Final Technical Report (Year 1)
IEC Grant No: 11-02
Title: Development of novel biofilm-based reactor systems for
enhancing mass transfer in syngas fermentation
Principal Investigator: Zhiyou Wen
Grantee Organization: Iowa State University
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Signature of PI
Public Abstract
Gasification of biomass into syngas (CO and H2) followed by fermentation of the syngas into
ethanol is a novel approach for producing biofuel from lignocellulosic biomass. Compared with
biochemical conversion, the gasification – syngas fermentation route has several advantages such
as (1) capability of using all components of biomass including lignin; (2) independence of
feedstock composition; (3) elimination of complex pretreatment and high enzyme costs; and (4)
independence of H2:CO:CO2 ratio in syngas fermentation.
Syngas fermentation, however, is limited by several factors, one of which is low gas-liquid mass
transfer efficiency. The goal of this project is to design a novel bioreactor to enhance the mass
transfer efficiency in syngas fermentation. We propose two bioreactor designs: hollow fiber
membrane reactor (HFM-BR) and monolithic biofilm reactor (MBR). The specific objectives are:
(1) to build the HFM-BR and MBR; (2) to evaluate the syngas fermentation performance of
Clostridium bacteria, and (3) to determine H2 and CO consumption rate and characterize mass
transfer coefficients of the HFM-BR and the MBR.
In the first year of this project, we completed the construction of the HFM-BR and MBR systems
and started the syngas fermentation in these two systems. The continuous operations of the
syngas fermentation in these two systems have also been established.
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Background
Biofuel (such as ethanol) production from lignocellulosic biomass can be achieved through a
hybrid thermochemical-biological processing, i.e., gasification of biomass to generate syngas (a
mixed gas containing carbon monoxide, hydrogen, carbon dioxide, nitrogen and small amounts
of hydrocarbons) with biochemical processing (fermentation) to convert the syngas into biofuels
or chemicals. As a hybrid process, gasification - syngas fermentation captures the benefits of
each pathway while mitigating some of their deficiencies, such as (1) utilization of the whole
biomass including recalcitrant lignin; (2) elimination of complex pretreatment steps and costly
enzymes; (3) higher specificity of the biocatalysts; (4) independence of syngas components ratio;
(5) bioreactor operation at ambient conditions; and (6) no issue of noble metal poisoning.
However, the efficiency of syngas fermentation is limited by low gas-liquid mass transfer
efficiency because both carbon monoxide (CO) and hydrogen (H2) are water-insoluble.
Enhancing the mass transfer efficiency in syngas fermentation is a major criterion in the design
of syngas fermentation bioreactors.
Goal and Objectives
The ultimate goal of this project is to develop an optimum bioreactor system with high gas-liquid
mass transfer efficiency for syngas fermentation purpose. With this IEC grant, we will evaluate
the applicability of two novel biofilm-based reactors (hollow fiber membrane-biofilm reactor
(HFM-BR) and monolithic biofilm reactor (MBR)) using Clostridium carboxidivorans strain P7
for ethanol production. Specific objectives and the subtasks for each objective are as follows:
Objective 1: To set up the HFM-BR and MBR systems.
Task 1-1: Set up HFM-BR system:
Task 1-2: Set up MBR system:
Objective 2: Evaluate the syngas fermentation performance of C. carboxidivorans P7 and
optimize fermentation parameters.
Task 2-1: Establish the syngas fermentation in the HFM-BR and MBR systems:
Task 2-2: Optimize syngas fermentation of C. carboxidivorans in HFM-BR and MBR
systems
Objective 3: Determine syngas consumption rate and mass transfer coefficients of the
HFM-BR and MBR.
Task 3-1: Develop the methods for determining syngas consumption rates and KLa:
Task 3-2: Evaluate the syngas consumption rates and their KLa values in the HFM-BR and
MBR systems
The timeline for each task is shown in the chart below:
Year 1 (Jul 2011- Jun 2012)
Task
1-1
1-2
Jul
Aug
Sep
Oct
Nov Dec
Set up the HFM-BR system
Set up the MBR system
2
Jan
Feb
Mar
Apr May
Jun
Establish syngas fermentation of the two
systems
2-1
Year 2 (Jul 2012- Jun 2013)
Jul
2-2
3-1
3-2
Aug
Sep
Oct
Nov Dec
Jan
Feb
Mar
Apr May
Jun
Optimize continuous syngas fermentation of the two reactor systems
Develop methods for determining KLa
Determine KLa at different conditions
Work conducted during this reporting period (July 2011 – June 2012)
Objective 1: To set up the HFM-BR and MBR systems
Task 1-1: Set up HFM-BR system
We evaluated several hollow fiber membrane materials and decided to use a Liqui-Cel®
membrane contactor MiniModule 1.7×8.75 (Membrana, Charlotte, N.C.) as the membrane
supported module in HFM-BR (Figure 1). This membrane module contains X50 microporous
hydrophobic polypropylene hollow fibers with 40% porosity and 0.04 μm pore size. The fiber
has an outer diameter of 300 μm and an internal diameter of 220 μm. The active membrane
surface area of the module is 1.4 m2. We are in the middle of contracting the HFM-BR using
this membrane module.
Task 1-2: Set up MBR system
We chose a cordierite ceramic monolithic column (Applied Ceramics, Atlanta, GA), with length
12 inches and diameter 3.66 inches, as the packing materials for the monolithic biofilm reactor.
The monolith channels are square with a width of 1.81 mm, termed as 200 cpsi (cells per square
inch) (Figure 2A). The monolith column was placed in a Plexiglass column (Figure 2B). As
shown in Figure 2B, two Plexiglass rings were used to secure the monolith column and to
prevent any by-pass of gas or liquid flow. Gas distribution was achieved with two diffuser
designs. The first design is to use aquarium wood air stone which were placed at the bottom the
reactor for generating bubbles. The gas and liquid flow are concurrent, i.e., both from the
bottom to the top. In this design, liquid is the continuous phase, while gas is discrete phase. The
small bubbles generated from the diffuser (air stones) will be “squeezed” into the channels of the
monolithic packing materials, forming Taylor flow inside the channels. A biofilm will form as
biocatalysts coating on the inner wall of the channel to improve the gas-liquid mass transfer. The
second design for gas distribution is to place a spray nozzle at the top of the column to spray a
tiny droplet of liquid (growth medium). These droplets will be mixed with syngas at the top of
the reactor to form a mist, and then flow through the channel downwardly. Similar to the first
design, the liquid flow and gas flow will also be in con-current mode. However, unlike the first
design, the liquid will become the discrete phase while the gas will be continuous phase. The
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monolithic column was connected to a BioFlo 110 fermenter (New Brunswick Scientific, Edison,
NJ) for controlling fermentation parameters such as temperature and pH (Figure 2B).
During the reporting period, we also investigated the attachment of C. carboxidivorans strain P7
to the inner channel wall of the monolithic packing materials. We built a mini MBR system and
run the system in batch fermentation mode (Figure 3A) for 14 days, the packing materials was
then taken out of the reactor and analyzed by SEM (Scanning Electron Microscope). As shown
in Figure 3B, it is clear indicated that C. carboxidivorans strain P7 could form a biofilm on the
surface of monolithic channels. Abundant rod-shaped bacteria were found in SEM image,
confirming the biofilm formation.
Objective 2: Evaluate the syngas fermentation performance.
This objective focuses on the implementation of the syngas fermentation into the two reactor
systems (HFM-BR and MBR). The strain use was C. carboxidivorans P7 (ATCC # PTA-7827)
which has been reported capable of fermenting syngas for ethanol production.
Task 2-1 Establish the syngas fermentation in the HFM-BR and MBR systems
Syngas fermentation in a control stir tank reactor: We first investigated the syngas fermentation
in an 11-liter BioFlo 110 Fermenter (New Brunswick Scientific, Edison, N. J.) as a control
system. The syngas was fed into the fermenter at 100 mL/min and at 3 psig head space pressure.
The syngas consisted of 20% CO, 5% H2, 15% CO2 and 60% N2, blended from individual gas
tanks. An oxygen trap column was used to remove any residual oxygen from the syngas entering
the bioreactor. The reactor had a working volume of 9 L with 400 mL C. carboxidivorans strain
P7 as the inoculum. The reactor temperature was kept at 37 °C and the agitation was controlled
at 500 rpm. The pH of the reactor was initially 5.78 and as the fermentation proceeded, the pH
was manually controlled with 20% NaOH solution.
We first conducted the syngas fermentation in a bath mode. Figure 4 shows the cell
concentration in terms of optical density (OD) during 14-day batch period. The P7 cells started to
grow after 1 day lag phase and increased exponentially. The cell concentration reached peak
value at day 2-3 with 1.08 OD units and was steady at ~ 1.0 for the next 5 days. The OD value
began to fall on day 7 and dropped to 0.25 units on day 13. Figure 5 shows the pH change
during the syngas fermentation. pH decreased from 5.78 to 4.56 on day 1 and recovered back to
6.0 at day 3, and then leveled off at ~ 5.9 for the remaining culture period. These observations
indicated that the acidogenesis phase ended on day 2-3 and switched into solventogenesis phase
with the resting P7 cells. The CO2 change during the syngas fermentation was also monitored.
As shown in Figure 6, CO2 in the inlet gas flow was kept at a constant level; however, the CO2
content in the outlet increased to 27.7% (v/v), corresponding to the maximum cell concentration
(1.08 OD, Figure 1). As the cell stationary phase ended after day 7, CO2 production ceased.
We further conducted an intensive study of the continuous syngas fermentation at different
operational parameters including gas flow rate, agitation speed, and the hydraulic retention time
(HRT). Table 1 shows the detailed arrangement of these three parameters for the entire
continuous fermentation period.
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The production of ethanol and acetic acid during this entire fermentation is presented in Figure 7.
As shown in the figure, the highest production of ethanol and acetic acid were on approximately
day 90, which fall into day 81-100 period (gas flow rate 500 mL/min, agitation 600 rpm, and
HRT 200 hours).
Syngas fermentation in a HFM-BR system: Syngas fermentation was also successfully
established in hollow fiber membrane biofilm reactor (HFM-BR). The reactor was first operated
in a batch mode to establish the attachment of the bacterial cells on the membrane surface.
Figure 8 showed the microbial attachment on the membrane surface on day 10.
After 15-day batch culture, the system was switched to continuous operation, with continuous
withdrawal of the fermentation broth and replenishment of fresh liquid medium. With the
continuous operation, suspended cells in the fermentation broth were gradually attached on the
hollow membrane surface; as a result, the broth of the fermenter vessel became clear. The
biofilm attached on the membrane surface continued to grow until the biofilm reached a
thickness equilibrated with the operating conditions. Because of the cell attachment, monitoring
the cell growth through the OD reading in the broth was not feasible. However, the pH
fluctuation (between 4.5 and 6.10) indicating the acetic acid formation in the syngas
fermentation. When pH was dropped to 4.5, the condition became favorable to ethanol
production. When pH decreased to below 4.5, NaOH solution (5 N) was added to the fermenter
to bring pH up to 6.10.
During this reporting period, we run the HFM-BR at a continuous mode at different experimental
settings including the gas flow rate, liquid recirculation rate and HRT (Table 2).
Figure 9 shows the ethanol and acetic acid production levels during this entire fermentation
period. It can be seen that the production of ethanol and acetic acid fluctuated significantly,
depending on the different operation parameters setting. There are two “peak” productions
during the entire fermentation, i.e., around day 25 and around day 70 (Figure 9), which fall into
the 200 ml/min gas flow rate, 150 ml/min liquid recirculation rate and 200 hours HRT; and 500
ml/min gas flow rate, 150 ml/min liquid recirculation rate and 200 hours HRT, respectively.
Compared with the ethanol and acetic acid production in the CSTR reactor (Figure 7), the HFMBR system produces a much higher concentration of ethanol and acetic acid (Figure 9)
Syngas fermentation in a MBR system: Operations of the monolithic biofilm reactor (MBR)
were the same as those in HFM-BR system. The reactor was first operated in a batch mode to
establish the attachment of the bacterial cells on the monolith inner channel wall. The attachment
was evident by the fact that the fermentation broth became clear, and the ethanol was detected in
the broth. Similar to HFM-BR, the MBR was also operated in a continuous mode, during which
the operational parameters were set at different levels to investigate the effect of gas flow rate,
liquid recirculation rate, and HRT on the syngas fermentation performance (Table 3).
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Figure 10 shows the ethanol and acetic acid production levels during this entire fermentation
period for the MBR operation. Overall, the production the ethanol and acetic acid production
level are much higher than the CSTR, but lower than the HFM-BR system.
CO and H2 consumption rate of HFM-BR and CSTR: During the project period, we also analyzed
the syngas (CO and H2) consumption efficiency of the HFM-BR and CSTR. This work will lay
the ground work for the mass transfer coefficient (KLa) measurement, which will be determined
in the second project year. As shown in Figure 11, both CO and H2 consumption efficiency of
HFM-BR were higher than those of the CSTR, indicating more syngas were consumed by this
type of reactor, this high consumption efficiency in HFM-BR was believed due to the enhanced
mass transfer performance of this type of reactor. This result further demonstrated the better
performance of HFM-BR than CSTR. It should be noted that the measurement of syngas
consumption efficiency of MRB was underway, and will be accomplished at the second project
year.
Summary
During the reporting period, we have completed the continuous operation of CSTR and HFM-BR
system for syngas fermentation. Different operational parameters for these two reactor systtems
have been studied. The results obtained indicate that HFM-BR is an excellent reactor system for
syngas fermentation, while MRB also shows an improved performance compared to the control
(CSTR). The major reason for the enhanced performance of these two reactor systems is due to
the enhancement of the mass transfer. In the second year of the project, we will quantitatively
determine and compare the mass transfer coefficients of these reactor systems to confirm our
hypothesis. The operation of these two reactors will also be optimized so a highest capability of
the reactor can be obtained.
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APPENDIEX: TABLES AND FIGURES
Table 1. Operational parameters for syngas fermentation in a CSTR fermenter
Day
0-39
40-50
51-60
61-70
70-80
81-100
101-107
108-113
114-119
120-124
125-140
141-150
151-160
161-170
Gas flow rate (mL/min)
200
200
50
100
300
500
500
500
500
500
500
500
500
500
Agitation (rpm)
600
600
600
600
600
600
800
400
300
200
600
600
600
800
7
HRT (hours)
batch
200
200
200
200
200
200
200
200
200
100
75
50
25
Table 2. Operational parameters for syngas fermentation in a HFM-BR system
Day
0-15
16-30
31-40
41-50
51-60
61-80
81-87
88-94
95-101
102-111
112-125
126-135
136-145
146-155
Gas flow rate (mL/min)
200
200
50
100
300
500
300
300
300
300
300
300
300
300
Liquid recirculation rate (mL/min)
150
150
150
150
150
150
50
100
250
500
150
150
150
150
8
HRT (hours)
batch
200
200
200
200
200
200
200
200
200
100
75
50
25
Table 3. Operational parameters for syngas fermentation in a MBR system
Day
0-15
16-40
41-55
56-65
65-80
81-90
91-95
96-100
101-105
106-110
111-120
121-130
131-145
146-160
Gas flow rate (mL/min)
200
200
50
100
300
500
200
200
200
200
200
200
200
200
Liquid recirculation rate (mL/min)
250
250
250
250
250
250
100
500
800
1000
250
250
250
250
9
HRT (hours)
batch
200
200
200
200
200
200
200
200
200
100
75
50
25
Figure 1. The membrane to be used in the construction of the HFM-BR
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(A)
(B)
Figure 2. MBR reactor for syngas fermentation. (A) the monolithic packing materials;
(B) the whole reactor system
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(A)
(B)
Figure 3. Systems for investigation of bacteria’s attachment on the inner wall of the monolithic
packing materials. (A) Mini-MBR system; (B) SEM image of monolithic column channel
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Figure 4. Cell concentration in batch mode syngas fermentation by C. carboxidivorans P7 in a
BioFlo 110 Fermenter
13
Figure 5. pH profile of a batch mode syngas fermentation of C. carboxidivorans P7 in a BioFlo
110 fermenter
14
Figure 6. CO2 content in the inlet and outlet gas flow during the batch mode syngas fermentation
by C. carboxidivorans P7 in a BioFlo 110 fermenter.
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Figure 7. Ethanol and acetic concentration in syngas fermentation by C. carboxidivorans P7 in a
CSTR reactor system.
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Figure 8. Microbial attachment on the hollow fiber membrane surface on day 10
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Figure 9. Ethanol and acetic concentration in syngas fermentation by C. carboxidivorans P7 in a
HFM-BR reactor system.
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Figure 10. Ethanol and acetic concentration in syngas fermentation by C. carboxidivorans P7 in
a MBR reactor system.
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Figure 11. Syngas (CO and H2) consumption efficiency of HFM-BR and CSTR operated at
different flow rate
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