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Energy Procedia 105 (2017) 895 900

The 8th International Conference on Applied Energy ICAE2016

Microbial community ability to adapt to altered temperature

conditions influences operating stability in anaerobic
digestion
Maria Westerholma*, Simon Isakssona, Li Suna, Anna Schnrera
a
Department of Microbiology, Swedish University of Agricultural Sciences, Uppsala BioCenter, SE-750 07 Uppsala, Sweden

Abstract

This study examined the influence of altered operating temperature on microbial community composition and how
this affects the ability of anaerobic degradation (AD) processes to cope with increasing loading rate. Four digesters
fed household and slaughterhouse waste, mimicking two large-scale processes, were operated in sets of two at 37C
or 52C, followed by a gradual increase or decrease in temperature in one digester in each set. All digesters were then
subjected to step-wise increases in organic loading rate (OLR) from 3 to 7 g VS/L/day, concurrently with decreased
hydraulic retention time (HRT). Temporal microbial changes were monitored by Illumina MiSeq analysis of bacterial
and archaeal 16S rRNA gene sequences. The digester transformed from thermophilic to mesophilic conditions failed
at 6 g VS/L/day, whereas the reference digesters and the mesophilic-to-thermophilic digester remained stable at all
OLR investigated. The bacterial community was characterised by relatively diversified structure dominated by
Bacteriodetes and Firmicutes in mesophilic conditions. Increasing temperature caused loss of complexity at phyla
level and enhanced the relative abundance of Firmicutes and Thermotogae. Temperature adaptation in the
thermophilic-to-mesophilic digester resulted in a bacterial community structure reflecting the communities observed
in digesters with initial temperature of 37C. However, certain populations found in the mesophilic digesters did not
appear at detectable levels during operation at 37C in the thermophilic-to-mesophilic digester.
Overall, the results showed that anaerobic process stability was highly dependent on the innate resilience of the
microbial community. The mesophilic microbiota formed from a thermophilic community had a slightly different
structure than its mesophilic counterpart and was considerably less resistant to increased OLR in digesters examined
within this study.

2017 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license
2016 The Authors. Published by Elsevier Ltd.
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
Selection and/or peer-review under responsibility of ICAE
Peer-review under responsibility of the scientific committee of the 8th International Conference on Applied Energy.

Keywords: Biogas; mesophilic; thermophilic; organic loading rate; microbial communtiy dynamics; Illumina sequencing

*Corresponding author. Tel.: +46-18-671000; fax: +46-18-672000.

E-mail address: Maria.Westerholm@slu.se

1876-6102 2017 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
Peer-review under responsibility of the scientific committee of the 8th International Conference on Applied Energy.
doi:10.1016/j.egypro.2017.03.408
896 Maria Westerholm et al. / Energy Procedia 105 (2017) 895 900

1. Introduction

According to commercial biogas producers, a major target in applied research must be increased
energy recovery through higher methane yields per unit organic matter for a particulate substrate.
Improved robustness to stress factors, such as altered operating conditions and inhibitory compounds, is
another valuable target for biogas producers, since it reduces the risk of process failure. Understanding
factors governing microbial ecology, in terms of community dynamics and diversity, provides
opportunities to improve the performance of anaerobic degradation (AD) systems [1]. The anaerobic
degradation of organic matter proceeds through a series of interlinked microbial processes (hydrolysis,
fermentation, acetogenesis, methanogenesis) and the stability and productivity of the process are strongly
dependent on balances between parallel metabolic pathways [2]. Among different process parameters,
temperature has been identified as a key factor influencing microbial community structure [3-6] and
hence the performance of biogas digesters. Compared with mesophilic AD (30-40C), thermophilic
processes (50-70C) are considered more effective [7] due to increased microbial activity and thus
enhanced biogas production. However, thermophilic digesters are more susceptible to inhibition and
sudden environmental changes [6]. A possible cause of this higher sensitivity to perturbation is
comparatively lower microbial diversity at higher temperatures [3,8] or restricted resilience of the
microbial community (i.e. its ability to adapt and remain active during altered operating conditions). In
AD of protein-rich material, an increased ratio of toxic ammonia (NH 3) to ammonium ( ) is another
possible source of instability at higher temperatures [9]. The organic loading rate (OLR) and hydraulic
retention time (HRT), which determine the average time spent by the substrate and microorganisms in the
digester, are also important parameters in AD operation [9,10]. These parameters are often interlinked and
rapid increases in OLR and decreases in HRT (i.e. shock loading) can cause instability and thereby
representing one way to evaluate the robustness of an AD process.
This study investigated the effects of altered operating temperature (to/from mesophilic to/from
thermophilic) on biogas digester performance and how this affects continuing operation under increasing
loading rate. Microbial community composition was determined under the period of temperature change
in order to assess the innate microbial resilience and identify key microorganisms related to efficiency
and stability of the AD process.

2. Material and methods

2.1. Biogas digester operation and analytical methods

Anaerobic digestion was conducted in four identical laboratory-scale continuously stirred tank
digesters (Belach Bioteknik, Stockholm, Sweden) with a working volume of 5 L. The digesters (D) were
operated in sets of two (D37 and Dinc; D52 and Ddec), where D37 and D52 served as reference digesters (the
labels inc and dec designate operation at increased and decreased temperature, respectively). The digester
parameters were initially set to mimic the operation of two industrial-scale biogas plants operated in two
different Swedish cities. Digesters D37 and Dinc were inoculated with sludge from a mesophilic (37C)
biogas plant and D52 and Ddec with sludge from a thermophilic (52C) biogas plant. Semi-continuous
operation was achieved by daily batch feeding six days a week with household, slaughterhouse and food
industry waste, supplemented with a mixture containing iron (Fe2+/Fe3+), cobalt, nickel, selenium,
tungsten and hydrochloric acid (BDP-865/866, Kemira Oyj, Helsingborg, Sweden). The substrate was
taken from the same biogas plants as the inoculum. During start-up (approx. 20-30 days) and the first
experimental period, the digesters were operated with an OLR of 3 g volatile solids (VS) per litre and day
and a HRT of 29-35 days. The processes were initiated with identical temperature as the source digester,
 Maria Westerholm et al. / Energy Procedia 105 (2017) 895 900 897

i.e. 37C for D37 and Dinc and 52C for D52 and Ddec. In the first period of operation (days 0-140 for D37
and Dinc and days 0-170 for D52 and Ddec) the temperature was kept constant for the two reference
digesters (D37, D52), but gradually increased (by 2C/week) in Dinc and decreased (by 2C/week) in Ddec to
reach an end-point of 52C and 37C, respectively (Fig. 1). All other operating parameters were kept
unchanged during this first period of operation. The processes were then run at the end-point temperature
conditions for 70 days (>2 HRT).
In the second experimental period (from day 141 or 171 to day 360), the OLR of all digesters was
gradually increased from 3 to 7 g VS/L/day by increasing the amount of substrate (Fig. 2), leading to a
simultaneous decrease in HRT from 29-35 days to 14-17 days. The processes were allowed to acclimatise
to the OLR change for at least 1 HRT after each 1 g VS/L/day increment.
Total gas production, pH and total solids (TS) and VS concentrations were measured as described
previously [11]. For calculation of degree of degradation, the rough estimation method recommended
elsewhere [12] was used in order to compensate for volatile fatty acid (VFA) losses in determination of
dry matter. Gas composition (methane (CH4) and carbon dioxide (CO2) concentration) and VFA
concentration were measured weekly, using gas chromatography and high performance liquid
chromatography (HPLC), respectively [11]. The volumetric gas data were converted to standard
conditions at pressure 1.01325 bar and temperature 273.2 K. Ammonium-nitrogen was determined
according to standard methods [13] and concentration of ammonia-nitrogen was calculated as described
elsewhere [14].

2.2. Molecular analysis of microbial community composition

Digester sludge samples for molecular analyses were collected on eight occasions between days 0 and
170 and stored at -20C until further use. DNA extraction, construction of 16S amplicon libraries and
Illumina sequencing were carried out on triplicate samples from each sampling point and digester, as
described previously [15].

3. Results

3.1. Anaerobic digester performance at changed temperature

In the first period of operation, digesters Dinc and Ddec demonstrated stable performance until the
temperature reached 40-44C (at about 40 and 70 days of operation, respectively), whereupon digester
performance became less stable, as shown by decreased specific methane yield (Fig. 1) and accumulation
of VFAs. The process in Dinc stabilised after continuing operation and increasing temperature, but for Ddec
actions were required (temporarily decreased OLR and ceased temperature alteration) at this point to
counteract the signs of disturbance. The increased temperature in D inc raised specific methane yield to
55128 Nml CH4/g VS, compared with 49328 Nml CH4/g VS in the reference digester D37. For Ddec the
decreased temperature lowered the methane yield from 56210 Nml CH4/g VS (at 52C) to 51114 Nml
CH4/g VS at the end-point temperature (37C). A slight increase in methane yield occurred in Ddec after
the instability period at 40-44C (Fig. 1). This peak coincided with degradation of VFA accumulated
during digester disturbance. Decreasing the temperature from 52 to 37C in Ddec resulted in VFA levels of
about 0.7-1.0 g/L. All digesters had total ammonia-nitrogen concentrations varying between 4.0-4.4 g/L
during the full experimental period. The increase in temperature in D inc raised the range of free ammonia-
nitrogen from 0.07-0.1 to 0.3-0.6 g/L, whereas the lower temperature in Ddec decreased it from 0.7-0.9 to
898 Maria Westerholm et al. / Energy Procedia 105 (2017) 895 900

0.07-0.09 g/L. In Dinc the degree of degradation was stable (~85%) in the first period of operation,
whereas in Ddec it decreased from 81 to 74% after transition from thermophilic to mesophilic temperature.

Fig. 1. Mean specific methane yield (Nml CH4/g VS/day) in the reference digesters (D37 in A and D52 in B) and the digesters with
increasing (Dinc, A) and decreasing (Ddec, B) temperature at constant OLR (3 g VS/L/day) in the first period of operation.

3.2. Perturbation of digesters by increased loading rate

The robustness of the digesters (after D37 and D52 had reached the end-point temperature) was assessed
by increasing the OLR in a step-wise manner (Fig. 2). Each step-wise increase resulted in some
deterioration in the performance, with a temporary lowering in specific methane production, but the
digesters were able to recover their efficiency after a few days of adjustment. However, by increasing
OLR to 6 g VS/L/day, Ddec failed as a result of VFA accumulation (6-10 g total VFA/L) and decreased
pH (<5.4), with subsequent cessation of methane production (Fig. 2). However, D37, D52 and Dinc were
successfully operated at the target OLR of 7 g VS/L/day, providing weekly average methane yield of
45357, 56915 and 56426 Nml CH4/g VS, respectively.

Fig. 2. Mean specific methane yield in the digesters with increasing OLR from 3 to 7 g VS/L/day during operation at 37C (D37 in A
and Ddec in B) and 52C (Dinc in A and D52 in B) in the second period of operation.

3.3. Microbial community dynamics at changed temperature and links to digester performance

The bacterial communities in the mesophilic reference digester D37 and in digester Dinc prior to
initiation of temperature changes (day 5) were characterised by a relatively diversified structure
 Maria Westerholm et al. / Energy Procedia 105 (2017) 895 900 899

dominated by Bacteriodetes, Firmicutes, Chloroflexi and members of the candidate division WWE1
(Waste Water of Evry 1). Increasing the temperature considerably lowered the complexity at phyla level
and enhanced the dominance of Firmicutes and Thermotogae (see bar 52C, days 117-131 for Dinc in Fig.
3). Bacteriodetes was only present at low relative abundance at this point (Fig. 3). In Ddec, Firmicutes and
Thermotogae were the most abundant phyla during operation at 52C (days 10-31 of Ddec in Fig. 3). The
gradual decrease in temperature from 52C to 37C in Ddec lowered the relative abundance of these phyla
and instead Bacteriodetes was considerably higher at 37C (days 142-167 of Ddec in Fig. 3). However, in
contrast to the mesophilic communities in D37 and Dinc (day 5) representatives of Chloroflexi were not
detected in Ddec at 37C (days 142-167).

Fig. 3. Composition and dynamics of the microbial communities in the mesophilic reference digester (37C, D37) and
in digesters with gradual increasing (37l52C, Dinc) and gradual decreasing temperature (52l37C, Ddec) in the first
period of operation. Archaeal sequences are included in the bar section designated minor phyla. Reads that could
not be classified are collectively referred to as unclassified.

4. Discussion

The results demonstrated that changing digester temperature from mesophilic to thermophilic and vice
versa without affecting process stability is feasible, except for temporary disturbances when passing
through 40-44C. Similar findings have been reported in previous studies [8,16], illustrating the
robustness of the AD system and the ability of microbial communities to adjust to new operating
conditions. The decrease in methane yield and the VFA accumulation observed during the gradual
temperature alteration indicate an imbalance between functional guilds within the microbial community at
this stage. However, previous studies have shown that acclimatisation of the microbial community can
enable stable operation in between mesophilic and thermophilic conditions [17-19] and occasionally even
increase methane yield compared with mesophilic temperature [17].
The community dynamics displayed in the digesters with increasing or decreasing temperatures in the
present study resulted in community profiles that largely resembled each other at the two end-point
temperatures (37 and 52 C; Fig. 3). The current result suggests that the performance disturbance in Ddec
during increasing OLR in the subsequent period of operation was a consequence of a community with
comparatively lower resilience than that initially adapted to mesophilic conditions.
900 Maria Westerholm et al. / Energy Procedia 105 (2017) 895 900

Acknowledgements

We gratefully acknowledge Peter Malmros, Uppsala Vatten, Anna Hrberg, Jnkping Energi and
Sara Frid. This work was financially supported by the Swedish Research Council for Environment,
Agricultural Sciences and Spatial Planning (Formas) through grant number 2012-807.

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Biography
Maria Westerholm started her biogas research in 2007 as a PhD student focusing on syntrophic
acetate oxidation in high-ammonia digesters. Following post-doctoral research about biogas
production at KU Leuven in Belgium, she now holds a position as post-doc researcher at the
Swedish University of Agricultural Sciences.