Winter Mesophilic and thermophilic anaerobic digestion of source-sorted organic wastes: effect of ammonia on glucose degradation and methane production Received: 26 March 1997 / Received
Trang 1ORIGINAL PAPER
C Gallert á J Winter
Mesophilic and thermophilic anaerobic digestion
of source-sorted organic wastes: effect of ammonia
on glucose degradation and methane production
Received: 26 March 1997 / Received revision: 13 May 1997 / Accepted: 19 May 1997
Abstract The wet organic fraction of household wastes
was digested anaerobically at 37 °C and 55 °C At both
temperatures the volatile solids loading was increased
from 1 g l)1day)1 to 9.65 g l)1day)1, by reducing the
nominal hydraulic retention time from 93 days to
19 days The volatile solids removal in the reactors at
both temperatures for the same loading rates was in a
similar range and was still 65% at 19 days hydraulic
retention time Although more biogas was produced in
the thermophilic reactor, the energy conservation in
methane was slightly lower, because of a lower methane
content, compared to the biogas of the mesophilic
re-actor The slightly lower amount of energy conserved in
the methane of the thermophilic digester was
presum-ably balanced by the hydrogen that escaped into the gas
phase and thus was no longer available for
methano-genesis In the thermophilic process, 1.4 g/l ammonia
was released, whereas in the mesophilic process only 1 g/l
ammonia was generated, presumably from protein
degradation Inhibition studies of methane production
and glucose fermentation revealed a Ki (50%) of 3 g/l
and 3.7 g/l ammonia (equivalent to 0.22 g/l and 0.28 g/l
free NH3) at 37 °C and a Ki(50%) of 3.5 g/l and 3.4 g/l
ammonia (equivalent to 0.69 g/l and 0.68 g/l free NH3)
at 55 °C This indicated that the thermophilic ¯ora
tolerated at least twice as much of free NH3 than the
mesophilic ¯ora and, furthermore, that the thermophilic
¯ora was able to degrade more protein The apparent
ammonia concentrations in the mesophilic and in the
thermophilic biowaste reactor were low enough not to
inhibit glucose fermentation and methane production
of either process signi®cantly, but may have been high
enough to inhibit protein degradation The data
indi-cated either that the mesophilic and thermophilic protein degraders revealed a dierent sensitivity to-wards free ammonia or that the mesophilic population contained less versatile protein degraders, leaving more protein undegraded
Introduction Anaerobic fermentation of the organic fraction of wastes
is a suitable method to reduce both volume and mass for deposition in sanitary land®lls Approximately 33% of household wastes (annual amount 70±140 kg/in-habitant; LuÈbben 1994) can be separated as bioorganic wet waste The residual semi-dry fraction (67%) may be disposed of either in sanitary land®lls or be incinerated, leading to an increased caloric energy output per mass unit, because of its decreased water content Biological treatment of the wet organic fraction can be performed either in the presence of air (composting) or in the ab-sence of air (biogas fermentation, sometimes in accu-rately, called anaerobic composting) In 1996, composting plants with a total capacity of 2.4 ´ 106
tonnes bioorganic wastes were in operation in Germany, whereas the total capacity for anaerobic fermentation was only 0.19 ´ 106tonnes (Biehler and Nuding 1996) Anaerobic fermentation signi®cantly reduces the total mass of wastes, generates solid or liquid fertilizer and yields energy It can be maintained at psychrophilic (12±16 °C, e.g in land®lls, swamps or sediments), mesophilic (35±37 °C, e.g in the rumen and in anaerobic digester) and thermophilic conditions (55±60 °C; e.g in anaerobic digesters or geothermally heated ecosystems) Disadvantages of thermophilic anaerobic fermentation are the reduced process stability and reduced dewatering properties of the fermented sludge and the requirement for large amounts of energy for heating, whereas the thermal destruction of pathogenic bacteria at elevated temperatures was considered a big advantage (Steiner and Kandler 1983; Winter and Temper 1987) The slightly higher rates of hydrolysis and fermentation
C Gallert (&) á J Winter
Institut fuÈr Ingenieurbiologie und Biotechnologie des Abwassers,
UniversitaÈt Karlsruhe, Am Fasanengarten,
D-76131 Karlsruhe, Germany
Tel.: 0721/608 3274
Fax: 0721/694826
e-mail: Josef.Winter@bau-verm.uni-karlsruhe.de
Trang 2under thermophilic conditions have not led to a higher
methane yield Hashimoto et al (1981) reported no
signi®cant change in the total methane yield from
organic matter for fermentation temperatures ranging
from 30 °C to 60 °C
Compared to mesophilic fermentation conditions, at
higher temperatures the pH increased through a
re-duced solubility of carbon dioxide, leading to a higher
proportion of free ammonia Ammonia is generated
during anaerobic degradation of urea or proteins
Livestock manure from pork and poultry contains
about 4 g N/l (Angelidaki and Ahring 1991), that from
cattle about 1.5 g N/l (Angelidaki and Ahring 1993) In
the organic fraction of household waste the organic
nitrogen that was released as ammonia during
anaero-bic fermentation amounted to 2.15 g N/l (Jager et al
1989) Free ammonia may be inhibitory for anaerobic
fermentation and may be toxic for methanogenic
bacteria (Angelidaki and Ahring 1993) Inhibitory
NH3 concentrations under mesophilic conditions of
80±150 mg N/l at a pH of 7.5 have been reported
(Koster and Lettinga 1984; Braun et al 1981) Under
thermophilic conditions at a pH of 7.2±7.3, for
ace-ticlastic methanogens, inhibitory concentrations of free
ammonia of 3.5 g/l NH4-N/l (250 mg/l NH3-N) were
found and, for hydrogenolytic methanogens, 7 g/l
NH4-N/l (500 mg/l NH3-N) (Angelidaki and Ahring
1993; Borja et al 1996)
In this study, we compare the fermentation of the wet
organic fraction of household wastes in laboratory scale
reactors under meso- and thermophilic conditions,
concentrating on the conversion of organic nitrogen into
ammonia and inhibition of carbon removal by
ammo-nia
Materials and methods
Source of wet organic waste
The bioorganic fraction of household wastes was manually sorted,
moistened with tap water (250 ml/kg waste) and homogenized with
an Ultraturrax homogenizer The homogenate was divided between
plastic bottles and frozen until required for anaerobic fermentation.
Each batch of homogenized waste slurry was analysed and the
results are shown in Table 1 For the experiments described here,
four dierent batches were thoroughly mixed and the analytical
parameters of the mixture separately determined (Table 1).
Continuous anaerobic fermentation experiments
in biogas reactors
Two reactors (glass columns, inner diameter 10 cm, total height
80 cm, liquid height 60 cm) with a working volume of 4.8 l were
used for anaerobic fermentation of biowaste To maintain
homo-geneity, biowaste slurry and fermentation gas (ratio approximately
1:1) were withdrawn from the surface of the liquid and recirculated
into the bottom part of the reactor with a pump (recirculation rate
20/h) The temperature of 37 °C (mesophilic reactor) or 55 °C
(thermophilic reactor) was maintained by circulating water from a
water-bath heater (B Braun, Melsungen) through a water jacket
surrounding the reactors The reactors were fed batchwise with
fresh waste once a day To maintain process stability at 19 days hydraulic retention time (t HR ) the feed had to be added at 12-h intervals twice a day Biogas production was measured continu-ously with a gas meter (Ritter, Hanau) and the pH was controlled on-line with a pH electrode (WTW, Weilheim) inserted into the recirculation line.
Initially the reactors were ®lled with anaerobic sludge from the municipal sewage treatment plant of Regensburg (Germany) After
an adaptation time of 2 weeks at 37 °C (mesophilic reactor) and
55 °C (thermophilic reactor), 240-ml aliquots were replaced by a 1:1 mixture of fresh sewage sludge and biowaste slurry Gas duction, pH and fatty acids were monitored When the gas pro-duction ceased and the pH reached 7.4, 240 ml digester content was again replaced, this time by only biowaste slurry When the gas production ceased again, a fed-batch feeding once per day to maintain an initial t HR of 93 days was started After process sta-bilization, the t HR in both reactors was reduced stepwise and concomitantly the loading was increased, as indicated later Batch digestion experiments in serum bottles
The euent of the mesophilic and the thermophilic biowaste re-actor was centrifuged in a WKF-50-K centrifuge (Gesellschaft fuÈr elektrophysikalischen Apparatebau, Brandau) at 830 g for 10 min
to separate most of the solid waste particles (about 80%) from the suspended biomass The supernatant fraction contained the sus-pended bacteria and the small-particulate sludge ¯ocs, including most of the Methanosarcina sp., as judged from microscopy with a phase-contrast ¯uorescence microscope It was used as an inoculum for testing the eect of ammonia on glucose fermentation and methanogenic activity during anaerobic fermentation by the me-sophilic and thermophilic biowaste ¯ora.
Batch experiments were performed in 120-ml serum bottles, supplemented with 45 ml potassium phosphate buer (50 mM,
pH 7.8), 4.5 mM glucose and 5 ml bacterial inoculum All experi-ments were prepared in an anaerobic chamber (Coy, Ann Arbor, Mich USA) Colourless resazurin guaranteed a redox potential of less than )350 mV Dierent ammonia or NaCl concentrations were obtained by addition of the respective portions of a concen-trated NH 4 Cl or NaCl solution, made oxygen-free by heating un-der vacuum and regassing with nitrogen Assays without addition
of ammonia or with addition of NaCl were run as controls Each experiment was done in duplicate.
Analytical methods Standard procedures (Deutsche Einheitsverfahren zur Wasser-, Abwasser und Schlammuntersuchung 1983) were used to determine total solids, volatile solids, alkalinity, Kjeldahl nitrogen and am-monia The chemical oxygen demand (COD) was measured after
Table 1 Carbon and nitrogen parameters of dierent batches of homogenized biowaste Minimum and maximum values diered because of the dierent compositions and moisture contents of the input material COD chemical O 2 demand, DOC dissolved organic
C, TKN total Kjeldahl N
for expt COD total (g/l) 118 215 176
Volatile solids (g/l) 121 178 172
NH
Trang 3oxidation of the organic material in the homogenized sample with
potassium dichromate/sulphuric acid according to Wolf and
Nordmann (1977) The dissolved proportion of the COD and the
dissolved organic carbon were measured after ®ltration (pore size
0.45 lm) of samples The dissolved organic carbon was analysed by
infrared spectroscopy with a Tocor 2 carbon analyser (Maihak,
Hamburg).
The methane content of the biogas and volatile fatty acids were
determined by gas chromatography For measurement of methane,
a Packard model 427 gas chromatograph with a
thermoconduc-tivity detector and nitrogen as the carrier and reference gas, at a
¯ow rate of 10 ml/min, was used For isothermal separation of
gases at 30 °C, a Poropack N (80±100 mesh; Sigma, Deisenhofen)
Te¯on column (1.8 m length, inner diameter 1.5 mm) was used.
Detector and injector temperatures were set at 100 °C.
Volatile fatty acids were analysed with a Packard model 437 gas
chromatograph and an automatic liquid sampler, model 911
(Chrompack, Frankfurt) The gas chromatograph was equipped
with a ¯ame ionization detector, supplied with H 2 (30 ml/min) and
oxygen (300 ml/min) Detector and injector temperatures were set
at 210 °C and the oven was heated to 180 °C Fatty acids were
separated on a 2-m Te¯on column (1.5 mm inner diameter), ®lled
with Chromosorb C101 (80±100 mesh; Sigma, Deisenhofen).
Samples were acidi®ed with phosphoric acid (5%) and clari®ed by
centrifugation before injection The concentration of ammonia
NH 3 NH
4 in the samples was determined with a colorimetric
test from Merck Co (Merck, 1974) In the test system ammonia
ions form indolphenol blue with salicylate and hypochlorite ions,
which can be quanti®ed spectrophotometrically at 655 nm in a
concentration range of 0.03±1 mg/l NH
4 The concentration of free ammonia (NH 3 -N) was calculated according to Anthonisen et
al (1976):
NH 3 -N NH4 -N 10 pH
K b =K w 10 pH K b =K w e 6344=273T
where N concentrations are in mg/l and T is in °C.
Glucose was measured photometrically according to Lever
(1972) as p-hydroxybenzoic acid hydrazide at 410 nm The
rela-tionship was linear for a concentration range of 0±250 mg/l.
The theoretical quantity of methane and carbon dioxide
pro-duced under anaerobic conditions was calculated from the C/H/O/
N ratio of the slurry according to Buswell and Mueller (1952), as
modi®ed by Richards et al (1991):
C n H a O b N c n ÿ 0:25a ÿ 0:5b 1:75cH 2 O
! 0:5n 0:125a ÿ 0:25b ÿ 0:375cCH 4
0:5n ÿ 0:125a 0:25b ÿ 0:625cCO 2
cNH
4 cHCO ÿ
3
Results
Biowaste fermentation at mesophilic
and thermophilic temperatures
Digested sewage sludge from the anaerobic reactor of
the city of Regensburg was acclimated to biowaste
di-gestion at 37 °C and 55 °C beginning with an apparent
tHRof 93-days as mentioned In the mesophilic reactor
the COD removal eciency during stepwise reduction of
the tHRto 80, 60 and 50 days was around 85% (Fig 1a)
When tHRwas further reduced to 19 days, equivalent to
a space loading of 9.65 g COD l)1 day)1, the
COD-re-moval eciency decreased to 64% In the thermophilic
reactor the COD-removal eciency was 95% at a tHRof
93 days During stepwise reduction of the retention time
to 19 days, equivalent to an increase of the space loading
to 9.65 g volatile solids l)1day)1, the COD removal decreased steadily from 95% to 67% (Fig 1b) The biogas production increased from 0.3 l l)1day)1 at 93 days tHRto 5.3 (37 °C) or 5.6 (55 °C) l l)1day)1 at 19 days tHR (Fig 1a, b) With every stepwise reduction of
tHR below 50 days, propionate accumulated, reaching
10 mM on some days in the mesophilic reactor, whereas the thermophilic reactor tended to accumulate acetate
up to 40 mM, before steady-state conditions, with less than 1 mM propionate or 4 mM acetate, were achieved again The reactor performance of anaerobic biowaste fermentation at 37 °C and 55 °C at tHR = 19 days is summarized in Table 2 At a COD loading of 9.65 g l)1day)163% or 67% of the COD was degraded
at 37 °C or 55 °C and the volatile solids reduction was 64% and 65% respectively Although the gas production per litre of reactor volume and per day was apparently slightly higher in the thermophilic reactor, because of a reduced solubility of CO2at the higher temperature, in total little more methane was produced in the mesophilic reactor This was due to a methane content of the biogas
of 67% in the reactor run at 37 °C but only 59% in the
Fig 1a,b Eciency of mesophilic (a) and thermophilic (b) biowaste fermentation at increasing loading rates A portion of the content of each fermenter was manually replaced once per day with fresh biowaste to maintain the respective loading rate/hydraulic retention time (t HR ) Biogas production and chemical O 2 demand (COD) removal were determined r Loading rate (VS volatile solids),
n COD removal, Ð hydraulic retention time, m biogas production
Trang 4reactor run at 55 °C (Table 2) The total energy release
by gases in both reactors may have been identical,
however, since the gas of the thermophilic reactor
con-tained some hydrogen (not quanti®ed) The ammonia
content of the euent of the thermophilic reactor was
notably higher, indicating that apparently more protein
was degraded at 55 °C than at 37 °C
The pH of the mesophilic and thermophilic reactors
during one feeding cycle at tHR = 22 days is shown in
Fig 2 Within 1 h after addition of the biowaste slurry,
preacidi®ed to a pH of pH 5.3, the pH of the reactor
content dropped from 7.5 to 6.75 and slowly increased
again to 7.4/7.5 within 24 h Due to the sharply
de-creasing pH after sludge addition, the CO2 content of
the biogas increased in the ®rst hours after feeding and
dropped later on, resulting in an average methane
con-tent for the biogas collected during one feeding cycle of
67% in the mesophilic reactor and 59% in the
thermo-philic reactor The alkalinity (Ks for a pH change from
7.5 to 6.5) was 17 mmol/l in the mesophilic reactor
and 25.6 mmol/l in the thermophilic reactor In both
reactors the buer capacity was too small to avoid acidi®cation after substrate addition (Fig 2)
Ammonia inhibition of the biogas process at 37 °C and 55 °C
Each 5-ml sample of a bacterial suspension from mesophically and thermophically fermented biowaste slurry, containing mainly the bacteria and some ®ne-particulate material, with a dry weight content of 11 g/l
in total (prepared as described in Materials and meth-ods), was suspended in 45 ml phosphate buer (50 mmol/l, pH 7.8, containing 4.5 mmol/l glucose) and supplemented with 0±35 g/l NH4Cl or NaCl Glucose degradation rates were determined for the time span necessary for 80%±90% degradation of the initial amount and are shown in Fig 3a With increasing concentration of ammonia, glucose degradation was slightly more inhibited at 55 °C than at 37 °C The Ki50
for ammonia-N was 3.7 g/l at 37 °C (=0.28 g NH3-N) and 3.4 g/l at 55 °C (=0.68 g NH3-N) The Ki50 was similarly determined for methane production (Fig 3b)
A 50% inhibition was seen at 3.0 g/l ammonia-N (=0.22 g/l NH3-N) in the mesophilic reactor and at 3.5 g ammonia-N (=0.69 g/l NH3-N) in the thermo-philic reactor (Table 3) In the presence of 5 g/l NaCl, glucose degradation and methane production were not signi®cantly in¯uenced, whereas in the presence of 15 g/l NaCl or more, methane was no longer produced, pre-sumably because of the high salinity
Discussion Manually fractionated biowaste was digested at 37 °C and 55 °C in a one-stage recirculated suspension reactor
at a minimal tHR of 19 days, equivalent to a volatile solids loading of 9.4 g l)1day)1 Similar short retention times for less concentrated biowastes were applied for the single-stage processes of WABIO, Bio-Stab or Ko-mpogas reactors, whereas the methane reactor in pro-cesses with a separate hydrolysis reactor and a
Table 2 Parameters of the
biowaste reactors at 37 °C and
55 °C VS volatile solids
a Including COD of
®ne-partic-ulate matter that was not
sedimented
fermentation Thermophilicfermentation
Space productivity (l biogas l )1 day )1 ) 5.3 5.6
Fig 2 pH pro®le of mesophilic and thermophilic biowaste
fermen-tation for one feeding cycle at 22 days hydraulic retention time.
Samples comprising 220 ml digested biowaste were replaced by fresh,
pre-acidi®ed waste (pH 5.3) After homogenization for 10 min, a pH
of 7.0 was obtained, which dropped further as a result of the ongoing
acidi®cation r 55 °C, d 37 °C
Trang 5separation unit for non-hydrolysed solids (e.g ®xed-bed
reactor of BTA or MAT) could be operated with only 5
days tHR at the same eciency (Scherer 1995)
The removal of volatile solids was in the same range
for mesophilic and thermophilic fermentation of our
biowaste slurry The ®nal volatile solids loading of
9.4 g l)1day)1 in the laboratory reactors was higher
than reported for full-scale plants (e.g 5±8 g l)1day)1;
Gessler and Keller 1995) The volatile solids removal
(64%±65%) was also higher in the laboratory reactors
under mesophilic and thermophilic fermentation
condi-tions, as compared to many full-scale plants, where it
was around 55% (KuÈbler 1994; Gessler and Keller
1995) Rivard et al (1995) reported a very high COD
removal eciency of 77% for municipal solid wastes and tuna processing wastes at mesophilic fermentation tem-peratures for loading rates up to 14 g volatile solids
l)1day)1 Kayhanian (1995) observed a degradation of 83% of the volatile solids fraction of wet waste in a fermentation with a high solids content at 30 days tHR
A highly ecient volatile solids removal may lead to
a reduced viscosity and a better separation of solids The supernatant of the euent of our mesophilic and ther-mophilic biowaste digester contained 13.2% (37 °C) or 30.7% (55 °C) of the non-degraded COD, respectively, after sedimentation of large solids Whereas the super-natant of the mesophilic reactor euent contained mainly soluble organic components, the supernatant of the thermophilic reactor euent in addition contained a high proportion of ®ne-particulate, suspended material The sediment of both euents came from non-degraded particles, such as lignin and ligni®ed plant material, which in municipal solid waste may comprise up to 15%
of the total COD (Barlaz et al 1989)
The space productivity of biogas was 5.3 l l)1 day)1
at 37 °C and 5.6 l l)1day)1at 55 °C KuÈbler (1994) re-ported biogas space productivities for wet waste fer-mentation of 4 l l)1day)1 for the thermophilic BTA process, whereas with cattle manure maximal space productivities of 5.58 l l)1day)1, (mesophilic fermenta-tion) and 6.67 l l)1day)1 (thermophilic fermentation) were reported by Mackie and Bryant (1995)
Although a little less biogas was produced in our mesophilic biowaste reactor, because of the higher methane content in the biogas the total amount of methane per litre of reactor volume and per day was higher than that in the gas of the thermophilic reactor (Table 2) A similar observation was reported by Mackie and Bryant (1995) for the digestion of cattle manure The advantage of thermophilic digestion was mainly that the euent was rendered hygienic by inactivation of bacteria (KuÈbler 1994) or viruses (Lund et al 1996) The speci®c gas production per gram of COD or per gram of volatile solids degraded was high in both of our reactors This was the result of a high lipid and fat content of the biowaste input material, caused by the addition of a batch of spoiled butter Consequently the speci®c methane productivity was also high: 0.59 l/g at
37 °C and 0.54 l/g volatile solids degraded at 55 °C (calculated from data of Table 2) For carbohydrates a theoretical methane yield of 0.35 l/g would be expected High speci®c methane yields of 0.4±0.59 l/g were also reported for thermophilic household solid waste diges-tion, by Rintala and Ahring (1994)
One obvious result of thermophilic biowaste diges-tion was the higher yield of ammonia: 1.4 g/l at 55 °C compared to 1 g/l at 37 °C fermentation temperature, presumably from protein degradation Angelidaki and Ahring (1993) reported a 25% inhibition of livestock waste digestion at thermophilic conditions in the pres-ence of 4 g ammonia-N/l, equivalent to 900 mg free
NH3/l at the respective pH The inhibitory eect was more pronounced on aceticlastic than on hydrogenolytic
Fig 3a, b Inhibiting eect of ammonia on mesophilic and
thermo-philic glucose fermentation (a) and methane production (b) The
bacterial ¯ora of the mesophilic and thermophilic biowaste digester
was separated from the euent by pelleting the solid particles Then it
was diluted in phosphate buer and supplemented with ammonium
chloride or NaCl as described under Materials and methods.
d Mesophilic incubation at 37 °C, r thermophilic incubation at 55 °C
Table 3 K i50 values for NH
4 =NH 3 and for NH 3 Up to 5 g/l NaCl there was no inhibition of glucose fermentation and methane
production
4 =NH 3 -N (g/l) K(g/l)i50NH3-N Methane production, 37 °C 3.0 0.22
Methane production, 55 °C 3.5 0.69
Glucose degradation, 37 °C 3.7 0.28
Glucose degradation, 55 °C 3.4 0.68
Trang 6methanogens Adaptation from 4 g to 6 g N/l required a
time span of 6 months We observed a 50% inhibition at
37 °C and 55 °C by 3±3.7 g ammonia/l for glucose
fer-mentation and methanogenesis as well The inhibitory
eect was presumably due to the free permeability of
NH3through the cell membrane At the actual pH of 7.6
in the reactors, 0.22±0.28 g free NH3/l caused a 50%
inhibition of mesophilic glucose fermentation and
methane production, whereas 0.68±0.69 g free NH3/l
caused a 50% inhibition of both processes at 55 °C
In the mesophilic reactor in the presence of 1 g total
ammonia at a pH of 7.6 the NH3 concentration was
calculated to be around 0.03 g and, in the thermophilic
reactor at 1.4 g total ammonia, around 0.126 g/l These
free ammonia concentrations were too low to in¯uence
methanogenesis signi®cantly On the other hand, from
the total Kjeldahl N of the biowaste during mesophilic
digestion, only about 1/3, and during thermophilic
di-gestion about 1/2, was converted to ammonia If this is
not a speci®c phenomenon of the mesophilic and
ther-mophilic population in our reactors, it may indicate that
protein degradation is inhibited by free ammonia, with a
higher sensitivity at mesophilic than at thermophilic
reactor temperatures
Acknowledgement We thank Martin StuÈtzel for his experimental
input during the preparation of his diploma thesis.
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