p ocesses
A icle
Case S udy o Anae obic Diges ion P ocess S abili y De ec ed
by Dissol ed Hyd ogen Concen a ion
Daniela Pla ošo á1,* , Jiˇ íRusín1, Jan Pla oš 2, Ka eˇ ina Smu ná3and Roman Bu yjan 4


Ci a ion: Pla ošo á, D.; Rusín, J.;
Pla oš, J.; Smu ná, K.; Bu yjan, R.
Case S udy o Anae obic Diges ion
P ocess S abili y De ec ed by
Dissol ed Hyd ogen Concen a ion.
P ocesses 2021,9, 106. h ps://
doi.o g/10.3390/p 9010106
Recei ed: 11 No embe 2020
Accep ed: 5 Janua y 2021
Published: 7 Janua y 2021
Publishe ’s No e: MDPI s ays neu-
al wi h ega d o ju isdic ional clai-
ms in published maps and ins i u io-
nal a ilia ions.
Copy igh : © 2021 by he au ho s. Li-
censee MDPI, Basel, Swi ze land.
This a icle is an open access a icle
dis ibu ed unde he e ms and con-
di ions o he C ea i e Commons A -
ibu ion (CC BY) license (h ps://
c ea i ecommons.o g/licenses/by/
4.0/).
1Ins i u e o En i onmen al Technology, VSB—Technical Uni e si y o Os a a, 17. Lis opadu 2172/15,
70800 Os a a, Czech Republic; [email p o ec ed]
2Depa men o Compu e Science, Facul y o Elec ical Enginee ing and Compu e Science,
VSB—Technical Uni e si y o Os a a, 17. Lis opadu 2172/15, 70800 Os a a, Czech Republic;
[email p o ec ed]
3Ins i u e o Languages, VSB—Technical Uni e si y o Os a a, 17. Lis opadu 2172/15,
70800 Os a a, Czech Republic; [email p o ec ed]
4GAS Con ol s. .o, No ýS ˇe 1407/59a, 73564 Ha íˇ o -P os ˇ edníSuchá, Czech Republic;
[email p o ec ed]
*Co espondence: [email p o ec ed]
Abs ac :
The pape p esen s he esul s o a labo a o y expe imen o mesophilic single-s age
anae obic diges ion pe o med o e i y he possibili y o ea ly de ec ion o p ocess ins abili y
and eac o o e load by e alua ing he cou se o dissol ed hyd ogen concen a ion o he main
in e media e. The diges ion p ocess was un in a Te a o s IS o a y d um bio eac o o 230 days.
The subs a e dosed on weekdays was ood le o e s om he uni e si y can een. A an a e age
empe a u e o 37
◦
C, an o ganic loading o ola iles o 0.858 kg m
−3
day
−1
and a heo e ical
e en ion ime o 259 days, biogas p oduc ion o 0.617 Nm
3
kg VS
−1
was achie ed wi h a CH4
con en o 51.7 ol. %. The alues o he es ablished FOS/TAC s abili y indica o anged om 0.26
o 11.4. The highes alue was eached when he eac o was o e loaded. The dissol ed hyd ogen
concen a ion measu ed by he ampe ome ic mic osenso anged om 0.039–0.425 mg dm
−3
. Da a
we e s a is ically p ocessed using Pea son’s co ela ion coe icien . The co ela ion o he hyd ogen
concen a ion wi h o he pa ame e s such as he concen a ion o o ganic acids was e alua ed. The
alue o Pea son’s co ela ion coe icien was 0.331 and co esponded o a p- alue o 0. The esul s
con i med a e y low limi o he hyd ogen concen a ion a which he mic obial cul u e, especially
me hanogens, was al eady o e loaded. The ampe ome ic mic osenso p o ed o be a he unsui able
o ope a ional applica ions due o insu icien sensi i i y and sho se ice li e. The newly designed
a io o dissol ed hyd ogen concen a ion o neu alizing capaci y was es ed bu did no wo k
signi ican ly be e han he es ablished FOS/TAC s abili y indica o .
Keywo ds:
anae obic diges ion; e men a ion; o a y bio eac o ; dissol ed hyd ogen; ampe ome -
ic senso
1. In oduc ion
Con olled anae obic diges ion wi h biogas p oduc ion is an es ablished and, in he
u u e, p omising way o ecological use o biomass and enewable ene gy p oduc ion. The
echnology o biogas plan s is based on he biological decomposi ion o o ganic subs ances
in an en i onmen wi hou access o ai . This is a bioene ge ic ans o ma ion o subs ances
in which he e is no signi ican educ ion in a e ilize alue. The p oduc s a e a biologically
s abilized ma e ial called diges a e, which is used mainly as a e ilize and biogas wi h a
me hane con en o 55–70% and a calo i ic alue o abou 18–26 MJ Nm−3.
Anae obic diges ion is a complex biochemical p ocess ha consis s o many pa s,
consecu i e and pa allel p ocesses; Guje and Zehnde illus a e his in a diag am [
1
]. To
simpli y he explana ion, mos au ho s di ide he p ocess in o ou p ima y phases [
2
]: hy-
d olysis is he i s s age o decomposi ion; mac omolecula o ganic subs ances a e b oken
P ocesses 2021,9, 106. h ps://doi.o g/10.3390/p 9010106 h ps://www.mdpi.com/jou nal/p ocesses
P ocesses 2021,9, 106 2 o 11
down in o low molecula weigh wa e -soluble subs ances. Decomposi ion akes place
using ex acellula hyd oly ic enzymes, which a e p oduced exclusi ely by e men a ion
bac e ia. In he second phase, acidogenesis, he p oduc s o hyd olysis decompose in o
simple o ganic subs ances, such as alcohols, acids, CO
2
, and hyd ogen, mainly by he
ac ion o bac e ia. The hi d phase is ace ogenesis, he o ma ion o ace ic acid. Syn ophic
ace ogenic mic oo ganisms decompose o ganic acids highe han ace ic acid, alcohols, and
a oma ic compounds. CO
2
and H
2
a e also p oduced. The las phase is me hanogenesis.
Commonly, p edominan ly ace o ophic me hanogenic mic oo ganisms decompose he
ace ic anion o o m me hane and CO
2
. Hyd ogeno ophic me hanogens consume H
2
and
CO
2
o p oduce me hane. CH
4
and CO
2
p edomina e in biogas om balanced diges ion in
a a io co esponding mainly o he composi ion o he incoming o ganic ma e in he sub-
s a e [
3
]. The in e ac ion o many g oups o mic oo ganisms is essen ial o he long- e m
main enance o he condi ions o e icien con e sion o o ganic ma e . Excess hyd ogen
in he whole sys em inhibi s he ac i i y o me hanogens, as well as ace ogenesis, and can
cause chaining p oblems leading o he collapse o he whole p ocess. The condi ions in
he e men e o he biogas plan wi h ega d o he one-s age p ocess a e main ained by
he ope a o so ha said diges ion phases a e in equilib ium o a long ime. I he e is a
mo e signi ican imbalance, es o ing equilib ium may no be easy a all o a sho pe iod.
1.1. Va iables Moni o ed du ing he Diges ion P ocess
I we conside a ma u e biogas echnology ensu ing minimal empe a u e luc ua ions
and minimal ai supply o he e men e , he ollowing a iables a e moni o ed o con inu-
ous e alua ion o p ocess s abili y: biogas p oduc ion is s ill some imes he only moni o ed
pa ame e . I does no desc ibe he s a e o he p ocess bu only i s esul , which depends
on he amoun o o ganic subs ances in oduced and on a high numbe o o he ac o s.
The concen a ion o CH
4
in biogas is also no he main indica o o p ocess s abili y. I is
a he a balance elemen . The amoun o CH
4
p oduced mus be in ela ion o he o ganic
load applied o o loads o he main g oups o subs a es, such as a s e sus ca bohy-
d a es e sus p o eins. A mo e sensi i e indica o o p ocess s abili y is he CH
4
/CO
2
concen a ion a io. This a io is de e mined by he composi ion o he eed mix u e and
does no change much du ing he s able ope a ion o he e men e . The hyd ogen con-
cen a ion in biogas is one o he mo e sensi i e indica o s o p ocess s abili y. In gene al,
i can be s a ed ha he appea ance o H
2
in biogas almos always signals he ins abili y
o he p ocess. Howe e , i is almos impossible o achie e an equilib ium dis ibu ion
o H
2
be ween he liquid and gas phases, which makes he e alua ion o his pa ame e
di icul . pH is no a su icien ly sensi i e indica o o p ocess s abili y. I he pH d ops,
his indica es dosing e o s made in he las ew days o weeks. I is necessa y o moni o
he pH, especially when diges ing subs a es ha do no p o ide su icien neu alizing
capaci y o he suspension in he e men e o subs a es ha a e oo ni ogenous. The
neu aliza ion capaci y (bu e ing capaci y) o he eac o consis s mainly o he equilib ium
sys em HCO
3−
/CO
2
oge he wi h he equilib ium sys em NH
4+
/NH
3
. Adequa ely high
neu aliza ion capaci y is he mos impo an ac o o p e equisi e o main aining he
s abili y o he p ocess a high o ganic loading. The con en o lowe ( ola ile) a y acids
(VFA) is one o he mos sensi i e indica o s o s abili y. Usually, he sum o C
2
–C
5
acids is
moni o ed, bu i is mo e app op ia e o moni o indi idual acids. E en a ela i ely high
concen a ion o ace ic acid can be emo ed quickly, bu highe acids usually pa alyze he
sys em o a longe ime [
4
]. The VFA/TIC (To al Ino ganic Ca bona e) pa ame e is he
a io o he sum o lowe a y acids exp essed by he equi alen o ace ic acid and he
neu alizing capaci y o med mainly by he bica bona e sys em. The i a ion pa ame e
de ec s a possible bu e de iciency and an excess o acids. I has become he mos aluable
indica o o p ocess s abili y, no only a he s a o he use o he echnology bu also
con inuously [
5
]. Méndez-Acos a e al. [
6
] used a Luenbe ge obse e o inc ease diges ion
s abili y by moni o ing ola ile a y acid (VFA) and o al alkalini y (TA) concen a ions in a
mul iple-inpu , mul iple-ou pu eedback con ol model. Fei kenhaue e al. [
7
] con i med
P ocesses 2021,9, 106 3 o 11
ha online ola ile a y acid (VFA) i a ion is a eliable me hod o measu ing subs a e
concen a ion wi hou he use o expensi e analy ical equipmen . They designed a eliable
measu ing cell o online i a ion o ola ile a y acids. Recalib a ion o he pH p obe
esis an o high sal concen a ions was only necessa y wice a week. Yuan and Zhu [
8
]
s udied a a ie y o inhibi o y subs ances and ound ha in e media e p oduc s a e he
p ima y cause o anae obic diges e upse o ailu e, including ee ammonia (FA), ola ile
a y acids (VFAs), and sul ide/sul a e. Howe e , hey a e essen ial nu ien s o bac e ial
g ow h and he anae obic diges ion p ocess.
1.2. Senso s and he Measu emen o he Pa ame e s
Many o he pa ame e s can be moni o ed, bu hey usually encoun e a mo e com-
plica ed ea men o he sludge sample be o e analysis. The esul s o e alua ion a e
a ailable wi h an imp ac ically long ime delay. I is ad isable o con inue looking o an
easily measu able and in o ma i e pa ame e , acco ding o which i would be possible o
eac immedia ely o he eme ging imbalance in he e men e . The p ocess biochemis y
sugges s he possibili y o apid cap u e o changes by moni o ing he dissol ed hyd ogen
concen a ion pa ame e . Acco ding o esea ch in he ield o was ewa e ea men and
om esea ch on e men e s o biogas plan s, he anae obic p ocess can be con olled in
connec ion wi h he measu emen o he concen a ion o he mos impo an chemical
in e media e-dissol ed dia omic hyd ogen H
2
(l). The li e a u e gi es se e al lab-scale
o pilo -scale examples, each using a di e en ype o H
2
(l) senso . H
2
(l) mos apidly
de ec s he upcoming o e load o he slowes g owing-me hanogenic mic obial biomass.
Dohányos s a es ha he as es de ec ion o o e load can be pe o med by measu ing H
2
(l)
in he ange o 0–200 mmol m
−3
, which co esponds o a pa ial equilib ium p essu e o H
2
up o 20,000 Pa [
9
]. Co d-Ruwisch e al. e i ied ha he pa ial p essu e o dissol ed H
2
in
he ange o 2–8 Pa co ela es linea ly wi h he o e load, and he no mal limi o s abili y
is no highe han 5–6 Pa. [
10
]. Pauss e al. indica e he common H
2
(l) concen a ion o
2–3.5
µ
mol dm
3
co ela ing o 200–350 Pa [
11
]. S ini asan ecommends in si u online
measu ing o he concen a ion o dissol ed H
2
wi h a Cla k oxygen p obe wi h e e sed
pola iza ion and a mo e powe ul elec ical signal ampli ie . I is a pola ized pla inum
elec ode, and he H
2
(l) sensi i i y should be linea be ween 1–50
µ
M co esponding o
100–5000 Pa [
12
]. The long- e m s abili y o he p obe and H
2
S in e e ence does no appea
o be known [
13
]. Al e na i ely, dissol ed hyd ogen can be en ained in he ca ie gas, and
he mix u e con inuously analyzed ch oma og aphically, bu his is no a sui able ope a ing
me hod o biogas plan ope a o s [
12
]. A che e al. e i ied he applicabili y o he
Exhaled Hyd ogen Moni o EHM (Gas Measu emen Ins umen s L d., Ren ew, Sco land)
in a biogas plan using a 6 m
3
e men e . The EHM can measu e gaseous hyd ogen in
biogas exac ly up o he limi o 0.1 Pa e en in indus ial condi ions. The esul s had o be
co ec ed acco ding o he H
2
S, CH
4
, and CO
2
c oss-sensi i i ies. Hyd ogen peaks in biogas
occu ed 3–6 h a e shock o e load. The esul s showed ha i would be mo e app op ia e
o measu e hyd ogen in he liquid phase [
14
]. S ong e al. desc ibed he cons uc ion and
use o a cheap senso , un o una ely wi h insu icien sensi i i y a he le el o 30 Pa [
15
].
Danne un e al. de eloped a sensi i e palladium me al-oxide-semiconduc o (Pd-MOS)
H
2
gas senso wi h a de ec ion limi o 0.1 Pa [
16
]. Appa en ly, his senso has no ye
been es ed o anae obic diges ion. Only a ew biogas plan s exis , which ely solely on
H
2
measu emen s o s abili y moni o ing. I hyd ogen is moni o ed a biogas plan s, i
is measu ed by elec ochemical senso s in he biogas. In he u u e, he measu emen s
o dissol ed H
2
could become an in e es ing al e na i e [
17
]. Wilcox e al. [
18
] es ed an
online bica bona e alkalini y (BA) senso and ecognized dis u bances by a neu al ne wo k.
They concluded ha he sys em is p ac ically usable and use ul in he case o an inle wi h
insu icien bu e ing capaci y. The au ho s [
19
] hypo hesize ha an imbalance in he AD
p ocess can lead o he accumula ion o sho -chain o ganic acids, as high concen a ions
o hese acids inhibi me hane o ma ion and, in ex eme cases, his can lead o he dea h
o he co esponding bac e ial popula ions. They a e based on he assump ion ha he e
P ocesses 2021,9, 106 4 o 11
is an analogy be ween he lo a o mic oo ganisms in he umen o a uminan and he
e men e eac o o a biogas plan . In a heal hy animal, he pan- lo a is p edominan ly
G am-nega i e and has a ious o ms. In he case o malnu i ion, his di e si y dec eases,
and G am-posi i e lac ic acid-p oducing s ep ococci soon p e ail and lac obacilli will
p edomina e. This condi ion is known as umen acidosis. Acco ding o he pa en , an
analogous change o he bac e ial popula ion om G am-nega i e o G am-posi i e in
he e men e is used as an indica o o p ocess in e up ion in a biogas plan . Bac e ial
de ec ion was pe o med by FT-MIR spec ome y.
Nguyen e al. [
20
] s udied wo signi ican s a egies o p omo e he s able pe o mance
o he AD p ocess, including moni o ing and con ol o e he p ocess. While he e is a con-
s an deba e on he impo ance o ins umen a ion e sus con ol s a egy, he expe ience
on bo h aspec s is limi ed in indus ial-scale AD ope a ions. Wi h he apid de elopmen
o ins umen a ion and au oma ic con ol, he implemen a ion and ope a ion cos o his
ad anced sys em is expec ed o dec ease. The op imis ic ou look o he upcoming decades
is ha small-scale AD plan s will be equipped wi h an au oma ic con ol sys em o be e
pe o mance. Meanwhile, a cen alized AD plan is s ill a easible op ion. Liu e al. [
21
]
in oduced a new con ol s a egy o ope a ing anae obic diges ion p ocesses e icien ly a
high load. The con ol sys em includes a cascade con olle embedded in o a ule-based
supe iso y sys em based on ex emum-seeking con ol. The con ol sys em measu es pH
and biogas p oduc ion a e and a ies he o ganic load by manipula ing he in luen low.
Good con ol pe o mances we e achie ed du ing he s a -up and s eady-s a e unning
ope a ions and du ing he ejec ion o dis u bances. The con ol sys em can un he p ocess
unde a high load condi ion and e icien ly ejec dis u bances wi hou explici measu e-
men o he in luen cha ac e is ics (Gaida e al. [
22
]). O e he las 40 yea s, many di e en
con ol me hodologies o subs a e eed con ol o anae obic diges ion p ocesses ha e been
p oposed in o de o inc ease plan e iciency and sus ainable long- e m ene gy p oduc ion.
This e iew shows ha al hough sophis ica ed con olle s exis , ull-scale biogas plan s a e
mos ly s ill ope a ed wi hou a closed-loop eed con ol. No ma e which applica ion, such
con ol always has o ind a comp omise be ween maximizing economic yield, minimizing
he ecological oo p in , and minimizing he isk o p ocess ailu e. Fo anae obic was ewa-
e ea men , con ol sys ems ha come close o his ideal exis , bu o ag icul u al as well
as indus ial biogas plan s, such con ol has no ye been de eloped, and nei he has been
success ully implemen ed and alida ed a ull-scale. The main challenges a e a lack o
obus and eliable p ocess moni o ing using online ins umen a ion and a conse a i e
indus y ha is eluc an o implemen ully au oma ed p ocess con ol s a egies.
Ou wo k aimed o e i y he possibili y o ea ly de ec ion o diges ion p ocess ins a-
bili y and eac o o e load by e alua ing he cou se o dissol ed hyd ogen concen a ion
using an ampe ome ic mic osenso .
2. Labo a o y Tes ing
Tes ing ook place in he Ins i u e o En i onmen al Technologies a VSB—Technical
Uni e si y Os a a, Czech Republic. One 230-day single-s age mesophilic mono-diges ion
expe imen was pe o med. The p ocess was conduc ed in a Te a o s IS o a y d um
bio eac o (INFORS HT, Bo mingen, Swi ze land); see he appa a us diag am in Figu e 1.
The d um eac o wi h a o al olume o 0.0187 m
3
was illed wi h 15.0 kg (app oxima ely
0.015 m
3
) o liquid inoculum, anae obic slu y, om he lab-scale (0.2 m
3
) psych ophilic
eac o p ocessing ood le o e s om uni e si y can een a psych ophilic condi ions
(18–22
◦
C). The inoculum was eed o pa icles la ge han 5 mm o educe he o ma ion
o loa ing c us s. Du ing he expe imen , he slu y was con inuously s i ed. The s i ing
was secu ed by o a ion o he eac o d um a ound he ho izon al axis. The o a ion speed
was se o 0.5 min
−1
. Diges ion empe a u e was se o 37
◦
C
±
1
◦
C. The main pa ame e s
o he inoculum a e lis ed in Table 1.
P ocesses 2021,9, 106 5 o 11
P ocesses 2021, 9, x FOR PEER REVIEW 5 o 11
was se o 0.5 min−1. Diges ion empe a u e was se o 37 °C ± 1 °C. The main pa ame e s
o he inoculum a e lis ed in Table 1.
Table 1. Inoculum, Subs a e, and Diges a e Pa ame e s.
Pa ame e Symbol, Uni Inoculum Subs a e Diges a e
Po en ial o Hyd ogen pH-H2O, - 7.63 3.4–5.6; mean 4.2 3.4–7.9, mean 7.1
To al Solids (105 °C) TS, w . % 2.96 11.2–24.9; mean 16.4 2.2–5.4; mean 3.6
Vola ile Solids (550 °C) VS, w . %TS 59.79 86.00–96.10; mean 92.90 50.00–70.60; mean 61.30
Vola ile Fa y Acids VFA, mg dm−3 3050 - 1999–15,856; mean 5352
To al Ino ganic Ca bona e TIC, mg dm−3 13,650 - 1226–14,489; mean 9773
Diges ion S abili y Ra io VFA/TIC, - 0.223 - 0.161–11.431; mean 1.323
Figu e 1. Appa a us diag am. 1. Ro a y d um eac o . 2. S a o connec ed o he base. 3. Liquid ba ch le el. 4. Po o
subs a e dosing and diges a e sampling. 5. Biogas ou le ube. 6. The mocouple. 7. Dissol ed hyd ogen senso . 8. Two
al es o sampling and e u n o biogas. 9. Gas low me e . 10. Po able gas analyze . 11. Wa e hea ed jacke .
Food le o e s o meals se ed in he uni e si y can een we e used as complex mono-
subs a e. Those le o e s we e no like common ki chen biowas e. I consis ed o unea en
Figu e 1.
Appa a us diag am. 1. Ro a y d um eac o . 2. S a o connec ed o he base. 3. Liquid ba ch le el. 4. Po o
subs a e dosing and diges a e sampling. 5. Biogas ou le ube. 6. The mocouple. 7. Dissol ed hyd ogen senso . 8. Two
al es o sampling and e u n o biogas. 9. Gas low me e . 10. Po able gas analyze . 11. Wa e hea ed jacke .
Table 1. Inoculum, Subs a e, and Diges a e Pa ame e s.
Pa ame e Symbol, Uni Inoculum Subs a e Diges a e
Po en ial o Hyd ogen pH-H2O, - 7.63 3.4–5.6; mean 4.2 3.4–7.9, mean 7.1
To al Solids (105 ◦C) TS, w . % 2.96 11.2–24.9; mean 16.4 2.2–5.4; mean 3.6
Vola ile Solids (550 ◦C) VS, w . %TS 59.79
86.00–96.10; mean 92.90
50.00–70.60; mean 61.30
Vola ile Fa y Acids VFA, mg dm−33050 - 1999–15,856; mean 5352
To al Ino ganic Ca bona e TIC, mg dm−313,650 - 1226–14,489; mean 9773
Diges ion S abili y Ra io VFA/TIC, - 0.223 - 0.161–11.431; mean 1.323
Food le o e s o meals se ed in he uni e si y can een we e used as complex mono-
subs a e. Those le o e s we e no like common ki chen biowas e. I consis ed o unea en
po ions o lunches, wi hou bones. The subs a e ob ained om he can een was sup-

P ocesses 2021,9, 106 6 o 11
plemen ed wi h a small amoun o d inking wa e o dec ease he con en o solids o a
pumpable le el. Subsequen ly, he mix u e was homogenized wi h TS-32T400V sc ew mill
(RM Gas o L d., P ague, Czech Republic) h ough a ma ix wi h ound holes (3 mm in di-
ame e ). The subs a e slu y was s o ed in a e ige a o a 4–6
◦
C o supp ess acidi ica ion.
The subs a e was dosed in o he d um eac o once e e y wo king day. The expe imen
las ed o 230 days, so we dosed he eac o 165 imes. The a e age dose was abou 80 g o
he subs a e. The egula size was 50 g, bu o he o e loading, we used a much la ge
amoun —up o 300 g. The main pa ame e s o he subs a e a e lis ed in Table 1.
The slu y empe a u e was con inuously measu ed by a he mocouple passing
h ough he ho izon al sha o he eac o in he cen e o he eac o . Da a we e manually
eco ded once daily. The biogas s eam om he eac o passed h ough a ube passing
om he gas space axially h ough he ho izon al sha o he eac o . Biogas p oduc-
ion was con inuously measu ed wi h a TG05 o a y d um gas me e (RITTER GmbH,
Schwabmünchen, Ge many). Gas olume inc emen s we e manually eco ded once daily.
The biogas composi ion was measu ed wi h a Biogas5000 po able analyze (GEOTECH
L d., Co en y, G ea B i ain) once a day in wo king days, p io o diges a e sampling
and subs a e dosing. The analyze used CH
4
and CO
2
in a ed senso s and O
2
and H
2
S
elec ochemical senso s. Du ing he gas composi ion measu emen s, he eac o o a ion
was s opped. The biogas sample was sucked h ough one al e and e u ned o he gas
space o he eac o by an adjacen al e. Immedia ely a e he analysis o he biogas
composi ion, a dissol ed hyd ogen senso was inse ed in o he liquid phase. A al e
(32 mm diame e ), commonly used o diges a e sampling and subs a e dosing, was used
o inse he H
2
senso . The senso was sealed wi h silicone ubbe . I usually ook 15 min
o s abilize he senso in he anae obic slu y. The o a ion o he eac o was esumed only
a e emo ing he senso and inse ing a dose o he subs a e.
The dissol ed hyd ogen senso was he MS 08 ampe ome ic mic osenso (AMT Anal-
ysenmess echnik GmbH, Ros ock, Ge many) wi h a s a ed de ec ion limi o 0.2
µ
g dm
−3
H
2
(l) and a ange o up o 1.5 mg dm
−3
H
2
(l); see Figu e 2. The H
2
senso used i s own
sepa a e empe a u e senso . The ips o bo h senso s we e always placed app oxima ely
30 mm below he su ace o he slu y. A e measu ing H
2
(l), a diges a e sample (app oxi-
ma ely 0.9
×
he olume o he subs a e dose) was d ained o analysis, and a dose o he
subs a e was in oduced in o he eac o . Thus, he diges a e sampling was pe o med a
he same equency as he subs a e sampling.
P ocesses 2021, 9, x FOR PEER REVIEW 6 o 11
po ions o lunches, wi hou bones. The subs a e ob ained om he can een was supple-
men ed wi h a small amoun o d inking wa e o dec ease he con en o solids o a pump-
able le el. Subsequen ly, he mix u e was homogenized wi h TS-32T400V sc ew mill (RM
Gas o L d., P ague, Czech Republic) h ough a ma ix wi h ound holes (3 mm in diam-
e e ). The subs a e slu y was s o ed in a e ige a o a 4–6 °C o supp ess acidi ica ion.
The subs a e was dosed in o he d um eac o once e e y wo king day. The expe imen
las ed o 230 days, so we dosed he eac o 165 imes. The a e age dose was abou 80 g
o he subs a e. The egula size was 50 g, bu o he o e loading, we used a much la ge
amoun —up o 300 g. The main pa ame e s o he subs a e a e lis ed in Table 1.
The slu y empe a u e was con inuously measu ed by a he mocouple passing
h ough he ho izon al sha o he eac o in he cen e o he eac o . Da a we e manually
eco ded once daily. The biogas s eam om he eac o passed h ough a ube passing
om he gas space axially h ough he ho izon al sha o he eac o . Biogas p oduc ion
was con inuously measu ed wi h a TG05 o a y d um gas me e (RITTER GmbH, Schwab-
münchen, Ge many). Gas olume inc emen s we e manually eco ded once daily. The
biogas composi ion was measu ed wi h a Biogas5000 po able analyze (GEOTECH L d.,
Co en y, G ea B i ain) once a day in wo king days, p io o diges a e sampling and sub-
s a e dosing. The analyze used CH4 and CO2 in a ed senso s and O2 and H2S elec o-
chemical senso s. Du ing he gas composi ion measu emen s, he eac o o a ion was
s opped. The biogas sample was sucked h ough one al e and e u ned o he gas space
o he eac o by an adjacen al e. Immedia ely a e he analysis o he biogas composi-
ion, a dissol ed hyd ogen senso was inse ed in o he liquid phase. A al e (32 mm
diame e ), commonly used o diges a e sampling and subs a e dosing, was used o inse
he H2 senso . The senso was sealed wi h silicone ubbe . I usually ook 15 min o s abi-
lize he senso in he anae obic slu y. The o a ion o he eac o was esumed only a e
emo ing he senso and inse ing a dose o he subs a e.
The dissol ed hyd ogen senso was he MS 08 ampe ome ic mic osenso (AMT
Analysenmess echnik GmbH, Ros ock, Ge many) wi h a s a ed de ec ion limi o 0.2 µg
dm−3 H2(l) and a ange o up o 1.5 mg dm−3 H2(l); see Figu e 2. The H2 senso used i s own
sepa a e empe a u e senso . The ips o bo h senso s we e always placed app oxima ely
30 mm below he su ace o he slu y. A e measu ing H2(l), a diges a e sample (app ox-
ima ely 0.9× he olume o he subs a e dose) was d ained o analysis, and a dose o he
subs a e was in oduced in o he eac o . Thus, he diges a e sampling was pe o med a
he same equency as he subs a e sampling.
Figu e 2. Ampe ome ic mic osenso and i s loca ion in he eac o .
Figu e 2. Ampe ome ic mic osenso and i s loca ion in he eac o .
The ollowing analyses we e egula ly pe o med on subs a e and diges a e sam-
ples: he de e mina ion o pH wi h a 340i me e a SenTix 41 p obe (WTW, Weilheim,
P ocesses 2021,9, 106 7 o 11
Ge many) [
23
], he de e mina ion o o al solids (TS, d ying a 105
◦
C in an O
2
a mosphe e
o cons an weigh , 2.0% RSD) by a DLB 160 3A mois u e analyze wi h a halogen lamp
(KERN, Balingen, Ge many) [
24
], he de e mina ion o he con en o o ganic subs ances,
namely loss on igni ion (Vola ile Solids, VS, igni ing a 550
◦
C in an O
2
a mosphe e o
cons an weigh , 5.0% RSD) by he mog a ime ic analyze TGA 701 (LECO, Ben on Ha -
bo , MI, USA) [
25
]. The VFA/TIC a io in he diges a e slu y was de e mined by a TIM
BIOGAS V02.2 au oma ic i a o (HACH Lange, Düsseldo , Ge many) [26].
3. Resul s and Discussion
Du ing he 230 days o mono-diges ion, he TS con en o he subs a e was mos
o en a ound 16%, wi h a loss on igni ion o abou 93% TS. The o ganic loading a e (OLR)
was pu pose ully changed so ha he e was a signi ican o e load wi h he e olu ion o
hyd ogen and subsequen eco e y. Calcula ed o all 230 days o he expe imen , he OLR
was a ied in he ange o 0–3.02 kg
VS
m
−3
d
−1
, wi h a mean o 0.858 kg
VS
m
−3
day
−1
.
Calcula ed jus o he days o eeding, he mean OLR was 1.396 kg
VS
m
−3
d
−1
, which is
s ill a a he low load o he mesophilic eac o . The mean alue o hyd aulic e en ion ime
(HRT) was 259 days. The ela ion be ween OLR and HRT is isible in Figu e 3. Due o he easy
o e load, i would be possible o conside sho ening he HRT only when co- e men ing
he subs a e in a mo e nu i ionally balanced mix u e.
P ocesses 2021, 9, x FOR PEER REVIEW 8 o 13
Figu e 3. Rela ion be ween o ganic loading and e en ion ime.
The no malized biogas p oduc ion om inoculum was high due o he ansi ion
om psych ophilic o mesophilic p ocess, bu wi hin 3 days i d opped apidly. This was
ollowed by a pe iod o g adual acclima iza ion and an inc ease in gas p oduc ion un il
day 107. The mos e icien p ocess ook place a a load o abou 2.5 kgVS m−3 d−1 and an
HRT o 65 days. Un il day 123, he CH4 con en emained high, bu gas p oduc ion was
al eady declining due o o e load. I was no un il a ound day 170 ha he p ocess s abi-
lized again due o he omission o subs a e doses and, subsequen ly, only low loading
(see Figu e 4). Sho ly a e ebalancing, CH4 p oduc ion was unusually high, which is
expec ed due o he me hana ion o accumula ed ola ile acids.
The main pa ame e s o he diges a e a e lis ed in Table 1. F om Figu e 5, i is clea
ha he pH alue in he eac o dec eased much la e han when o e loading s a ed, and
VFA accumula ion s a ed, which is also well-known in o ma ion. The VFA/TIC limi
alue de e mining s abili y o a gi en p ocess appea s o be app oxima ely 0.4. I is he e-
o e in line wi h he commonly s a ed ange [26].
Figu e 3. Rela ion be ween o ganic loading and e en ion ime.
The no malized biogas p oduc ion om inoculum was high due o he ansi ion
om psych ophilic o mesophilic p ocess, bu wi hin 3 days i d opped apidly. This was
ollowed by a pe iod o g adual acclima iza ion and an inc ease in gas p oduc ion un il day
107. The mos e icien p ocess ook place a a load o abou 2.5 kg
VS
m
−3
d
−1
and an HRT
o 65 days. Un il day 123, he CH
4
con en emained high, bu gas p oduc ion was al eady
declining due o o e load. I was no un il a ound day 170 ha he p ocess s abilized again
due o he omission o subs a e doses and, subsequen ly, only low loading (see Figu e 4).
Sho ly a e ebalancing, CH
4
p oduc ion was unusually high, which is expec ed due o
he me hana ion o accumula ed ola ile acids.
The main pa ame e s o he diges a e a e lis ed in Table 1. F om Figu e 5, i is clea ha
he pH alue in he eac o dec eased much la e han when o e loading s a ed, and VFA
accumula ion s a ed, which is also well-known in o ma ion. The VFA/TIC limi alue
de e mining s abili y o a gi en p ocess appea s o be app oxima ely 0.4. I is he e o e in
line wi h he commonly s a ed ange [26].
P ocesses 2021,9, 106 8 o 11
P ocesses 2021, 9, x FOR PEER REVIEW 8 o 13
Figu e 3. Rela ion be ween o ganic loading and e en ion ime.
The no malized biogas p oduc ion om inoculum was high due o he ansi ion
om psych ophilic o mesophilic p ocess, bu wi hin 3 days i d opped apidly. This was
ollowed by a pe iod o g adual acclima iza ion and an inc ease in gas p oduc ion un il
day 107. The mos e icien p ocess ook place a a load o abou 2.5 kgVS m−3 d−1 and an
HRT o 65 days. Un il day 123, he CH4 con en emained high, bu gas p oduc ion was
al eady declining due o o e load. I was no un il a ound day 170 ha he p ocess s abi-
lized again due o he omission o subs a e doses and, subsequen ly, only low loading
(see Figu e 4). Sho ly a e ebalancing, CH4 p oduc ion was unusually high, which is
expec ed due o he me hana ion o accumula ed ola ile acids.
The main pa ame e s o he diges a e a e lis ed in Table 1. F om Figu e 5, i is clea
ha he pH alue in he eac o dec eased much la e han when o e loading s a ed, and
VFA accumula ion s a ed, which is also well-known in o ma ion. The VFA/TIC limi
alue de e mining s abili y o a gi en p ocess appea s o be app oxima ely 0.4. I is he e-
o e in line wi h he commonly s a ed ange [26].
Figu e 4. Biogas p oduc ion and i s CH4con en .
P ocesses 2021, 9, x FOR PEER REVIEW 9 o 13
Figu e 4. Biogas p oduc ion and i s CH4 con en .
Figu e 5. VFA and TIC e sus pH.
Thanks o a signi ican educ ion in dosing, he ex eme o e load (VFA/TIC peak o
11.4) was o e come in abou 55 days. The cou se o he dissol ed hyd ogen concen a ion
is shown in Figu e 6. The dissol ed hyd ogen concen a ion measu ed by he ampe ome -
ic mic osenso anged om 0.039 mg dm−3 o 0.425 mg dm−3. Apa om o e load, he
ypical H2(l) concen a ion was 0.12 ± 0.04 mg dm−3, co esponding o abou 6000 Pa pa ial
p essu e. When o e loaded, i eached 0.40 mg dm−3 H2(l), co esponding o abou 20,000
Pa pa ial p essu e. Simila ly, high alues we e measu ed e en a e he ini ial hea ing o
he inoculum a he beginning o he expe imen . I he inc eased hyd ogen pe sis ed o
se e al days, he acid con en inc eased apidly. The maximum H2(l) concen a ion meas-
u ed by us he e o e app oxima ely co esponds o he alue gi en by Dohányos [6]. The
small peak o he H2(l) ha appea s a ound day 105 was caused by he ou days o eac o
s a a ion due o Eas e b eak ollowed by he high doses o subs a e.
Figu e 5. VFA and TIC e sus pH.
Thanks o a signi ican educ ion in dosing, he ex eme o e load (VFA/TIC peak o
11.4) was o e come in abou 55 days. The cou se o he dissol ed hyd ogen concen a ion is
shown in Figu e 6. The dissol ed hyd ogen concen a ion measu ed by he ampe ome ic
mic osenso anged om 0.039 mg dm
−3
o 0.425 mg dm
−3
. Apa om o e load, he
ypical H
2
(l) concen a ion was 0.12
±
0.04 mg dm
−3
, co esponding o abou 6000 Pa
pa ial p essu e. When o e loaded, i eached 0.40 mg dm
−3
H
2
(l), co esponding o abou
20,000 Pa pa ial p essu e. Simila ly, high alues we e measu ed e en a e he ini ial hea -
ing o he inoculum a he beginning o he expe imen . I he inc eased hyd ogen pe sis ed
o se e al days, he acid con en inc eased apidly. The maximum H
2
(l) concen a ion
measu ed by us he e o e app oxima ely co esponds o he alue gi en by Dohányos [
6
].
The small peak o he H
2
(l) ha appea s a ound day 105 was caused by he ou days o
eac o s a a ion due o Eas e b eak ollowed by he high doses o subs a e.
An al e na i e p ocess s abili y pa ame e was p oposed as he a io o dissol ed
hyd ogen concen a ion o neu aliza ion capaci y; see Figu e 7. Howe e , so a , his
pa ame e does no appea o be signi ican ly mo e sensi i e han he VFA/TIC a io
used. A e he heigh co ec ion o he peaks, he on o bo h peaks is si ua ed on he
same days o he p ocess. I will be necessa y o es his beha io in se e al di e en
co- e men a ion p ocesses. In he case o he p ocess moni o ed he e, eaching he alue
o 1.0 o he new pa ame e (H
2
(l)/TIC) * 48,000 announced he onse o ins abili y, bu
his happened app oxima ely on he same day when he VFA/TIC a io inc eased abo e
P ocesses 2021,9, 106 9 o 11
0.4. The acid o ma ion is a apid p ocess. Some p ocesses a e likely o equi e o e load
supp ession a an ea ly s age. Gi en ha he de ec ion limi o H
2
(l) o he ampe ome ic
senso is 0.5
µ
g dm
−3
, which co esponds o a pa ial p essu e o H
2
(g) o 10 Pa, and
some li e a u e epo s an incipien o e load no mally om 2 Pa [
9
], i is app op ia e
o sea ch o he mo e sensi i e senso . Fu he mo e, he obus ness and se ice li e o
he ampe ome ic senso in he sludge en i onmen is no e y sa is ac o y. The he mal
conduc i i y de ec o should mee he equi emen s, bu he e may be a p oblem wi h
sensi i i y. Fu he mo e, he cou se o H
2
(l) concen a ion should be co ela ed wi h he
concen a ion o acids de e mined by GC-MS o iso achopho esis.
P ocesses 2021, 9, x FOR PEER REVIEW 10 o 13
Figu e 6. Dissol ed H2 concen a ion.
An al e na i e p ocess s abili y pa ame e was p oposed as he a io o dissol ed hy-
d ogen concen a ion o neu aliza ion capaci y; see Figu e 7. Howe e , so a , his pa-
ame e does no appea o be signi ican ly mo e sensi i e han he VFA/TIC a io used.
A e he heigh co ec ion o he peaks, he on o bo h peaks is si ua ed on he same
days o he p ocess. I will be necessa y o es his beha io in se e al di e en co- e -
men a ion p ocesses. In he case o he p ocess moni o ed he e, eaching he alue o 1.0
o he new pa ame e (H2(l)/TIC) * 48,000 announced he onse o ins abili y, bu his hap-
pened app oxima ely on he same day when he VFA/TIC a io inc eased abo e 0.4. The
acid o ma ion is a apid p ocess. Some p ocesses a e likely o equi e o e load supp es-
sion a an ea ly s age. Gi en ha he de ec ion limi o H2(l) o he ampe ome ic senso is
0.5 μg dm−3, which co esponds o a pa ial p essu e o H2 (g) o 10 Pa, and some li e a u e
epo s an incipien o e load no mally om 2 Pa [9], i is app op ia e o sea ch o he
mo e sensi i e senso . Fu he mo e, he obus ness and se ice li e o he ampe ome ic
senso in he sludge en i onmen is no e y sa is ac o y. The he mal conduc i i y de ec-
o should mee he equi emen s, bu he e may be a p oblem wi h sensi i i y. Fu he -
mo e, he cou se o H2(l) concen a ion should be co ela ed wi h he concen a ion o ac-
ids de e mined by GC-MS o iso achopho esis.
Figu e 6. Dissol ed H2concen a ion.
P ocesses 2021, 9, x FOR PEER REVIEW 10 o 11
Figu e 7. Dissol ed H
2
e sus VFA/TIC a io.
The g aph in Figu e 7 depic s he alues o he VFA/TIC and H
2(l)
a iables du ing
he expe imen . We ha e compu ed he Pea son co ela ion coe icien o hese a iables,
and he esul is 0.331, wi h he p- alue almos 0. The linea co ela ion be ween hese wo
a iables is e y weak, as may be seen om he g aph. The alues o he H
2
(l)
a iable do
no ollow he VFA/TIC a iable cou se. The amoun o dissol ed hyd ogen is no a ec ed
by he eac o o e loading, and o he ac o s play a ole in i s concen a ion change.
4. Conclusions
The labo a o y expe imen con i med he possibili y o using an ampe ome ic mi-
c osenso o dissol ed hyd ogen o de ec he ins abili y o he anae obic diges ion p o-
cess. Un o una ely, he de ec ion limi o he speci ic senso used was no low enough o
ully e eal he beginnings o o e loading. So a , i has no been possible o p o e ha
he newly p oposed p ocess s abili y pa ame e in he o m o he a io o dissol ed hy-
d ogen concen a ion and neu aliza ion capaci y would signi ican ly help o de ec o e -
load. The i s s a is ical e alua ion o he ela ionship shows ha he e should be a linea
co ela ion be ween he VFA/TIC and H
2
(l), bu i is a ec ed by he o e loading o he
eac o . Deep expe imen s need o be pe o med o e alua e he ela ionships.
Au ho Con ibu ions: D.P. was esponsible o he labo a o y wo k, pe o ming he expe imen s.
J.R. is esponsible o he s udy design and he main ideas. J.P. was esponsible o da a analysis,
K.S. was esponsible o he o e all pape p epa a ion and language, R.B. ocused on he o e all
expe imen and i s ele an ness. All au ho s ha e ead and ag eed o he published e sion o he
manusc ip .
Funding: The wo k was suppo ed by he Minis y o Educa ion, You h and Spo s o he Czech
Republic unde he p ojec s ERDF “Ins i u e o En i onmen al Technology—Excellen Resea ch”
[No. CZ.02.1.01/0.0/0.0/16_019/0000853], La ge Resea ch In as uc u e ENREGAT [No.
LM2018098], and he Ope a ional P og amme Resea ch, De elopmen and Educa ion [No.
CZ.02.1.01./0.0/0.0/17_049/0008419 COOPERATION.
Ins i u ional Re iew Boa d S a emen : No applicable.
In o med Consen S a emen : No applicable.
Figu e 7. Dissol ed H2 e sus VFA/TIC a io.
The g aph in Figu e 7depic s he alues o he VFA/TIC and H
2(l)
a iables du ing
he expe imen . We ha e compu ed he Pea son co ela ion coe icien o hese a iables,
and he esul is 0.331, wi h he p- alue almos 0. The linea co ela ion be ween hese wo
a iables is e y weak, as may be seen om he g aph. The alues o he H
2
(l) a iable do
no ollow he VFA/TIC a iable cou se. The amoun o dissol ed hyd ogen is no a ec ed
by he eac o o e loading, and o he ac o s play a ole in i s concen a ion change.