupont
and Statoil have used FCLBs with static mixers for bioconversion
of methane to SCP which were capable of producing 10,000 ton
SCP per year [46]. In addition, multiple patent applications for
modied FCLBs were recently led [45,47,51], indicating sustained
interest in this conguration, most likely due to their superior
performance in delivering high gas to liquid mass transfer rates.
Therefore, FCLBs appear to be one of the best choices for methane
fermentation, particularly in large scale processes.
Airlift reactor is another major group that has been utilized
for methane conversion. Two commonly used airlift reactors are
bubble column and external airlift loop bioreactors (EALB). These
reactors have been applied in methane fermentation via methan-
otrophs for PHB [43], SCP [52], and biomass production [50,53].
However, poor liquid circulation has rendered them a less viable
option for methane fermentation [43,50,52].
Stirred tank reactors (STRs), operating in batch, fed-batch or
continuous mode, are the most commonly used reactors in research
laboratories, and have been utilized for methane fermentation;
however, the required power input, heat removal, and usually
large shear stress have made this reactor conguration less attrac-
tive for industrial methane conversion applications [23,42,5457].
Besides being widely available, the mass transfer rate in STRs can
be adjusted through the choice of the gas sparger, energy input
(agitator rate), and choice of the propeller. This exibility is often
desirable for academic and industrial research and development.
These aspects are discussed in more detail in Section 3.2.
In addition to the major reactor congurations listed in Table 1,
membrane aerated bioreactors (MABR) have been utilized for
methane bioremediation as well. These reactors introduce sub-
strate gases via membranes to avoid gas bubbles. To date, MABR
application has been limited to methanol production on bench scale
85
systems [58,59], and membrane fouling caused by cell blocking the
pores can signicantly reduce mass transfer rate.
Since the mass transfer from gas to liquid is the major bottle-
neck in biochemical methane conversion, the reported kL a values
of methane and oxygen are used as a major criterion for comparing
different reactor congurations. Based on the reported kL a values,
the operational pros and cons, and the industrial adoptions, Table 1
indicates that the forced circulation loop bioreactor (FCLB) delivers
the best performance. However, it is important to notice the dis-
parity in reported kL a values for FCLBs they range from 25.7 to
7700 h1 . The disparity is probably due to the fact that some FCLBs
utilize static mixers to facilitate interphase mass transfer between
the gas and liquid phases, and these static mixers could improve kL a
up to two orders of magnitude [60]. In addition, it has been shown
that operation conditions such as supercial gas and liquid veloci-
ties can have a signicant impact on the effectiveness of these static
mixers [61]. Other factors that affect the kL a achievable by FCLBs
include the length of static mixer, the number of mixers utilized,
spacing between mixers, and the location of the mixers in the loop
[62]. While static mixers can signicantly enhance the mass trans-
fer from gas to liquid, their usage results in increased energy input
to circulate the liquid, which can increase dramatically with high
cell density. Additional discussions on static mixers can be found in
[62]. It should be noted that the kL a reported in [51] and [52] were
measured in water that is cell-free, and the rheological and inter-
facial properties of water is different to that of a real fermentation
broth, which would result in quite different mass transfer perfor-
mance. Finally, for large scale bioreactors where the gas phase is
closer to plug-ow instead of well-mixed, there could exist a gra-
dient in gas-phase methane and oxygen concentrations. Therefore,
there could be high dissolved oxygen concentrations at locations
close to the gas inlet, which is typically inhibitive to methanotrophs
and could also alter product selectivity. It has been suggested to
inject air and/or other nutrient gases such as methane or natural
gas at different locations of a reactor to even-out the dissolved gas
proles [47,48,50,54].
3.1.2. For low concentration methane feed
More than half of the sources of anthropogenic methane,
including landlls and animal husbandry, contain less than 3%
methane in their gas emissions [63]. Therefore, various enhance-
ments (both structural and non-structural) have been reported in
recent literature for bioconversion/abatement of methane from low
concentration methane emission (LCME) sources. The lower part
of Table 1 summarizes the congurations utilized for biological
abatement of LCME. As methane removal is often the primary objec-
tive in these applications, removal efciency has been reported
where applicable. Removal efciency is essentially the percentage
of methane consumed (or removed) from the inlet gas concentra-
tion [64].
Biolters and biotrickle lters (BTFs) are the two most com-
monly used reactors in this category. Biolters (or conventional
biolters) utilize immobilized cells to convert methane, and they
usually operate in batch or fed-batch mode. Typical biolters con-
sist of methanotrophs embedded on a carrier material, which may
be organic or inorganic. Waste gas containing methane is contin-
ually fed to the lter, while the liquid phase containing essential
nutrients is fed intermittently (usually on a per day basis). A com-
prehensive discussion of conventional biolters, their operating
conditions, and methane abatement capabilities can be found in
[65]. For reactor designs utilizing mixed consortium of bacteria,
low inlet loads, or large empty bed resonance times (EBRT) methane
removal efciencies as high as 100% [65] have been observed; how-
ever, the highest reported methane removal efciency in a lab scale
biolter with practical EBRT is 41% [66].
Table 1
Bioreactors for Methane Bioconversion.
Reactor Type Sub Categories
Forced circulation loop Horizontal tubular loop
bioreactor (FCLB) [49,50]
Vertical tubular loop
[43,48,50]
U-loop [44,45,47,51]
Airlift External loop airlift
[50,53,72]
Bubble column [43,52]
Stirred tank reactor (STR) Fed-batch and continuous
stirred tank reactor
Biolters Conventional biolters Often involves
[65,66,96]
Biotrickle lters (BTFs) Can have a solid support
[63,6769] packing or cells can be
a
14,400 when sodium dodecyl sulfate (SDS) added as a surfactant.
b
Calculated using kL aCH4 = 0.855kL aO2 .
Characteristics
Consist of three tube
segments in a circular loop.
A peristaltic pump pumps
the broth up the riser
segment through static
mixing elements and into
the degassing unit from
which the broth proceeds
through the downcomer
segment and back to the
pump.
Consist of closed
circulation loops with riser
and downcomer zones
with a degassing unit
separating the zones.
Liquid circulation is
achieved via gas bubbles
rising in the riser segment.
Substrate gases are
pumped into the reactors
through gas spargers
located at the bottom of
the reactor. The rising
bubbles induce agitation in
the reactor
Generally consist of a
vessel in which the liquid
broth and cells are
mechanically agitated by
an impeller as substrate
gases are pumped into the
vessel via a sparger located
at the bottom of the vessel
immobilized
methanotrophic cells. The
immobilization matrix can
be one of a variety of
materials but must be
porous to allow for
moisture to drain and
substrate gas to rise within
the reactor
suspended in the liquid
medium. The liquid phase
is allowed to trickle down
over the packing while the
substrate gas is pumped
into the reactor at the
bottom
86
Oxygen kL a (h1 ) Methane kL a (h1 ) Removal Efciency Pro Con
a
9000 [46] b
7700 [46] Low shear stress; high High cell density may lead
reactant utilization; low to higher pumping cost and
130 [48] 70 [48] capital and operational lower mass transfer of
costs; can recycle gas and substrate gases
57 [49] 25.7 [49] liquid
99 [72] 85b [72] 16% [72] Compressed gas is the only Difcult to achieve full
source of energy input; low utilization of substrate
capital and operational gases in single pass; bubble
costs; extremely low shear coalescence happens more
K.A. Stone et al. / Biochemical Engineering Journal 128 (2017) 8392
stress; higher kL a per unit frequently;
power than CSTR
27 [53] 15 [53] Poor liquid circulation may
result in non-homogenous
distribution of nutrients
18 [29] 15b [29] 59% [29] Empirical models available High power to volume
14 [95] 12 [95] for mass transfer; power ratios; poor mixing and
input can be used to adjust heat removal for large scale
kL a; uniform distribution of reactors; higher shear
substrate stress
41% [66] Low capital and Typically requires
31.5% [65] operational costs; relatively high residence
19.7% [96] methanotrophic consortia times
from site can be utilized;
works at ambient pressure
and temperature
280 [63] 78% [68] Low liquid and gas ow Typically not well mixed;
35% [69] rates; packing could be microbial growth build up
13.1% [63] used as a matrix for cell between packing
immobilization; low
investment and
operational costs
 K.A. Stone et al. / Biochemical Engineering Journal 128 (2017) 8392
Biotrickle lters (BTFs) are similar to conventional biolters in
the sense that they also contain solid packing material, although
cells can be immobilized or free oating. The key difference is that,
in BTFs, the liquid phase is continuously pumped to the top of
the reactor and allowed to trickle down over the solid packing.
Such difference contributes to higher methane removal efciency
in BTFs on average for similar residence times. BTFs have been used
for methane abatement with removal efciencies as high as 78%
[63,6769].
A capillary column sequenced with a continuous STR (CSTR)
utilizing Taylor (segmented) ow has been reported for LCME
abatement [70]. Taylor ow of mixed gas and liquid in the capil-
lary column can improve kL a by an order of magnitude compared
to CSTR alone, but cell growth could easily cause clogging of the
column and the operation was limited to batch mode [70].
3.2. Gas transfer enhancements in STRs
Although stirred tank reactors (STRs) seem to provide the lowest
kL a compared to other bioreactor types as shown in Table 1, they
are the most commonly used bioreactors in academic and industrial
R&D labs. In this section, we discuss ways to enhance gas transfer
in STRs, in particular, agitation and gas spargers.
It is a common belief that higher agitation rates lead to higher
kL a values; however, it has been shown that an optimal agitation
rate exists in the presence of cells, and when exceeding these lim-
its, kL a actually decreases with increased agitation [27,29]. Similar
ndings have been obtained in our cell-free experiments to study
the effect of agitation rate on kL a for three different gas spargers
in a 3 L Bioo 115 reactor with cell-free NMS medium (Fig. 1). In
addition, higher agitation requires higher energy input and results
in higher shear stress. It is worth noting that the purpose of the
experiments is not to recommend a specic optimal condition,
but rather to illustrate that over agitation could negatively impact
kL a. Because kL a depends on many factors besides agitation rate and
sparger type, such as reactor conguration, operation conditions,
medium composition and cell density, it is recommended to per-
form tests on the actual system to determine the optimal agitation
rate and sparger type.
Another approach to enhance kL a in STRs is through optimizing
impeller congurations. Moucha et al. [71] tested 18 impeller con-
gurations in STRs and discovered signicant variance in kL a caused
by impeller congurations used at the same power consumption
values. The largest mass transfer rate was observed with two Rush-
ton turbines on a common shaft yielding a kL aO2 = 1000 h1 at
800 W/m3 and supercial gas velocity (Ug ) 8.48 m/s. Although this
study was conducted with pure oxygen as gas feed, methane mass
transfer improvement of similar scale is expected due to the similar
physical properties of methane and oxygen, as discussed in Section
2.
Gas spargers can also have a signicant impact on gas-liquid
mass transfer by directly affecting the size of gas bubbles in the
reactor. Bouai et al. [23] tested the effects of three different sparger
designs on oxygen mass transfer rate in a bubble column and
found that a glass sintered porous plate and a perforated exible
membrane produced the smallest bubbles and highest kL a val-
ues for each supercial gas velocity (Ug ) tested. The highest kL aO2
achieved was around 360 h1 by the glass sintered porous plate
at Ug of 0.04 m/s. However, the effect of different gas spargers on
mass transfer rate could be altered by the mixing condition in the
bioreactor. For example, Fig. 1 indicates that the effect of different
spargers on kL a in a Bioo 115 is not signicant, even though the
microsparger produced bubbles that were noticeably smaller than
the other two spargers. A patent [56] was led recently in which a
new design for gas spargers with both horizontal and vertical dif-
fusers was presented with the goal of improving oxygen absorption
87
by microorganisms, revealing a continuing desire for more efcient
gas sparger designs for fermentation processes.
3.3. Operation safety
Methane is an explosive gas. Therefore, the bioconversion of
methane via methanotrophs poses serious safety concerns that
must be addressed. The combustible range of methane/air mixtures
is 515% volume of methane. Although most methane fermentation
process are outside of this range, the following safety consider-
ations and practices are very important to ensure the safety of
operation. The reactor should be designed in a manner that pre-
vents the accumulation of un-utilized substrate gases [12]. This
can be achieved via gas recycling [63,72], enhanced mass trans-
fer that enables full utilization of substrate gas (usually for LCME)
[73], as well as proper venting. This also stresses the need for in-line
measurement of oxygen and methane in the reactor [47].
For lab scale methanotroph fermentations, four safety practices
are recommended ([9] and personal communications with Dr. Mary
Lidstrom, University of Washington and Dr. Christopher Roberts,
Auburn University). 1) Proper gas ventilation of any unused/non-
recycled methane and oxygen by placing the fermentation system
in a fume hood or under proper metal snorkel tubes, so efuent
gases can be removed safely. 2) A fail-safe, auto shut off gas system
should be implemented to handle possible exhaust failure or gas
leaks. The system usually consists of a ammable gas detector with
a relay that is connected to the electronic mass ow controllers
and solenoid valves in the gas lines. If the power fails or if there is
a detectable ammable gas, the system will automatically shut off
the ow controllers and valves. 3) Static mixers should be utilized
to mix methane and oxygen. 4) Finally, both the equipment sys-
tem and the researchers should be properly grounded. This can be
achieved by using an anti-static mat for the equipment system and
having the researcher wear an anti-static wrist band.
4. Promoting agents of gas transfer
Besides bioreactor conguration and operation, adding com-
ponents with higher afnity to methane and oxygen to enhance
mass transfer has been studied. In this section we review the use of
vectors, polymers, nanoparticle, electrolytes, and non-ionic surfac-
tants to enhance mass transfer of methane. Table 2 summarizes
different promoting agents that have been applied to enhance
methane mass transfer in the literature, and each group of pro-
moting agent is discussed below.
4.1. Vectors
Vectors are non-aqueous, non-toxic, non-volatile, and non-
biodegradable organic phases added to the fermentation broth
that can induce signicant increases in gas to liquid mass trans-
fer [7477]. Commonly used vectors and their enhancements are
summarized in Table 2. The vector-enhanced mass transfer is
contributed to the signicantly higher solubility of methane and
oxygen in the organic phase, as well as the increased specic inter-
facial area due to the addition of the organic phase. However,
addition of vectors can cause an increase in the broth viscosity,
which could negatively impact mass transfer and operation costs.
In addition, it has been reported that methanotrophs were found in
contact with or even trapped within the vectors [67,78,79]. Stud-
ies for the separation of cells from the organic phase as well as
recycling the vector have not been published to date.
 88
Table 2
Promoting Agents that Enhance Gas-Liquid Mass Transfer.

Promoting Agents Examples Reactor Setup Gas Flow Rate Amount Added Mass Transfer Enhancement

Vectors a
Silicone oil [29,64,72,73] STR, 800 rpm 0.42 L min1 (0.21 vvm) 10% vol/vol Removal efciency increased from 34% to 57%. The elimination
capacity (g CH4 m3 h1 ) increased from 75 to 106 (41% increase). [64]
Trickle bed reactor 0.21 L min1 (EBRT of 4.8 min) 10% vol/vol Removal efciency increased from 15% to 40%. The elimination
capacity (g CH4 m3 h1 ) increased from 22 to 51 (132% increase). [64]
STR, 500 rpm 0.42 L min1 (0.21 vvm) 10% vol/vol kL aCH4 increase from 11.7 h1 to 36.3 h1 (210% increase).
Removal efciency increased from 28 to 44%. [29]
Airlift reactor Recirculation of 0.1 vvm. 10% vol/vol kL aCH4 increase from 84.4 h1 to 164.9 h1 (95% increase). [72]
Three parallel horizontal 6078 vvm 10% vol/vol Average methane removal (g CH4 m3 h1 ) increaseb [73]:

K.A. Stone et al. / Biochemical Engineering Journal 128 (2017) 8392

ow bioreactors HFBR 1 increased from 4.2 (control) to 5.5 (31% increase)
HFBR 2 increased from 3.1 (control) to 5.5 (77% increase)
HFBR 3 increased from 2.3 (control) to 4.0 (74% increase)
Parafn oil [78] STR 1.140 L min1 (0.38 vvm) 5% vol/vol Cell concentration was 6 g/L with parafn, compared to the control
2 g/L without oil (200% increase). [78]
Polymers Kraton (TM G1657) [77] STR, 300 rpm 0.8 vmm 10% vol/vol OTR (mgO2 L1 h1 ) increased from 770 to 946 (23% increase). [77]
Airlift reactor 0.1 vmm 10% vol/vol OTR (mgO2 L1 h1 ) increased from 1046 to 1252 (20% increase). [77]
Desmopan (DP9370A) STR, 300 rpm 0.8 vmm 10% vol/vol OTR (mgO2 L1 h1 ) increased from 770 to 2083 (171% increase). [77]
[72,77,82] Airlift reactor 0.1 vmm 10% vol/vol OTR (mgO2 L1 h1 ) increased from 1046 to 3710 (255% increase). [77]
Airlift reactor 0.1 vmm 10% vol/vol kL aO2 increased from 98.6 h1 to 232.8 h1 (136% increase) [72].
STR, 300 rpm 0.45 L min1 (0.5 vvm). 5% vol/vol Specic area of oxygen bubble increased by 3 times and enhanced OTR
by 2.5 times. [82]
Elvax (TM 360) [77] STR, 300 rpm 0.8 vmm 10% vol/vol OTR (mgO2 L1 h1 ) increased from 770 to 997 (29% increase). [77]
Airlift reactor 0.1 vmm 10% vol/vol OTR (mgO2 L1 h1 ) increased from 1046 to 1131 (8% increased). [77]
Nanoparticles CuO in SDSb [97] STR, 400 rpm Injected as batch system 0.1% wt of nanouid 40% enhancement of methane transfer as quantied by the partial
pressure of methanec . [97]
Methyl-modied Silica Closed media bottles Injected as batch system 0.25% wt particles Dissolved methane concentration increased from 6.190 to 7.902 mg/L
[87,98] stirred at 200 rpm (28% increase).
The kL aCH4 increased from 22.68 to 41.88 h1 (85% increase). [87]
STR, 200 rpm 1.0 L min1 0.5% wt particles Calculated kL aCH4 increased from 69.1 to 126.4 h1 (83% increase). [98]
Saturation concentration increased from 21.7 ppm to 25.2 ppm (16%
increase). [98]
Electrolytes KNO3 [92] STR, 300 rpm 3.0 L min1 5% wt The kL aCH4 increased from 103 to 233 h1 (126% increase). [92]
MgSO4 [92] STR, 300 rpm 3.0 L min1 5% wt The kL aCH4 increased from 103 to 711 h1 (590% increase). [92]
NaCl [92] STR, 300 rpm 3.0 L min1 5% wt The kL aCH4 increased from 103 to 401 h1 (289% increase). [92]
Non-ionic surfactants Tween 20 [94] Biolter 4.2 L min1 (EBRT 4.25 min)d 0.5% wt Removal efciency increased from 36% to 65% with surfactant. [94]
Brij 35 [69,94] Biolter 4.2 L min1 (EBRT 4.25 min) 0.5% wt Removal efciency increased from 36% to 42% with surfactant. [94]
Biotrickling lter Varied 0.5% wt Removal efciency increased from 15% to 33% with surfactant. [69]
Non-ionic surfactant and Brij 35 + Silicone oil [73] Parallel horizontal ow 6078 vvm 1 g/L Brij 35 Removal rate of CH4 increased 110174% compared to control and 55%
organic vector bioreactors 10% vol/vol Silicone Oil compared to just silicone oil added [73]
a
All kL a values are for methane calculated using kL aCH4 = 0.855kL aO2 [29].
b
SDS is a surfactant that allows the nanoparticles to be dispersed in water.
c
Enhancement was seen in systems at elevated pressures of 10 bar and 20 C. Even higher enhancement was seen with lower temperatures.
d
Tween 20 was added every 4 days with the medium. In between those additions, 2 mL of emulsion of methanol + soy oil was added.
 K.A. Stone et al. / Biochemical Engineering Journal 128 (2017) 8392
Fig. 1. Effect of gas ow rate, sparger type, and agitation speed on kL a in an Eppendorf Bioo 115 bioreactor.
Green solid line with squares: the conventional stainless steel sparger equipped in Eppendorf Bioo 115 reactors; Blue dashed line with circles: Alitas silicone tube punctured
throughout with 1 mm slits; Red dotted line with triangles: Eppendorf microsparger.
4.2. Polymers
Polymer particles with high afnity and specicity towards
gases can be used as solid vectors. These polymers are
non-biodegradable, inexpensive (unlike their liquid vector coun-
terparts), and can be tailored on shape, size, and gas target [80].
Also, studies have shown that polymers can be made in a way that
little to no cell adhesion would occur, which helps reduce the loss
in cellular biomass and the ability to have complete recovery of the
polymers themselves [81]. The reported studies on polymers are
summarized in Table 2 with their effect on mass transfer enhance-
ment. It has been suggested that the increased specic interfacial
area, caused by the addition of polymer, is the most inuential
factor for the mass transfer enhancement [8284]. However, all
studies listed in Table 2 were carried out in abiotic systems, and
the effects of the polymer particles on methanotrophs growth and
viability have not been fully examined.
4.3. Nanoparticles
Nanoparticles have been used to enhance mass transfer from
gas to liquid for the past decade. The level of mass transfer
enhancement is affected by particle size and surface characteris-
tics, especially the hydrophobic or hydrophilic properties [8587].
Although the enhancement of nanoparticles to oxygen transfer
has been studied and documented [88,89] using nanoparticles to
enhance methane transfer has been limited. Table 2 lists the studies
that focus on enhancing mass transfer of methane using nanopar-
ticles. Similar to polymers, all available studies were carried out
in abiotic systems and focused on evaluating the mass transfer
enhancement. The potential effect of the nanoparticles on cells,
as well as how to separate nanoparticles from cells has not been
examined. In addition, it should be noted that nanoparticles can
change the optical reading of the broth, which can interfere with
cell density measurements if not separated [90].
4.4. Electrolytes
Electrolytes have been shown to have a positive effect on the
mass transfer rate and kL a of carbon monoxide, mainly due to the
89
electrical double layer around the gas bubbles and reduced bubble
coalescence due to electro-repulsive forces between bubbles [91].
Methane transfer has recently been investigated with several salts
commonly found in the mineral medium used by methanotrophs
including, MgSO4 , MgCl2 , NaCl, and KNO3 , and the results are sum-
marized in Table 2 [92,93]. The anions in the salts had the largest
inuences on kL a, with SO4 2 containing compounds consistently
reporting the highest kL a values.
Compared to the promoting agents discussed above, elec-
trolytes are inexpensive and can be easily separated from cells. The
enhancement of methane mass transfer is signicant despite the
decreased solubility of methane in liquid phase due to salting-out
effects. However, the studies listed in Table 2 were conducted in
abiotic systems, and the signicant enhancement was seen at high
salt concentrations, much higher than those reported in a typical
methanotrophs liquid medium (0.1 wt% KNO3 and 0.75 wt% NaCl)
[42]. It is unclear whether methanotrophs can tolerate such high
salt concentrations.
4.5. Non-ionic surfactants
Surfactants are chemical agents that decrease the surface ten-
sion of a liquid. Non-ionic surfactants are the specic agents that
can be used in water containing salts (such as NMS medium) as
they have no interactions with the ions. Additionally, these sur-
factants are biodegradable and are non-toxic to microorganisms at
low enough concentrations [69,73,94]. Limited studies have been
done with methanotrophs and non-ionic surfactants in bioler or
biotrickle lter reactors for LCME conversion as listed in Table 2. It
is worth noting that these results were obtained with the presence
of methanotroph cells, and the improvement in methane removal
rate was signicant as shown in Table 2. It is also worth noting that
surfactants can also serve as an emulsifying agent to oils, effectively
dispersing the vector to further increase transfer of methane [73],
which is also listed in Table 2.
However, the application of non-ionic surfactants to other types
of bioreactors and high methane feed has not been reported. In
addition, like with other enhancing agents, there is a lack of study
on separation of surfactants from biomass.
90 K.A. Stone et al. / Biochemical Engineering Journal 128 (2017) 8392
5. Conclusions
Driven by the desire for sustainable fuels and chemicals,
methanotrophs that utilize methane as their sole carbon and energy
source have drawn renewed research interest. In this work, we pro-
vide a mini review on the recent progress in process considerations
with biological methane conversion, with focuses on bioreactor
congurations and mass transfer promoting agents.
Although many factors affect the mass transfer rate of gas sub-
strate, we found that bioreactor congurations have the most
profound impact. For high concentration methane feed, among dif-
ferent types of reactors, the forced circulation loop reactors show
the most promising mass transfer results, i.e., the highest kL a for
methane. For low concentration methane emissions (LCME) feed,
biotrickle reactors deliver the highest methane removal efciency.
For the most commonly used stirred tank reactors in research labs,
agitation speed, the types of propeller, and gas sparger design could
affect mass transfer rate signicantly and should be optimized.
It is worth noting that methanotrophs can generate a range
of excreted organic products such as extracellular polysaccharides
(EPS), ectoine (a cyclic amino acid) and acetate, depending on spe-
cic growth conditions [42]. These excreted byproducts, especially
EPS, could cause signicant increase in broth viscosity and severe
foaming, which could greatly limit the operability of bioreactors.
Currently, the common practice to address this challenge is adding
antifoam to the bioreactor as needed to control foaming. However,
how to effectively handle this challenge for high cell density culture
is an important issue for methane bioconversion commercializa-
tion; further research and new solutions are needed to examine and
control the effects of excreted byproducts to the process operation.
Regarding mass transfer promoting agents, addition of vectors,
polymers, nanoparticles, electrolytes or non-ionic surfactants all
show different level of improvements on kL a; however, study is
lacking on how the promoting agents could affect biocatalyst per-
formance. In addition, further research into the effects of the agents
on the overall system performance, and downstream processing
(such as the separation and recycle/reuse of the enhancing agent to
support the economic feasibility of such applications) are needed
before application of promoting agents become practical.
With the projected increase in natural gas and biogas pro-
ductions in the US and the world, biological methane conversion
technologies could have signicant economic and societal impacts,
particularly on the Earths ecosystem and environment sustain-
ability. As discussed in this mini review, besides biocatalyst
development, process development is a major technical hurdle that
prevents industrialization of biological methane conversion. Fur-
ther research and advancements in bioreactor design, operation,
and gas transfer enhancement are needed to make the biological
process an economically feasible route for methane conversion.
Acknowledgements
The authors would like to thank Prof. Mary Lidstrom (University
of Washington) for helpful discussions. The authors also would like
to thank the anonymous reviewers for their suggestions and com-
ments. Financial supports from Department of Education GAANN
Fellowship (ED12-P200A120228 for KAS), NSF IGERT (1069004 for
MVH), and NSF (1264861 for QPH and JW) are gratefully acknowl-
edged.
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