2017,
the straw output of China was estimated to be about 884
million tons, of which 736 million tons were collectible, and nearly 30% of it
was contributed by corn stalk (National Bureau of Statistics of China 2014). As an important renewable biomass resource, the total
utilization rate of corn stalks was less than 40%, and most of them were burned
without recycling, causing environment pollution and waste of resources (Wang et al. 2014). In view of the
environmental issue and increasing energy crisis, more attention has been paid
to anaerobic digestion of cellulosic materials such as straws and the
production of biogas. Ensiling of corn stalks is a pretreatment method and preservation
technology. The deterioration in quality and effective components of corn
stalks during storage can be inhibited by ensiling, which also prolongs the
storage of biogas raw materials thus, favoring continuous biogas production (Hansen et al. 2004; Vervaeren et al. 2010).
Because corn stalk is mainly composed of lignin, cellulose, and hemicellulose,
the cross-linking and entangling of these components limits the degradation and
utilization abilities of hydrolytic microorganisms. Therefore, the anaerobic
digestion of corn stalks treated with different ensiling methods shows issues
including poor stability, long start-up time and extended digestion period
(Wang et al. 2014). Therefore, corn
stalks should be pretreated reasonably before anaerobic digestion and biogas
production. To date, more common effective pretreatment methods involve
physical, chemical and biological techniques; among which, ammoniation is an
effective chemical approach (Ma et al.
2011). During the ammoniation pretreatment, breakdown of fibrous structures and
formation of ammonium salts occurs simultaneously. The waste liquid after the
pretreatment can serve as a nitrogen fertilizer, avoiding secondary pollution,
which usually occurs during chemical pretreatment processes (Luo et al. 2015) ammoniated rice straws with
urea solutions. After this pretreatment, they reported an increase by
20.67–38.20% in the cumulative biogas output compared with the control group.
The output reached a maximum under the urea content of 4 wt%. Likewise, Kim
(Kim and Lee 2006) pretreated straws with an ammonia solution, and reported
about 62% lignin removal rate at 60ºC, 5 wt% ammonia, a solid-liquid ratio of
1:6, and after 12 h treatment, it could significantly increase methane
production.
The ammoniation pretreatment of raw corn stalks has been
widely studied, although the focus was mainly on the palatability and nutrition
for livestock. Few studies have also evaluated the effects of this pretreatment
on the anaerobic digestion and biogas producing characteristics of
ensiling-treated corn stalks. In this research, corn stalks, pretreated with
three different ensiling approaches, were ammoniated with a 20 wt% ammonia
solution. The start-up period, anaerobic digestion period, biogas production efficiency
and volatile fatty acids (VFAs) content during the anaerobic digestion
processes, with or without the ammoniation pretreatment, were analyzed. Based
on the comparative results, the feasibility of ammoniation pretreatment for
anaerobic digestion of ensiling-treated corn stalks was discussed for the
improvement of efficiency and continuity of biogas production.
Experimental apparatus
A controllable thermostatic fermentation apparatus was
used in the experiment, as illustrated in Fig. 1. The apparatus mainly
consisted of a heating water tank, a temperature controller, a cylindrical 304
stainless steel fermenter with an effective volume of
Experimental methods
The anaerobic digestion experiment was carried out in a
Analysis approaches
The TS and VS values were determined via the drying methods
(TS was heated at 105ºC for 24 h and VS was heated at 550ºC for 4 h) as
previously reported (Zhao 2006). The pH of slurry, biogas composition, and
output were measured every day, and the chemical oxygen demand (COD), VFAs and
ammonia nitrogen (NH4+-N) were measured at 5-day
intervals. The biogas composition was determined with a Biogas Check device
with methane and carbon dioxide measurement at ± 3.0% accuracy. The pH was
determined with a PHS-3C-type portable pH meter (Oleron Company). The COD and NH4+-N
were measured with a 5B-3C (V8) instrument (Lianhua Technologies Company)
(Jiang 2001). The VFAs were determined by spectrophotometry (Wang et al. 2008).
Results
Effect of ammoniation pretreatment on the anaerobic
digestion and biogas production of ensiled corn stalks
The biogas output data in
the anaerobic digestion of ensiled corn stalks with and without ammoniation
pretreatment are presented in Fig. 2. The start-up stages of three different
ensiled corn stalks after ammoniation were remarkably accelerated compared to
the control group, and no delay was observed. In other words, the ammoniation
pretreatment can effectively accelerate the start-up processes in the anaerobic
digestion of ensiled corn stalks. Throughout the reaction time, the biogas
output per day increased largely and reached a maximum on days 5 to 9.In
detail, the output of the ammoniated groups CB, ME and LB reached a maximum of
11.02 L·d-1 on day 5, 9.77 L·d-1 on day 7, and 9.03 L·d-1
on day 9, respectively. In contrast, the biogas output of the un-ammoniated
ensiled corn stalks reached a maximum on day 20 to 23. In
detail, the output of the un-ammoniated groups of LB, ME and that of CB reached
a maximum of 12.19 L·d-1 on day 20, 11.59 L·d-1 on day
20, 8.21 L·d-1 on day 23, respectively. These results thus show
that the ammoniation pretreatment could effectively shorten the anaerobic
digestion time of ensiled corn stalks by about 15 days. This may be because
ammoniation can effectively destroy the crystalline structures of lignin and
cellulose in ensiled corn stalks, accelerate the start-up of anaerobic
digestion, thus shortening the anaerobic digestion period.
Fig. 1:
Schematic illustration of the controllable thermostatic fermentation apparatus
1.Temperature
controller 2. Thermal insulation 3. Fermenter① 4. Fermenter②5.
Fermenter③ 6. Fermenter④ 7. Data acquisition instrument 8. Computer 9. Biogas
analyzer10. Water tank 11. Biogas tank 12. Inner water tank 13. Solenoid valve
14. Hot water pump 15. Thermostatic water
tank16. Heating wire 17. Temperature sensor
Table 1: Properties
of feedstock for fermentation
Feedstock |
TS %) |
VS (%) |
pH before the treatment |
pH after the treatment |
AH-CB |
22.56 |
20.13 |
2.4 |
8.4 |
AH-ME |
28.75 |
26.30 |
3.8 |
8.3 |
AH-LB |
31.10 |
28.47 |
3.1 |
8.3 |
Cow Manure |
20.82 |
14.47 |
- |
7.3 |
Inocula |
9.01 |
5.60 |
- |
7.6 |
AH: Ammonium hydroxide TS: total
solid VS: volatile
solid
Table 2: Composition
of each group
Group |
Straw (g) |
Cow manure (g) |
Inocula (g) |
Water (g) |
AH-CB |
455.06 |
1091.95 |
1200.00 |
1252.99 |
AH-LB |
449.03 |
1091.95 |
1200.00 |
1259.01 |
AH-ME |
510.19 |
1091.95 |
1200.00 |
1260.60 |
CK |
0.00 |
0.00 |
1200.00 |
2800.00 |
Fig. 2: Effect of ammoniation pretreatment on the biogas output
of different ensiling-treated corn stalks
The cumulative biogas output in those anaerobic digestion
processes of ensiled corn stalks, with and without ammoniation pretreatment, is
presented in Fig. 3. In the start-up stages, the three kinds of ensiled corn
stalks pretreated by ammonia showed a much higher output compared to the control
group, and exhibited different biogas production characteristics. The pretreatment with
ammonia could increase the hydrophilicity of straw surface, widen the cellular
surface voids, broaden the area for microbial attachment, perturb the
crystalline structure of lignin to release encapsulated cellulose, and improve
the contact between hydrolytic acid producing bacteria and corn stalks (Zhou et al. 2012). Through the course of
anaerobic digestion, methanogens are enriched continuously, and the cumulative
biogas output of ensiled corn stalks increases (Li et al. 2009). However, we observed that due to the ammoniation
pretreatment, the biogas output of ensiled corn stalks reduced by 32.54–48.27%
compared to the control group in the presence of the same amount of feedstock.
Among these three ammoniated ensiled groups, the biogas output of the ME corn
stalks (with no additive) was the highest, at 108.97 L. On the other hand, the
un-ammoniated ensiled corn stalks showed an extended start-up period but a
higher cumulative biogas output. Over the reaction time, the cumulative biogas
output of different ensiled corn stalks increased, and the un-ammoniated CB
group presented the highest value of 167.95 L, followed by161.55 L for the ME
group and 156.82 L for the LB group. Thus, compared with the control group, the
ammoniation pretreatment significantly shortened the anaerobic digestion period
of corn stalks that were treated with different ensiling methods, but because
this pretreatment reduced the concentrations of VFAs in the slurry, the biogas
output of ensiling-treated corn stalks declined to a certain extent.
Effects of pretreatment with ammonia on the pH and VFAS
in the anaerobic digestion of corn stalks at moderate temperatures
pH is an important index
and parameter in the monitoring anaerobic digestion (Latif et al. 2017). The changes in pH can directly reflect the acid-base
environment of an anaerobic digestion system and the state of various
biochemical reactions of microorganisms in the reactor. The pH changes in the
anaerobic digestion of different ensiling-treated corn stalks with and without
pretreatment are mentioned in Fig. 4. In the start-up stages, all the systems
were naturally acidified and the lowest pH of 5.6 was observed in the un-ammoniated LB group on day 3.
During the start-up stages of the anaerobic digestion, the microorganisms in
the reactor gradually adapted to the environment. Cellulose and hemicellulose
were gradually hydrolyzed into cellobiose and glucose, in the presence of
hydrolytic bacteria. Acid producing bacteria rapidly converted these small
organic molecules into VFAs. The production of VFAs was faster than their
utilization by methanogens and thus, the C/N ratios in the slurry increased,
resulting in a lack of nitrogen and insufficient nutrients for methanogens in
the start-up stages. As a result, the VFAs were accumulated and pH decreased in
these anaerobic digestion systems, leading to the acidification phenomena (Zhen
et al. 2015). Along with the
reaction, the methanogens in the slurry of anaerobic digestion systems utilized
un-ammoniated ensiled corn stalks, gradually adapted to the environment, and
the pH steadily elevated to a normal level at around 7.5. As shown in Fig. 4,
the pH of systems containing ammoniated ensiled corn stalks decreased slightly
in their start-up stages. These results indicate that ammoniation pretreatment
can suppress the pH change in the anaerobic digestion of ensiled corn stalks,
significantly improving the stability of the anaerobic digestion systems.
Fig. 3: Effect of ammoniation pretreatment on the cumulative
biogas output ofdifferent ensiling-treated corn stalks
Fig. 4: Effect of ammoniation pretreatment on the pH in the
anaerobic digestion of different ensiling-treated corn stalks
Fig. 5: Effect of ammoniation pretreatment on the
concentrations of VFAs in the anaerobic digestion of different ensiling-treated
corn stalks
VFAs are important intermediates in anaerobic digestion,
and they can be directly converted by methanogens (Feng et al. 2018). The concentrations of VFAs in the anaerobic digestion
systems with and without pretreatment are mentioned in Fig. 5. The
concentrations of VFAs in the ammoniated digestion systems were significantly
lower than that of the control group during the entire anaerobic digestion, and
the lowest concentration was 5.1 mg·L-1. The concentrations of VFAs
were low from beginning to end because the residual ammoniation straws neutralized a part of VFAs in the digestion
slurry, and the high concentrations of ammonia nitrogen in the slurry in the
initial stages suppressed the formation of small-molecule VFAs by
acid-producing bacteria (Elmashad et al.
2004). In the initial stages of anaerobic digestion of ammoniated corn stalks,
the concentrations of VFAs decreased slightly and finally reached a stable
level. A similar trend was observed in the change of VFAs concentrations in the
un-ammoniated corn stalks. In the early stages of anaerobic digestion, along
with the reaction, some organic macromolecules were gradually converted into
VFAs, while other smaller organic molecules did so in the presence of
hydrolytic acid producing bacteria (Gan et
al. 2017). The methanogens did not completely adapt to the environment in
the early stages, thus, the utilization rates of VFAs were lower than their
rates of production rates acid-producing bacteria, leading to the increase in
VFAs in the slurry. On day 15, the concentration of VFAs in the digestion
slurry of the un-ammoniated ME group reached a maximum
of 13.19 g·L-1. The biogas output of different un-ammoniated
ensiling-treated corn stalks reached the maximum after day 15. Throughout the
reaction time, the VFAs concentrations decreased gradually and finally reached a stable level in the digestion
slurry of different un-ammoniated ensiled corn stalks.
Effect of pretreatment with ammonia on the COD concentration
(ammonia nitrogen) in the anaerobic digestion of corn stalks at moderate
temperatures
Fig. 6 shows the ammonia
nitrogen concentrations of ammoniated and un-ammoniated ensiled corn stalks. In
the initial stages of the anaerobic digestion, the ammoniated corn stalks
showed a very high concentration of ammonia nitrogen, which may correlate with
the residual dilute ammoniain the straws after the ammoniation pretreatment.
Compared with the control group, the ammoniation pretreatment reduced the
biogas output of ensiled corn stalks to a certain extent. This may be because
the VFAs concentrations were low in the early stages of the anaerobic digestion, and excess ammonia nitrogen consumes the
hydrogen ions produced in the acidification processes, thus affecting the
anaerobic digestion performance and reducing the methane output (Xu et al. 2012). Along with the reaction,
the concentrations of ammonia nitrogen in the digestion slurry of ammoniated,
ensiled corn stalks decreased gradually. With the release of nitrogen in
straws, the concentrations of nitrogen in the digestion slurry was balanced
dynamically with the amount of nitrogen required for the reproduction and
metabolism of anaerobic microbial flora. Finally, the concentrations of ammonia
nitrogen in the ammoniated groups reached a stable level of 700–1200 mg·L-1,
and that of the digestion slurry of un-ammoniated
groups increased gradually. On day 15, the concentration of ammonia nitrogen in the ME group digestion slurry reached a maximum of 2802 mg·L-1, and then decreased slightly. Eventually,
the concentrations of ammonia nitrogen in the digestion slurry remained constant.
Fig. 6: Effect of ammoniation pretreatment on theammonia
nitrogen concentrations in the anaerobic digestion of different
ensiling-treated corn stalks
Fig. 7: Effect of ammoniation pretreatment on the COD
concentrations in the anaerobic digestion of different ensiling-treated corn
stalks
Fig. 7 shows the COD concentrations of ammoniated and
un-ammoniated ensiling-treated corn stalks. The trends of variation in COD
concentrations of the ammoniated and un-ammoniated three different ensiled corn
stalks were similar, in accordance with a volcanic trend. In the early stages
of the fermentation, the cellulose, hemicellulose, and other macromolecular
organics in corn stalks were gradually decomposed into water-soluble, smaller organic
molecules. The rates of hydrolysis of macromolecular organics were higher
compared to their microbial consumption, which led to the gradual increase of
COD concentrations in the digestion slurry. The reaction was accompanied by
gradual enrichment and adaptation of methanogens to the environment, consuming
a large amount of small water-soluble organic molecules. This led to decrease
in concentrations of COD, in accordance with the fast increase of biogas
output. The high COD concentrations in the initial stages of the fermentation
of ammoniated corn stalks indicates that the ammoniated ensiled corn stalks can
function as the feedstock for anaerobic digestion, and the maximum values were
achieved in the first 5 days, proving that this feedstock is easy to decompose.
Moreover, these values achieved their maximum at 60% earlier compared to the
untreated materials, demonstrating that the pretreatment approach is
appropriate for large-scale applications.
Effect of ammonia pretreatment on the methane concentration
in the anaerobic digestion of corn stalks at moderate temperatures
The effect of ammoniation
pretreatment on the methane concentration of ensiling-treated corn stalks is
illustrated in Fig. 8. The substances in the fermentation flask had produced a
large amount of H2 and CO2 in the first place, thus, the
methane contents in the biogas were generally low at the initial stages of the
anaerobic digestion. The start-up stages of different ammoniated ensiled corn
stalks were obviously faster than those of the control group. The ammoniation
pretreatment degraded the lignin on the surface of corn stalks, and hence the
hydrolytic acid producing bacteria could quickly contact the cellulose and
hemicellulose inside the straws. Thus, the hydrolysis stages were no longer the
rate-determining steps of the anaerobic digestion courses of corn stalks (Yuan et al. 2015). Along with the reaction,
the number of methanogens increased gradually. These methanogens converted VFAs
into methane, and led to increase in methane concentrations. The maximum
methane concentrations of 62.71, 62.59 and 60.6%, respectively, occurred at day
5–8. In the final stages of the reaction, the contents of available organic
matter in the digestion slurry decreased gradually, leading to decrease in
methane concentrations to 45–50%. The start-up stages of un-ammoniated ensiled
corn stalks were slow, and a delay was observed in the start-up stages. Over
the reaction time, gradual hydrolysis of organic macromolecules such as
cellulose occurred, and a steady increase was observed in the concentrations of
VFAs in the digestion slurry, in the number of methanogens, and in the methane
concentrations that reached the maximum values of 71.10, 71.08 and 67.2%,
respectively, on day 19. On comparing, it was found that the ammoniation
pretreatment accelerated the start-up stages of the anaerobic digestion and
shortened the anaerobic digestion periods, but the maximum methane
concentrations decreased by about 10%.
Discussion
Fig. 8: Effect of ammoniation pretreatment on the methane
concentrations in the anaerobic digestion of different ensiling-treated corn
stalks
In this experiment, the effect of ammoniation
pretreatment on the methanogenic performance in anaerobic digestion of corn
stalks with different ensiling methods was studied. The effect of ammoniation
pretreatment was reflected by the cumulative gas production and other
parameters change in anaerobic digestion of corn stalks with different ensiling
methods before and after pretreatment. It can be seen from the trend of daily
biogas production in Fig. 2. The ammoniation pretreatment can significantly
destroy the fiber component of corn stalks, and promote the separation of
cellulose and hemicellulose from lignin, which is beneficial to the utilization
of easily fermentable substances by anaerobic fermentation microorganisms, and
improve the start-up speed of anaerobic digestion of corn straw with different
silage methods. Kim and Lee (2006) used ammonia soak to pretreat corn straw and found that at 60°C, w=15%
ammonia water, the solid-liquid ratio of 1:6 soaked straw for
12 h, lignin can reach 62% removal rate, which is beneficial to improve the
contact of anaerobic digestion microorganisms on corn stalks. It can be
concluded from Fig. 3 that the ammoniation pretreatment can slightly reduce the
cumulative yield of anaerobic fermentation biogas in different silage corn
stalks to a certain extent, but it can significantly improve the anaerobic
fermentation efficiency. During the 0–15 d, and the cumulative gas production
of ME, CB and LB three kinds of silage corn stalks accounted for 78.48, 74.11
and 70.35% of the cumulative gas production, respectively. It can be seen from
Fig. 4 and 5 that the pH undergone a process of first falling and then rising.
When the pH is low, the microorganisms need more energy to maintain the neutral
environment of their own cytoplasm, thus inhibiting the activity of
microorganisms and reducing the biogas production of anaerobic digestion of
corn stover with different silage methods. Ammoniation pretreatment
significantly reduced the concentration of organic acids in the digestion
broth, which may be the main reason for the reduction of biogas accumulation in
anaerobic digestion of different silage corn stalks. Ammonia pretreatment can
improve the stability of anaerobic digestion process, keep the pH between 7.2–7.5,
which is beneficial to increase the activity of anaerobic microorganisms,
increase the hydrolysis rate of anaerobic microorganisms and the rate of methanogenesis,
and shorten the anaerobic Oxygen digestion cycle. It can be seen from the trend
of COD in the anaerobic fermentation liquid of Fig. 7. The ammoniation
pretreatment can break the ester bond between lignin and cellulose and
hemicellulose, and it can damage the surface of siliceous cells and increase
the release rate of organic matter in corn straw with silage methods.
At present, domestic and international methods for
pretreatment of corn straw anaerobic fermentation can be divided into physical
methods, chemical methods and biological methods. Ammonia pretreatment as an
effective way to treat corn stover can destroy the fiber component of corn
stover to some extent, and cause some functional groups to break, thus
separating lignin from cellulose and hemicellulose, which is beneficial to
improve the actual biogas engineering. It is beneficial to improve the
utilization of digestion substrate by anaerobic digestion microorganisms in the
actual biogas engineering, improve the efficiency of anaerobic fermentation,
and thus improve economic benefits.
Conclusion
The anaerobic digestion
start-up stages of these ensiled corn stalks were accelerated and their
anaerobic digestion periods were significantly shortened. Compared to the
control group, the anaerobic digestion periods were shortened by 15 days. The
biogas output per day and cumulative biogas output of the ammoniated with 20
wt% CB group were the highest, was 11.02 L·d-1 and 167.95 L,
respectively. The pH of these anaerobic digestion systems was stabilized at
around 7.5. The ammoniation pretreatment significantly improved the stability
of pH in the anaerobic digestion of ensiled corn stalks. The ammoniation
pretreatment effectively improved the methane production of different ensiled
corn stalks in the early stages of anaerobic digestion processes. Furthermore, the methane concentrations can rapidly
increase to 50% in the early stages of anaerobic digestion.
Acknowledgments
This work was funded by
the National Natural Science Foundation of China (51706128,51509122), and Gansu
Provincial Higher Education Science and Technology Achievements Transformation
Project (2018D-04), Gansu Natural Science Foundation (18JR3RA154), 2018
Yangling Demonstration Zone Collaborative Innovation Major Project
(2018CXY-14), Scientific Research Program Funded by Shaanxi Provincial Education
Department (17JS018).
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