2012 Wiley Periodicals, Inc. Biotechnology and Bioengineering, Vol. xxx, No. xxx, 2012 1
hydrogenotrophic archeae, microorganisms which can cultures. Initially, mixtures (380 mL) of mesophilic or
bind CO2 with H2 and convert them to methane. thermophilic sludge and basal anaerobic (BA) medium
(Angelidaki and Sanders, 2004) with VSS concentration 5 g/L
were added in 1,140 mL serum bottles. The BA medium
4H2 CO2 CH4 2H2 O;
(1) contained 10 mM PBS buffer. The bottles were then closed
DG0 130:7 kJ=mol with butyl-rubber stoppers and aluminum crimps and
ushed with pure H2. After that, pure CO2 (190 mL) was
injected into the bottles to achieve a ratio of H2/CO2 4:1. The
Hydrogenotrophic methanogens belong to the orders bottles were incubated in shakers at 378C (bottles inoculated
Methanobacteriales, Methanococcales, Methanomicrobials, with mesophilic sludge) and 558C (bottles inoculated with
and Methanosarcinaceae (Karakashev et al., 2005). Anaerobic thermophilic inoculum) with shaking speed 300 rpm.
sludges from anaerobic reactors contain hydrogenotrophic Twenty-ve milliliter of the bottle content was daily
methanogens (Demirel and Scherer, 2008). replaced with fresh BA medium using 100 mL syringe and
Nevertheless in order for the whole process to be at the same time the gases were also refreshed by ushing
considered as renewable, the H2 needed for the biogas with pure H2 and then adding CO2 as described above.
upgrading should also be provided by renewable sources. This procedure continued for approximately 2 months.
One obvious way is to use excess electricity from wind mills, The experiment was performed with duplicate bottles. To
for water electrolysis to produce hydrogen as we have determine the kinetics of H2 consumption, enriched
mentioned previously (Luo et al., 2012). In addition, H2 can mesophilic or thermophilic cultures (10 mL cultures)
be also obtained by other sources, including coal gasica- were transferred to serum vials (60 mL serum vials) and
tion, petroleum renery, petrochemical plants, and soda pre-incubated for 1 h under the atmosphere of pure N2, and
manufacture (Ni et al., 2011). Utilization of H2 for biogas then given the appropriate H2 partial pressure (00.2 atm)
upgrading provides several advantages, such as utilization by injecting pure H2 and corresponding CO2 using syringe.
of the existing infrastructure of biogas plants and energy All the bottles were pressurized to 1.5 atm by adding
conversion for H2 utilization. additional N2. The experiment was conducted for 1h to
We have proposed an innovative method for in situ ensure that the initial hydrogen concentration was not
biogas upgrading by the addition of hydrogen to anaerobic decreased signicantly (Ahring and Westermann, 1987).
reactor treating cattle manure (Luo et al., 2012). This in situ All the bottles were prepared in fume cupboards to avoid
upgrading method is very simple, but it has negative effect the possible explosion of H2.
on the anaerobic process due to pH increase. Therefore, it
requires special solutions, such as codigestion with acidic Continuously Fed Reactor Experiment
substrates or pH control, to keep the pH within appropriate
limits. Therefore, in the present study we investigated an After enrichment for half a month in the previous
innovative process for biogas upgrading, in a separate experiment, thermophilic condition was shown to be
reactor enriched with hydrogenotrophic methanogens, and more efcient than mesophilic condition. So the continu-
fed with biogas and hydrogen. The biomethanation ously fed reactor was immediately started up and
efciencies and microbial community compositions of operated at thermophilic temperature. The reactor was
both mesophilic- and thermophilic-enriched cultures were 1 L bottle with 600 mL working volume. The reactor was
investigated. Furthermore, the biogas upgrading and reactor lled with thermophilic inoculum from full-scale biogas
performance was studied in a continuously fed thermophilic reactor and BA medium containing 10 mM PBS buffer.
anaerobic reactor under varying operation conditions. The initial VSS concentration was 5 g/L. The feeding gas
was composed of H2, CH4, and CO2 with the ratio 60:25:15.
The gas was injected to the bottom of the reactor through
Materials and Methods
ceramic gas diffusers. The initial gas ow rate was 3 L/
(L day) and then increased gradually. Forty milliliter
Inocula
mixture in the reactor was daily replaced with fresh BA
As sources of inoculum, mesophilic anaerobically digested medium. The reactor was magnetically stirred at 500 or
sewage sludge (Wastewater treatment plant, Lundtofte, 800 rpm. Steady-state was dened as a period of 6
Denmark) and thermophilic anaerobically digested manure consecutive days with daily variation of biogas production
(Biogas Plant, Snertinge, Denmark) were used. rate of <10%.
Figure 1. Proles of H2, CO2, and CH4 at the beginning of the enrichment (a) thermophlic (b) mesophilic and the end of the enrichment (c) thermophlic (d) mesophilic.
Period (day) 010a 1143 (I) 4473 (II) 7496 (III) 97135 (IV)
Gas injection rate (L/(Lreactor day)) 3 6 12 12 24
Gas retention time (h) 8 4 2 2 1
Mixing speed (rpm) 500 500 500 800 800
Biogas production rate (L/(Lreactor day)) 1.4 0.3 2.5 0.4 5.1 0.6 4.9 0.5 10.1 1.3
Biogas composition
CH4 (%) 93.5 4.4 95.4 2.8 89.9 4.1 94.2 2.8 90.8 2.4
CO2 (%) 4.2 2.5 0.7 0.4 2.6 1.5 1.9 0.5 2.2 1.3
H2 (%) 2.3 2.4 3.9 0.8 7.5 1.2 3.9 0.7 7 1.8
H2 consumption rate (L/(Lliquid day)) 2.9 0.5 5.9 0.4 11.3 0.7 11.6 0.8 22.8 2.1
CH4 production rate (L/(Lliquid day)) 0.9 0.2 1.5 0.3 2.6 0.5 2.7 0.6 5.3 1.4
Yield CH4/H2 0.31 0.26 0.23 0.23 0.23
Acetate concentration (mM) 5.9 1.2 8.8 0.7 9.8 1.3 10.5 1.1 9.4 1.6
a
The data are not in steady-state for period 110.
Biogas Upgrading Performance of Thermophilic operation, the methane contribution originating from
Anaerobic Reactor residual organic matter in the inoculum was eliminated.
The CO2:H2 ratios were 0.26, 0.23, 0.23, 0.23 during the
The biogas upgrading potentials were then tested in steady-states of phase I, II, III, and IV, respectively. It
continuously stirred tank reactor (CSTR) at 558C since showed that the consumed H2 was almost stoichiometrically
the conversion of H2 and CO2 into CH4 was signicantly converted to CH4.
faster under thermophilic condition compared to meso- Acetate was also detected as the main metabolite in
philic operation. The operation parameters and results are the liquid phase with the concentration between 5.9 and
shown in Table II. The inoculum had obvious hydrogeno- 10.5 mM.
trophic methanogenic activity, and therefore H2 in the During the experiment, pH was relatively stable (around
feeding gas was efciently consumed even during the start- 7.8), which is within the pH range appropriate for
up period of the reactor. At day 10, the CH4 content could methanogens (OFlaherty et al., 1998).
be increased from 25% of the feeding gas to around 93% of The microbial communities at days 50 and 100 were
the upgraded gas. After that, the gas injection rate increased analyzed by PCRDGGE, and similar distribution of
to 6 L/(L day) and the reactor was operated under this dominant bands as that found from thermophilic-enriched
condition until steady-state was achieved. The CH4 content culture in the rst experiment were observed (data not
was as high as 95.4%, and there were only small amount shown).
of H2 and CO2 left. The quality of the upgraded biogas In the biogas upgrading process, gasliquid mass transfer
was adequate for utilization as natural gas. At day 44, the is crucial since it determines the available substrate for
gas injection rate was further increased to 12 L/(L day). methanogens. Therefore, the dissolved H2 during the steady-
However, the CH4 content in the upgraded gas decreased state of each operation condition was measured. Figure 5
to around 90% even after around 1-month operation. The shows both measured dissolved H2 (H2l, mol/L) and the
gasliquid mass transfer has been found as the limiting calculated dissolved H2 equivalent to gas phase (H2gTh, mol/
factor for bioconversion of gaseous substrate, especially L) according to Henrys law. The H2gTh were always three to
when the gas (such as H2) has low solubility (Pauss et al., eight times higher than H2l in the whole operation periods,
1990). Therefore, the mixing speed was increased from 500 which indicated that there are gasliquid mass transfer
to 800 rpm at day 74 to lower the gasliquid mass transfer limitations in the process. It is consistent with the fact
limitations. The increase of mixing speed resulted in the that H2 is a kind of gas with low solubility. The H2gTh
increase of CH4 content to around 95%. It seems that changed with the variations of the operation parameters
intensive mixing of the liquid reduced the H2 content in the while H2l were relatively stable. To describe the gasliquid
biogas, and thereby resulted in higher CH4 content. After mass transfer, Equation (2) is used.
day 97, the gas injection rate increased to 24 L/(L day).
Though a small decrease of CH4 content was observed again, rt 22:4kL aH2gTh H2l (2)
the methane content still remained around 90%. The above
results demonstrated that biogas upgrading was achieved in where rt (L/(L day)) is the H2 gasliquid mass transfer rate,
thermophilic anaerobic reactor with high gas injection rate. 22.4 (L/mol) represents that 1 mol gas is 22.4 L (STP), kLa
Theoretically, the molar ratio of CH4 production rate (1/day) is the transfer coefcient. It is obvious rt is
to H2 consumption rate is 0.25. Nevertheless, the measured determined by the difference between gaseous and liquid
ratio was higher than 0.31 during the initial 10 days. It may H2 [assuming kLa is constant for the sample operating
be explained by the excess CH4 production from residual conditions (i.e., mixing)]. Higher gasliquid mass transfer
organic matter contained in the inoculum. After long-term rate could be obtained with larger difference between H2gTh
and H2l. In our experiment, rt is the same as H2 microbial biomass, but also other residual organic matter.
consumption rate during the steady-states of the reactor. ATP has been reported as an effective method to assess the
The H2 consumption rate in phase II was almost two-times cell concentration in the literature (Hwang and Hansen,
higher than that in phase I (Table II), therefore, the 1998). Therefore, ATP concentrations under different
difference between H2l and H2gTh should also be increased operation parameters were measured (Fig. 6). It is obvious
to obtain a higher H2 gasliquid mass transfer rate, which the increase of ATP was in correspondence with the increase
explained the higher H2 content in the upgraded gas of of H2 consumption rate.
phase II. However, the H2 content in the upgraded gas Ex situ biogas upgrading by anaerobic digestion has not
decreased from 7.5% in phase II to around 3.9% in phase III. been studied before, and the results from this study showed
This may be due to the increase of mixing speed resulting that CH4 content in the biogas around 90% or even higher
in an increase of the kLa (Kramer and Bailey, 1991), and was achieved under thermophilic condition with gas
thereby decreased the difference between H2l and H2gTh. It is injection rate up to 24 L/(L day). In Denmark, there are
expected that by increasing kLa in phase IV, the CH4 content more than 20 centralized biogas plants and cattle manure is
can be further increased. Besides increasing the mixing the main substrate. The daily average production of biogas is
speed, other methods to increase kLa include efcient gas 140 m3 per 100 m3 reactor tank (Raven and Gregersen,
diffusion equipment (such as hollow ber membrane; Kim 2007). If the biogas upgrading concept proposed in the
et al., 2011), biogas recirculation (Guiot et al., 2011), etc. present study is applied in these biogas plants, an anaerobic
Table II shows that H2 consumption rates were increased reactor for biogas upgrading with volume 1/10 of the biogas
with the increase of gas injection rates. The H2 consumption reactor is needed. If more efcient gasliquid mass transfer
rates under steady-states can be described by the following method is adopted, it is expected that even smaller reactor
equation: for biogas upgrading can be used.
rc mYX (3)
Conclusions
kd 1
m (4) The present study proposed and demonstrated a method for
SRT
biogas upgrading in an anaerobic reactor. Both mesophilic
where rc (L/(L day)) is the H2 consumption rate, Y (L/g) is and thermophilic sludges had obvious biomethanation
the yield coefcient (STP), X (g/L) is the cell concentration, potential from H2 and CO2. Enrichment under thermophilic
m (1/day) is the specic growth rate of the microorganism, condition provided higher CH4 production rate from H2
kd (1/day) is the specic decay rate of the microorganism and CO2. Microbial community analysis showed that
(constant), SRT (day) is the sludge retention time. As shown different archaeal species were involved in mesophilic-
in Equation (4), m is constant since kd and SRT are constant and thermophilic-enriched cultures, but the dominant
in our study. Besides, Y is also constant. Therefore, it archaeal species were all belonged to the order
is expected that X was increased with the increase of gas Methanobacteriales. Biogas upgrading in a thermophilic
injection rate. VSS is usually assumed to represent biomass anaerobic reactor showed that CH4 content higher than 90%
cell concentration (Hwang and Hansen, 1998). Nevertheless, was obtained with the gas injection rate as high as 24 L/
in reality this is not always the case, because as was (L day). The study also revealed that gasliquid mass transfer
previously mentioned VSS does not only represent is the rate limiting factor for efcient H2 utilization.