Conventional mesophilic vs. thermophilic anaerobic digestion: A trade-off between performance and stability? Rodrigo A. Labatut*, Largus T. Angenent, Norman R. Scott Department of Biological and Environmental Engineering, Cornell University, Ithaca, NY 14853, USA article info Article history: Received 7 October 2013 Received in revised form 6 January 2014 Accepted 18 January 2014 Available online 28 January 2014 Keywords: Co-digestion Manure Temperature Loading rate Mixing intensity LCFA Adsorption abstract A long-term comparative study using continuously-stirred anaerobic digesters (CSADs) operated at mesophilic and thermophilic temperatures was conducted to evaluate the influence of the organic loading rate (OLR) and chemical composition on process perfor- mance and stability. Cow manure was co-digested with dog food, a model substrate to simulate a generic, multi-component food-like waste and to produce non-substrate spe- cific, composition-based results. Cow manure and dog food were mixed at a lower e and an upper co-digestion ratio to produce a low-fiber, high-strength substrate, and a more recalcitrant, lower-strength substrate, respectively. Three increasing OLRs were evaluated by decreasing the CSADs hydraulic retention time (HRT) from 20 to 10 days. At longer HRTs and lower manure-to-dog food ratio, the thermophilic CSAD was not stable and eventually failed as a result of long-chain fatty acid (LCFA) accumulation/degradation, which was triggered by the compounded effects of temperature on reaction rates, mixing intensity, and physical state of LCFAs. At shorter HRTs and upper manure-to-dog food ratio, the thermophilic CSAD marginally outperformed the biomethane production rates and sub- strate stabilization of the mesophilic CSAD. The increased fiber content relative to lipids at upper manure-to-dog food ratios improved the stability and performance of the thermo- philic process by decreasing the concentration of LCFAs in solution, likely adsorbed onto the manure fibers. Overall, results of this study show that stability of the thermophilic co-digestion process is highly dependent on the influent substrate composition, and particularly for this study, on the proportion of manure to lipids in the influent stream. In contrast, mesophilic co-digestion provided a more robust and stable process regardless of the influent composition, only with marginally lower biomethane production rates (i.e., 7%) for HRTs as short as 10 days (OLR ¼ 3 g VS/L-d). ª 2014 Elsevier Ltd. All rights reserved. 1. Introduction Thermophilic anaerobic digestion (55e60 C) has the potential to produce higher biomethane yields, and a more organically- stable, pathogen-free effluent compared to conventional mesophilic digestion (35e40 C). However, up until now most commercial-scale anaerobic digesters are operated at meso- philic temperatures. In addition to the higher energy input, poor stability and reliability of the thermophilic process are * Corresponding author. 225 Riley-Robb Hall, Cornell University, Ithaca, NY 14853, USA. Tel.: þ1 607 339 9429. E-mail address: [email protected](R.A. Labatut). Available online at www.sciencedirect.com ScienceDirect journal homepage: www.elsevier.com/locate/watres water research 53 (2014) 249 e258 0043-1354/$ e see front matter ª 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.watres.2014.01.035
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wat e r r e s e a r c h 5 3 ( 2 0 1 4 ) 2 4 9e2 5 8
Available online at w
ScienceDirect
journal homepage: www.elsevier .com/locate/watres
Conventional mesophilic vs. thermophilicanaerobic digestion: A trade-off betweenperformance and stability?
Rodrigo A. Labatut*, Largus T. Angenent, Norman R. Scott
Department of Biological and Environmental Engineering, Cornell University, Ithaca, NY 14853, USA
Table 4 e Concentrations of volatile fatty acids (VFA) andlong-chain fatty acids (LCFA) in the digestate of themesophilic and thermophilic CSAD at steady-stateconditions for each period; concentration is g COD/Lunless otherwise stated; VFA and LCFA correspond to thesum of individual fatty acids; no data obtained at P II forthermophilic CSAD due to process failure.
CSAD Mesophilic Thermophilic
Period I II III IV I II III IV
Acetate 0.04 0.03 0.01 0.02 0.62 F 0.06 0.07
Propionate 0.00 0.00 0.00 0.02 0.59 F 0.01 0.01
Isobutyrate 0.00 0.13 0.00 0.00 0.06 F 0.00 0.00
Butyrate 0.00 0.00 0.00 0.00 0.00 F 0.00 0.00
Isovalerate 0.00 0.38 0.00 0.00 0.10 F 0.00 0.00
Valerate 0.00 0.14 0.01 0.01 0.01 F 0.00 0.03
Isocaproate 0.00 0.06 0.02 0.04 0.00 F 0.02 0.05
Caproate 0.00 0.51 0.00 0.06 0.01 F 0.00 0.06
Heptanoate 0.00 0.00 0.00 0.11 0.00 F 0.00 0.11
Capric acid 0.29 0.00 0.00 0.00 0.04 F 0.00 0.00
Lauric acid 0.40 0.06 0.00 0.00 0.73 F 0.07 0.00
Myristeate 0.44 0.00 0.00 0.00 0.57 F 0.00 0.00
Palmitate 0.55 0.70 0.13 0.17 0.26 F 0.17 0.15
Stearate 0.37 0.31 0.14 0.16 0.12 F 0.14 0.14
Oleate 0.30 0.14 0.04 0.05 0.23 F 0.05 0.03
Linoleate 0.23 0.00 0.04 0.08 0.06 F 0.00 0.06
VFA 0.06 1.27 0.04 0.26 1.39 F 0.08 0.33
LCFA 2.57 1.23 0.35 0.46 2.00 F 0.43 0.38
Acetate/Propionate
(g/g)
16.7 7.3 e 1.3 1.0 F 11.1 4.9
Palmitate/LCFA (g/g) 0.21 0.57 0.37 0.37 0.13 F 0.39 0.40
g COD-LCFA/kg TS 243 87 16 22 159 F 20 19
g COD-LCFA/kg VS 301 105 19 27 189 F 24 23
F: digester failure.
wat e r r e s e a r c h 5 3 ( 2 0 1 4 ) 2 4 9e2 5 8 253
were 76% and 64% of the theoretical (Buswell) SMY of the
mesophilic and thermophilic CSADs, respectively (Table 5).
Consequently, VS destruction in the thermophilic CSAD was
significantly lower compared to the mesophilic CSAD during
Period I, which was also reflected in the higher total COD of
the thermophilic CSAD (see Table 3). The observed SMY of the
mesophilic digester was only slightly affected by shortening
the HRT from 20 days to 15 days (Period II), representing 72% of
the theoretical SMY. This is not surprising considering that a
small proportion of the influent substrate (i.e., 25%) consisted
of slowly-degradable, high-lignin cow manure.
Table 5 e CSAD performance parameters obtained at steady-s
CSAD Mesophilic
Period I II I
Biomethane production rate (L/L-d) 0.665 0.849 0.5
Biomethane content (% in biogas) 61.7% 61.6% 62
Specific methane yield, SMY (L/g VS added)
CSAD (This study) 444 424 25
BMP (Labatut, 2012) 503 503 31
Theoretical, Buswell 587 587 52
Volatile solids destruction (%) 71.5% 60.9% 39
F: CSAD Failure.
During Periods III and IV biomethane production rates
were 5% and 7%, respectively, higher in the thermophilic
digester (p < 0.01) (Table 5). A considerable decrease in the
biomethane production rate was observed in the mesophilic
CSAD when the co-digestion ratio was changed from 75% to
25% dog food at a constant OLR of 2 g/L-d (Period III) (Table 5).
The slightly higher VS destruction achieved by the thermo-
philic digester during Period III was not statistically significant
(p > 0.05). However, during Period IV, a significantly higher VS
destruction was attained by the thermophilic CSAD relative to
the mesophilic (p < 0.05), as the effects of faster reaction rates
(particularly hydrolysis) become more noticeable at higher
OLRs (shorter HRTs) under thermophilic temperatures.
Regardless of the operating temperature, VS destruction effi-
ciency decreased significantly in both CSADs (p < 0.01) as a
result of the increase of less degradable cow manure.
3.2. Stability of CSADs throughout the study
3.2.1. Ability of mesophilic and thermophilic digesters toreach steady-state conditionsThe mesophilic digester was highly stable and reached
steady-state conditions in each of the four study periods
(Table 6), and biomethane production rates increased with
each increase in OLR, regardless of the co-digestion ratio
(Fig. 1). The thermophilic digester, however, experienced two
major upsets (Fig. 1), which resulted in underperformance
during Period I and precluded it from reaching steady-state
conditions in Period II (Table 6). Key to both upsets were the
influence of temperature on physical properties and the
chemical composition of the influent, specifically the high
lipid content and lower fiber-to-lipid ratio of the co-digestion
mixture during Periods I and II (Table 6). As discussed below,
during both upsets biogas production essentially stopped as a
result of high VFA concentrations and resulting low pH levels
(Fig. 1).
3.2.2. Acetate accumulation and free-energy limitations forpropionate oxidation at thermophilic temperaturesAcetate and propionate accounted for most of the VFA accu-
mulated at the time of the thermophilic CSAD first upset on
day 210 (Fig. 2). Even-carbon LCFAs are primarily degraded by
H2-producing bacteria to acetate and H2 via b-oxidation (Jeris
and McCarty, 1965; McInerney, 1988), while odd-carbon
LCFAs are mainly degraded to acetate and propionate
thermophilic (solid line) temperatures; the limits where all
the reactions are possible at mesophilic and thermophilic
temperatures are enclosed in the lighter and darker boxes,
respectively. The plot was built assuming 1 mM for all fatty
acids, 100 mM for bicarbonate, 0.7 atm for methane, and
0.3 atm for carbon dioxide.
wat e r r e s e a r c h 5 3 ( 2 0 1 4 ) 2 4 9e2 5 8 255
the solid matrix to determine the biomass-associated LCFAs
(adsorbed), whereas in the present study, LCFAs were
extracted from the bulk, i.e., liquid and solid phases, thereby
TS includes all the solid matter in the digestate.
3.2.3. Higher transient accumulation of LCFAs atthermophilic temperaturesWe characterized the thermophilic CSAD with faster lipid
hydrolysis rates through increased LCFA accumulation.
Although both CSADs showed accumulation of LCFAs over
time, the concentration of LCFAs in the thermophilic digester
was 40% higher before the mixing perturbation compared to
itsmesophilic counterpart, i.e., 4.5 vs. 2.8 g COD/L (355 vs. 261 g
COD-LCFA/kg TS), respectively (Fig. 2). This is not surprising
considering that overall digestion rates can be up to 2.25 times
faster at thermophilic temperatures relative to mesophilic
(O’Rourke, 1968). In lipid-containing substrates, degradation
of LCFAs via b-oxidation can be the slowest conversion step
and control the overall kinetics of the digestion process
(Novak and Carlson, 1970; O’Rourke, 1968; Pavlostathis and
Giraldo-Gomez, 1991; Rinzema et al., 1994). Thus, differences
between the rates of hydrolysis of lipids and b-oxidation of
long chain fatty acids can result in a reactant-product imbal-
ance and accumulation of LCFAs over time, resulting in inhi-
bition. Hanaki et al. (1981) reported that LCFA accumulation
could also be accentuated at concentrations greater than 1.4 g
COD/L, because at this level LCFAs were inhibitory for the b-
oxidizing organisms themselves. Furthermore, at a concen-
tration of 2.9 g COD/L the authors observed a lag phase of
nearly 10 days for LCFA degradation, which consequently
extended that ofmethanogenesis to over 20 days. On the other
hand, Angelidaki andAhring (1992), found that concentrations
as low as 0.6 g COD/L of unsaturated oleic acid (C18:1) and
1.5 g COD/L of saturated stearic acid (C:18:0) increased the lag
phase of methanogenesis in batch tests conducted at 55 �C.Koster and Cramer (1987) observed a sharp decrease in the
methanogenic activity at concentrations over 1.3e1.4 g COD/L
for capric (C10:0), myristic, (C14:0) and oleic acids, and of over
0.5 g COD/L for lauric acid (C12:0). Based on above reports and
the work of Neves et al. (2009a), both CSADs in this study were
within the inhibitory range during the period before the
perturbation; however, no apparent decreased biomethane
production rates or differences between the mesophilic and
thermophilic CSADs before the perturbation were observed
(see Figs. 1 and 2). Instead, b-oxidizers seemed to have been
more affected, and particularly at thermophilic temperatures,
as suggested by the greater accumulation of LCFAs observed
in the thermophilic CSAD and higher concentration of acetate
observed in the mesophilic.
3.2.4. The influence of temperature on physical properties andtheir impact on process stabilityIt was apparent that the first upset of the thermophilic CSAD
was triggered by an increase in the mixing intensity per-
formed during days 148e149 of the study (Fig. 1). Although
both CSADs observed an increase in LCFA degradation after
the mixing perturbation, the extent of degradation was more
than two times higher in the thermophilic CSAD compared to
the mesophilic (Fig. 2). It is evident that, such LCFA degrada-
tion was the cause of the sudden and sustained increase in
VFAs observed after the mixing perturbation in the thermo-
philic CSAD (Fig. 1). Mixing intensities were unintentionally
increased in both CSADs from 100 to 1500 RPM in repeated
occasions during days 148e149 (Figs. 1 and 2). It is known that
high mixing intensities can disrupt the syntrophic associa-
tions between H2-producing bacteria and H2-utilizing metha-
nogens and have a detrimental effect on digester stability
(Speece et al., 2006; Hansen et al., 1999; McMahon et al., 2001;
Hoffmann et al., 2008; Vavilin and Angelidaki, 2005; Stroot
et al., 2001). Schmidt and Ahring (1993) observed that after
physically disintegrating granules of a propionate- and n-
butyrate-fed UASB, the PH2 increased to over 10�4 atm,
causing propionate and butyrate degradation rates to
decrease 30 and 20%, respectively. They concluded that, by
disintegrating the granules, syntrophic relationships were
disrupted by the increased distance between H2-producing
and H2-utilizing organisms, which in turn increased the H2
mass transfer resistance. We believe that in this study, high
mixing intensities produced a similar effect for the thermo-
philic digester, affecting H2 transport, increasing PH2, and
halting propionate oxidation.
The fact that the thermophilic CSAD observed higher LCFA
degradation rates compared to the mesophilic can be
explained by the effect of temperature on two main physical
properties, which in turn exacerbated the effect of the
increased mixing intensity. First, the lower water viscosity of
the thermophilic CSAD due to the 18 �C difference with its
mesophilic counterpart resulted in a mixing intensity that
(described by the Reynolds number) was 36% higher in the
former. This translated into a mixing rate that was actually
closer to 2000 RPM during the perturbation, rather than
1500 RPM (See Supporting Information). With high concen-
trations of LCFAs in the digestate, the naturally-occurring lipid
emulsions could have been homogenized by the repeated
wat e r r e s e a r c h 5 3 ( 2 0 1 4 ) 2 4 9e2 5 8256
vigorous mixing. The increased LCFA-to-water interface area
resulting from the dispersed, smaller-diameter emulsions
would favor the contact between substrate and fatty-acid
oxidizing bacteria, and increase degradation rates. Second,
because fatty acids have different melting points depending
on their degree of saturation and chain length, their physical
state changes with temperature. At thermophilic tempera-
tures, LCFAs were in a more liquid form e thereby, a higher
extent of emulsification should have been achieved through
mixing, especially at 36% higher mixing rate, which could
have further increased LCFA availability for b-oxidizers and
therefore their rate of degradation. For example, palmitic acid
(C16), which constituted 42% of the total LCFA pool of the
thermophilic digester before the mixing perturbation, has a
melting point of 62.9 �C, suggesting that palmitic acid should
have been in a practically liquid state in the thermophilic
digester, whereas closer to a solid state in the mesophilic
digester (See Supporting Information).
3.2.5. Fiber-to-lipid ratio, rather than lipid loading rate e themain control of stability in the thermophilic CSADThe second upset of the thermophilic digester occurred after
the overall OLR was increased from 1.5 to 2 g VS/L-
d (0.36e0.49 g lipid/L-d) (Table 5). LCFAs accumulated to high
levels within one HRT cycle in the thermophilic digester
(Fig. 2), and acetate and propionate increased as the pH
concurrently dropped, bringing biogas production to a halt
(Fig. 1). To recover the digester, only cow manure was fed on
day 352 and for the following three feeding cycles; dog food
was re-introduced on day 360 at a decreased portion of 25%,
which forced the premature start of Period III for the ther-
mophilic CSAD (see Fig. 1). Within 24 h of manure-only
feeding (day 354), the concentration of LCFAs decreased by
50% (Fig. 2). A similar behavior was observed by Hanaki et al.
(1981), who reported accumulation of LCFAs onto the solid
matrix (and decrease from the aqueous phase) within a day of
exposure to neutral fats. Indeed, the fact that acetate or biogas
production did not increase during the 24-h period, also sug-
gests that LCFAs were adsorbed on the manure matrix.
Particularly, the non-polar, hydrophobic surfaces of the
manure fibers have been reported to effectively reduce LCFAs
from solution, which in turn reduce their bioavailability and
therefore inhibitory effects (Nielsen and Ahring, 2006). It was
apparent that for Periods I and II of this study, the ratio of
manure-to-lipid-containing substrate (i.e., dog food) was
insufficient for the amount of hydrolyzed lipids in the ther-
mophilic CSAD (Table 5). This was evidenced by the accu-
mulation of LCFAs in the liquid phase, which produced
inhibition, instability, and ultimately digester failure. An
excess of adsorbent material (e.g., manure fibers) should
decrease the bioavailability of free (non-adsorbed) LCFAs,
which in turn would reduce possible inhibition and increase
the stability of the process. If not enough adsorbentmaterial is
available for the amount of adsorbate (i.e., LCFAs), the adsor-
bent will saturate and LCFAs will accumulate in the liquid
phase. Indeed, even though the lipid loading rates of Periods I
(unstable) and IV (stable) were comparable, no accumulation
of LCFAs was observed in Period IV e i.e., the higher fiber-to-
lipid ratio provided enough adsorbent surface area to main-
tain the thermophilic reactor exceptionally stable compared
to Periods I and II (Table 5). Therefore, it is evident that the
amount of LCFAs that can be absorbed onto the manure fibers
will primarily depend on the proportion betweenmanure and
the lipid-rich material, rather than the lipids loading rate.
Furthermore, it is apparent that relatively higher manure-to-
lipid ratios should be required at thermophilic temperatures
as compared to mesophilic temperatures due to the increased
hydrolysis and b-oxidation rates at thermophilic tempera-
tures. The increase of the manure-to-lipid ratio is not the only
factor improving the resilience of the thermophilic digester in
periods III and IV; the continuous exposure of the digester to
fats from the beginning of the study could also have modified
the microbial community structure to be more efficient at
degrading fats, particularly LCFAs.
4. Conclusions
Results of this study confirm that digester temperature has an
important role on determining process performance and sta-
bility by directly influencing key physical properties of the
system and the substrate. Specifically, increased hydrolysis
rates of thermophilic digesters can produce significant accu-
mulation of LCFAs over time when lipid-rich wastes are co-
digested with inadequate amounts of manure. Rather than
the lipid loading rate, it is the fiber-to-lipid ratio that appears to
control the stability of the process. Manure fibers provide the
surface area for LCFAs to adsorb and decrease their concen-
tration in solution, thereby providing stability to the thermo-
philic process. In addition, mixing intensities of CSADs are
significantly higher at thermophilic temperatures; hence,
excessive mixing should be avoided not only to minimize
operational costs, but also because it can increase the extent of
homogenization of lipid emulsions, and thus the rate of hy-
drolysis. The process can be further exacerbated by the fact
that LCFAs are in a more liquid state at thermophilic temper-
atures, and the bioenergetics for b-oxidation are more favor-
able, making thermophilic digesters more prone to inhibition
and instability due to molecular hydrogen accumulation and
consequential shock loads of acetate and propionate.
Overall, provided that lipid-containing wastes are co-
digested with adequate amounts of manure, thermophilic
wat e r r e s e a r c h 5 3 ( 2 0 1 4 ) 2 4 9e2 5 8 257
for partial financial support in completing this study. Kristen
Vitro, an undergraduate student at Cornell University, is
recognized for her continued assistance and thorough labo-
ratory analyses in conjunction with this study.
Appendix A. Supplementary data
Supplementary data related to this article can be found at
http://dx.doi.org/10.1016/j.watres.2014.01.035.
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