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Enhanced ni ogen emo al o low ca bon was ewa e in deni i ica ion bio eac o s by
u ilizing indus ial was e owa d ci cula economy
© Else ie L d.
Accep ed e sion (Final d a )
Kiani, Sepideh; Kujala, Ka ha ina; Pulkkinen, Jani; Aal o, Sanni L.; Suu näkki, Su i;
Kiu u, Tapio; Tii ola, Ma ja; Klø e, Bjø n; Ronkanen, Anna-Kaisa
Kiani, S., Kujala, K., Pulkkinen, J., Aal o, S. L., Suu näkki, S., Kiu u, T., Tii ola, M., Klø e, B., &
Ronkanen, A.-K. (2020). Enhanced ni ogen emo al o low ca bon was ewa e in deni i ica ion
bio eac o s by u ilizing indus ial was e owa d ci cula economy. Jou nal o Cleane P oduc ion,
254, A icle 119973. h ps://doi.o g/10.1016/j.jclep o.2020.119973
2020
Jou nal P e-p oo
Enhanced ni ogen emo al o low ca bon was ewa e in deni i ica ion bio eac o s by
u ilizing indus ial was e owa d ci cula economy
Sepideh Kiani, Ka ha ina Kujala, Jani Pulkkinen, Sanni L. Aal o, Su i Suu näkki,
Tapio Kiu u, Ma ja Tii ola, Bjø n Klø e, Anna-Kaisa Ronkanen
PII: S0959-6526(20)30020-2
DOI: h ps://doi.o g/10.1016/j.jclep o.2020.119973
Re e ence: JCLP 119973
To appea in: Jou nal o Cleane P oduc ion
Recei ed Da e: 30 Augus 2019
Re ised Da e: 12 Decembe 2019
Accep ed Da e: 2 Janua y 2020
Please ci e his a icle as: Kiani S, Kujala K, Pulkkinen J, Aal o SL, Suu näkki S, Kiu u T, Tii ola M, Klø e
Bjø, Ronkanen A-K, Enhanced ni ogen emo al o low ca bon was ewa e in deni i ica ion bio eac o s
by u ilizing indus ial was e owa d ci cula economy, Jou nal o Cleane P oduc ion (2020), doi: h ps://
doi.o g/10.1016/j.jclep o.2020.119973.
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Sepideh kiani: Concep ualiza ion, Me hodology, Valida ion, Fo mal analysis, In es iga ion, Resou ces, W i ing
- O iginal D a , W i ing - Re iew & Edi ing, Visualiza ion
Anna-Kaisa Ronkanen: Concep ualiza ion, Me hodology, Valida ion, Fo mal analysis, In es iga ion,
Resou ces, W i ing - O iginal D a , W i ing - Re iew & Edi ing, Visualiza ion, Supe ision, P ojec
adminis a ion
Bjö n Klö e: Concep ualiza ion, Me hodology, Valida ion, Fo mal analysis, In es iga ion, Resou ces, W i ing -
O iginal D a , W i ing - Re iew & Edi ing, Visualiza ion, Supe ision, P ojec adminis a ion
Ka ha ina Kujala: Me hodology, Valida ion, Fo mal analysis, In es iga ion, Resou ces, W i ing - O iginal
D a , W i ing - Re iew & Edi ing, Visualiza ion, Supe ision
Jani Pulkkinen: Valida ion, Fo mal analysis, In es iga ion, Resou ces, W i ing - Re iew & Edi ing,
Tapio Kiu u: In es iga ion, Resou ces, P ojec adminis a ion
Sanni L. Aal o: Me hodology, Valida ion, Fo mal analysis, In es iga ion, Resou ces, W i ing - Re iew &
Edi ing, Visualiza ion
Su i Suu näkki: Me hodology, Valida ion, Fo mal analysis, In es iga ion, Resou ces, W i ing - Re iew &
Edi ing, Visualiza ion
Ma ja Tii ola: Me hodology, Valida ion, Fo mal analysis, In es iga ion, Resou ces, W i ing - Re iew &
Edi ing, Visualiza ion, Supe ision, P ojec adminis a ion
1
Enhanced ni ogen emo al o low ca bon was ewa e in
deni i ica ion bio eac o s by u ilizing indus ial was e owa d
ci cula economy
Sepideh Kiani
a
*, Ka ha ina Kujala
a
, Jani Pulkkinen
b
, Sanni L. Aal o
c,d
, Su i Suu näkki
c
, Tapio Kiu u
b
, Ma ja
Tii ola
c
, Bjø n Klø e
a
and Anna-Kaisa Ronkanen
a
a
Wa e , Ene gy and En i onmen al Enginee ing Resea ch Uni , Facul y o Technology, P.O. Box 4300, FI-
90014 Uni e si y o Oulu, Finland
b
Na u al Resou ces Ins i u e Finland, Su on ie 9A, 40500 Jy äskylä, Finland
c
Depa men o Biological and En i onmen al Science, Nanoscience Cen e , 40014 Uni e si y o Jy äskylä,
Finland
d
Depa men o En i onmen al and Biological Sciences, Uni e si y o Eas e n Finland, P.O. Box 1627,
70211 Kuopio, Finland
*Co esponding au ho : Sepideh Kiani (Email: sepide[email p o ec ed])
1
Abs ac 1
Aquacul u e needs p ac ical solu ions o nu ien emo al o achie e sus ainable ish p oduc ion. Passi e 2
deni i ying bio eac o s may p o ide an ecological, low-cos and low-main enance app oach o was ewa e 3
ni ogen emo al. Howe e , inno a i e o ganic ma e ials a e needed o enhance ni a e emo al om he low 4
ca bon e luen s in in ensi e eci cula ing aquacul u e sys ems (RAS). In his s udy, we es ed h ee 5
addi ional ca bon sou ces, including biocha , d ied Sphagnum sp. moss and indus ial po a o esidues, o 6
enhance he pe o mance o woodchip bio eac o s ea ing he low ca bon RAS was ewa e . We assessed 7
ni a e (NO
3-
)
emo al and mic obial communi y composi ion du ing a one-yea in si u column es wi h eal 8
aquacul u e was ewa e . We ound no signi ican di e ences in he NO
3-
emo al a es be ween he 9
woodchip-only bio eac o and bio eac o s wi h a zone o biocha o Sphagnum sp. moss (maximum emo al 10
a e 31-33 g NO
3-
-N m
-3
d
-1
), bu po a o esidues inc eased NO
3-
emo al a e o 38 g NO
3-
-N m
-3
d
-1
, wi h 11
s able annual educ ion e iciency o 93%. The eadily a ailable ca bon eleased om po a o esidues 12
inc eased NO
3-
-N emo al capaci y o he bio eac o e en a highe in low concen a ions (>52 mg L
-1
). The 13
mic obial communi y and i s p edic ed unc ional po en ial in he po a o esidue bio eac o di e ed ma kedly 14
om hose o he o he bio eac o s. Adding po a o esidues o woodchip ma e ial enabled smalle bio eac o 15
size o be used o NO
3-
emo al. This s udy in oduced indus ial po a o by-p oduc as an al e na i e ca bon 16
sou ce o he woodchip deni i ica ion p ocess, and he encou aging esul s may pa e he way owa d 17
g ow h o blue bioeconomy using he RAS. 18
19
Keywo ds: Reci cula ing aquacul u e sys em, woodchip bio eac o , ca bon sou ce, po a o esidues, ni a e, 20
mic obial communi y 21
22
23
24
25
26
27

2
1 In oduc ion
28
Reci cula ing aquacul u e sys ems (RAS) a e en i onmen ally iendly solu ions ha aim o achie e ze o 29
was e om ish p oduc ion. Al hough RAS ha e been used o mo e han 10 yea s in di e en coun ies, 30
including wo la ges RAS in Finland wi h a p oduc ion capaci y o o e 4000 ons, ni a e (NO
3-
)
emo al is 31
s ill a c i ical challenge (Pulkkinen e al., 2018). Remo al o NO
3-
is a challenge as aquacul u e was ewa e 32
has low ca bon (C) bu high ni ogen (N) concen a ions. A ew p e ious s udies ha e examined he use o 33
deni i ying bio eac o s o ea ing aquacul u e e luen . So a , such s udies ha e ocused on RAS e luen s 34
wi h high chemical oxygen demand (COD) (Lepine e al., 2016), added bica bona e (HCO
-3
) o inle wa e 35
( on Ahnen e al., 2016b) and dilu ed e luen om an ou doo ish a m wi h low eci cula ion in ensi y and 36
low NO
3-
-N concen a ion (~6 mg L
-1
) ( on Ahnen e al., 2018, 2016a). In con as , ea men o highly 37
in ensi e indoo RAS e luen s wi h low COD (12.9 ± 1.8 mg L
-1
) and high NO
3-
-N concen a ion (>50 mg 38
L
-1
) has ecei ed li le a en ion. 39
In deni i ying bio eac o s, ni ogen (N) is emo ed by he e o ophic deni i ie s con e ing NO
3-
o ni ogen 40
gas unde anoxic condi ions. Unde ni a e- ich condi ions, his p ocess depends on he a ailabili y o he 41
ca bon sou ce as he o ganic elec on dono (Wang and Chu, 2016). Ex e nal ca bon sou ces, such as ace a e 42
o me hanol, a e o en supplied o he sys em o achie e e icien deni i ica ion (Che chi e al., 2009). 43
Howe e , he cos o ca bon addi ion is ypically high (Zhang e al., 2016) and he p ocess needs egula ion 44
o p e en o e - o unde -dosing o he liquid ca bon sou ces (Roche e al., 2015). Solid ca bon sou ces can 45
p o ide a cos -e ec i e al e na i e o he classical ca bon sou ces men ioned abo e. In ecen yea s, esea ch 46
has ocused on solid ca bon sou ces wi h high quali y, op imal e iciency and slow- elease abili y in he 47
ea men o excessi ely ni a e-con amina ed wa e , pa icula ly su ace wa e (Beu el e al., 2016) and 48
g oundwa e (Zhang e al., 2012). Wood-pa icle p oduc s (e.g. woodchip and sawdus ) ha e been widely 49
used, due o hei abili y o supply ca bon o he deni i ica ion p ocess o 5-15 yea s and hus allow good 50
NO
3-
emo al wi h minimum bio eac o main enance (Schippe e al., 2010). Howe e , he la ge space 51
equi emen o ull-scale woodchip bio eac o s has p omp ed e o s o enhance he deni i ica ion a e by 52
using inno a i e na u al ca bon sou ces (Tangsi e al., 2017). Inexpensi e indus ial ood by-p oduc s, such 53
3
as indus ial po a o esidue, could ha e high po en ial o be u ilized in iden i ying bio eac o o enhance 54
ni a e emo al. Po a o indus ies can gene a e 20-25 % was e om peeling, imming and cu ing p ocesses 55
(Liang and McDonald, 2014). 56
This s udy examined he use o a deni i ying bio eac o o ea indoo in ensi e RAS e luen wi h low 57
COD and high NO
3-
concen a ion, as pa o he unique RAS esea ch pla o m (see Pulkkinen e al., 2018), 58
and compa ed di e en ca bon sou ces, including po a o esidue, o imp o ing he ni ogen emo al 59
pe o mance o woodchip bio eac o s. The o e all aim was o e alua e he pe o mance o deni i ying 60
bio eac o s in emo ing NO
3-
om aquacul u e was ewa e wi h low COD o a pe iod o o e one yea . 61
Speci ic objec i es we e o (1) s udy he sui abili y o wood-based bio eac o s o ea ing RAS e luen , (2) 62
assess whe he he NO
3-
emo al pe o mance o woodchip p ocess can be enhanced by addi ional ca bon 63
sou ces, (3) o assess he e ec o di e en ca bon sou ces on he mic obial communi y composi ion in 64
di e en compa men s o he bio eac o s, and (4) o iden i y dominan bac e ia and hei unc ional po en ial 65
in he bio eac o s s udied. The in en ion was o ind solu ions o imp o ing wa e ea men and o 66
enhancing NO
3-
emo al in he eci cula ing aquacul u e sys ems. 67
2 Ma e ial and me hods
68
2.1 RAS e luen wa e quali y
69
The s udy was conduc ed a he Laukaa ish a m o he Na u al Resou ces Ins i u e Finland (LUKE) in 70
cen al Finland, in he esea ch pla o m examining RAS. The RAS design is desc ibed in de ail in Pulkkinen 71
e al. (2018). In b ie , e luen was ob ained om a RAS consis ing o a eed collec o uni , swi l sepa a o , 72
d um il e (60 µm mesh) and ixed bed bio eac o , ollowed by a mo ing bed bio eac o and a ickling 73
il e . In o de o p e en any changes in wa e chemis y, mic obiology o wa e empe a u e, all es s we e 74
pe o med using he na u al RAS e luen . The e luen is cha ac e ised by low ca bon (15.3 mg L
-1
on 75
a e age), bu high N con en (mean NO
3-
-N con en 34.7 mg L
-1
) (Table 1). Due o he e icien ni i ica ion 76
uni be o e he bio eac o s, NO
3-
is domina ing N ac ion. 77
Table 1. Mean in low wa e quali y pa ame e s (SD = s anda d de ia ion, n = numbe o sample) 78
4
Wa e quali y pa ame e s In low (mean ± SD) n
To al o ganic ca bon (mg L
-
1
) 15.3 ± 2.1 5
Dissol ed o ganic ca bon (mg L
-
1
) 14 ± 1.3 5
Chemical oxygen demand (mg L
-
1
) 12.9 ± 1.8 5
Biological oxygen demand (mg L
-
1
) 3.8 ± 2.2 13
Ni a e-ni ogen (mg L
-
1
) 34.7 ± 15.6 27
Ni i e-ni ogen (mg L
-
1
) 0.1 ± 0.06 30
Ammonium-ni ogen (mg L
-
1
) 0.5 ± 0.2 30
Dissol ed oxygen (mg L
-
1
) 8.1 ± 1.7 29
pH 6.9 ± 0.2 28
Oxida ion
-
educ ion po en ial (
Eh
,
mV)
178.6 ± 60.4 35
Alkalini y (mg CaCO
3
L
-
1
) 54.2 ± 18 25
Sulpha e (mg L
-
1
) 10.5 ± 3.2 24
2.2 Bio eac o design
79
The pe o mance o deni i ying bio eac o s was s udied in ou anspa en ac ylic columns (0.1 m diame e 80
× 0.32 m high) wi h upwa d low di ec ion applying a heo e ical e en ion ime (HRT) o 48 h a con olled 81
empe a u e (15.5±0.8°C) (Fig. 1). In each column, he eac i e media we e placed on op o an ine qua z 82
g a el bed, om which hey we e sepa a ed by plas ic ne ing wi h 2 mm po e size, o p e en clogging wi h 83
ma e ials con aining o ganic ma e . A cons an in low a e o 0.6 mL min
-1
was applied o each bio eac o 84
o 346 days, using a pe is al ic pump. The upwa d low di ec ion and he qua z g a el laye a he base o 85
he columns p e en ed he de elopmen o p e e en ial low pa hways and ensu ed uni o m dis ibu ion o 86
low in o he columns. The columns consis ed o packed-media zones (zone 1, zone 2, zone 3) con aining 87
woodchips, indus ial po a o was e, biocha o d ied Sphagnum sp. moss in he a ios shown in Fig. 1. The 88
packed-media has no been eplaced du ing he s udy pe iod. All bio eac o s wi h addi ional laye con ain 89
same o al olume o woodchips. Howe e , Sphagnum sp. moss was mixed wi h woodchips in he zone 2, 90
due o i s di e en cha ac e is ic and small pa icle size dis ibu ion. I is well known ha na u al pea has 91
5
ypically low hyd aulic conduc i i y (e.g. Ronkanen and Klø e 2005), which could cause isks in longe 92
HRT o e en clogging o he bio eac o . In o de o a oid his, moss was mixed wi h woodchips. The 93
packed-media zones we e sepa a ed om he ou le ee wa e zone by a ixed pe o a ed PVC pla e 94
( hickness 5 mm) a a heigh o 4.5 cm om he op o he column. The columns we e sealed a bo h ends o 95
p o ide con olled condi ions. 96
The selec ed ca bon sou ces had di e en C/N a ios, anging om 28 o 249 (Table 2). Woodchips had he 97
highes C/N a io, bu biocha con ained he highes amoun o ca bon. The used woodchips we e ob ained 98
locally om esh bi ch ees (p o ided by he ene gy company Vapo G oup). The a e age woodchip size 99
was a ound 3 cm × 1.5 cm × 0.4 cm and mean po osi y 63%. The Sphagnum sp. moss used was common 100
mi e lo a p o ided by Vapo G oup. The biocha (po osi y 46%) was ob ained om RPK Hiili Oy. The 101
po a o ma e ial es ed comp ised indus ial esidues om POHJOLAN PERUNA Oy wi h a d y ma e 102
con en o 12% (de e mined a e d ying he ma e ial a 105°C o 24 h). 103
P io o he expe imen s, solid ma e ials (woodchips and biocha ) we e washed wi h dis illed wa e and 104
sa u a ed o 48 h. In o de o p e en e men a ion, he po a o esidues we e kep in he eeze p io o use. 105
The ozen po a o esidues we e de os ed a oom empe a u e o 8 h be o e he es . 106
12
s a , bu only a e s able deni i ica ion a es a e es ablished and low ni i e concen a ions a e de ec ed in 211
he ou low. 212
The in low NH
4+
-N concen a ion anged be ween 0.17-1.0 mg L
-1
(Table 1; Fig. S1b). Low NH
4+
-N 213
p oduc ion was de ec ed in all bio eac o s, wi h ou low concen a ions o 0.8±0.5 mg L
-1
, 0.9±0.5 mg L
-1
, 214
0.9±0.6 mg L
-1
and 3.8±3.4 mg L
-1
in BR1, BR2, BR3 and BR4, espec i ely. Less han 2 mg L
-1
o NH
4+
-N 215
was eco ded in he i s h ee weeks in BR1-BR3 (Fig. S1b). Howe e , he bio eac o wi h po a o esidues 216
(BR4) showed ela i ely high NH
4+
-N, wi h a mean concen a ion o 10 mg L
-1
, in he i s 10 days o he 217
expe imen , bu i hen declined o lowe han 4 mg L
-1
o each he backg ound le el. The con inuous 218
p oduc ion o ammonium in BR4 indica es he occu ence o dissimila i y ni a e educ ion o ammonium 219
(DNRA). In gene al, a educing en i onmen and high TOC/NO
3-
a io (1400/15-110/16 in BR4; days 1-70) 220
can indica e he occu ence o DNRA (K a e al., 2014; an Rijn e al., 2006). DNRA has also been 221
obse ed in p e ious woodchip bio eac o s udies (Lu e al., 2013; Zhao e al., 2018). Reducing condi ions, 222
indica ed by Eh alues, we e also seen in his s udy, which led he sys em o SO
42-
educ ion (Fig. 4). 223
In he s a -up phase, all bio eac o s eleased DOC. The a e o elease was highes in BR4, wi h ou low 224
concen a ions o 1380 mg L
-1
measu ed on day 6 a e s a -up (Table. S1). The DOC elease om he o he 225
bio eac o s was much lowe (<100 mg L
-1
; Table S1). Wi hin 70 days a e s a -up, ou low DOC 226
concen a ion dec eased o 81 mg L
-1
in BR4 and o he backg ound le el (14 ± 1.3 mg DOC L
-1
) in BR1-227
BR3 (Table S1).
Ini ial ca bon con en lush-ou is common in bio eac o s. The s a -up COD concen a ion 228
in he ou low anged 59-940 mg L
-1
in BR1-BR4 (Table. S1) exceeding empo a ily he maximum 229
concen a ion o 42 mg L
-1
obse ed in Finnish i e s (Niemi and Raa eland, 2007). Howe e , s a -up phase 230
o he woodchip bio eac o is sho compa ed o es ima ed li e ime (5-15 yea s), so he po en ial pollu ion o 231
ca bon is mino compa ed o he amoun o ni ogen emo ed. Lepine e al. (2016) epo ed an 232
app oxima ely 50-day lush-ou pe iod o a plywood bio eac o ea ing aquacul u e e luen a HRT o 42 h. 233
Somewha highe ca bon leaching (200 mgL
-1
) has been epo ed o bio eac o s packed wi h esh 234
woodchips and a mix u e o woodchips and biocha (Hassanpou e al., 2017; Hoo e e al., 2016). Release 235
o high DOC concen a ions o ecipien wa e bodies om use o bio eac o s as an end-o -pipe ea men 236
can ad e sely a ec aqua ic ecosys ems, e.g. by causing a DO concen a ion educ ion, ligh and empe a u e 237

13
changes (P ai ie, 2008; Solomon e al., 2015), esul ing in lowe ish p oduc ion (S asko e al., 2012). Hence, 238
a si es go e ned by s ic egula ions o when ecycling ou low o ish a ms, high DOC migh need o be 239
con olled. Schippe e al. (2010) iden i ied HRT as a ac o con olling he ini ial magni ude o DOC 240
deple ion and i s du a ion in wood-based bio eac o s. Howe e , he ac ha ca bon was mo e eadily 241
eleased om po a o esidues han om he o he ca bon sou ces used in his s udy p o es ha HRT is no 242
he only con olling ac o and ha ca bon quali y also plays a key ole. In he p esen s udy, he e was 243
signi ican ly lowe ou low DOC concen a ion o 53, 68 and 81 mg L
-1
in bio eac o s BR1, BR2 and BR3, 244
which can be pa ly explained by highe ni a e loading (Hassanpou e al., 2017) and pa ly by he ype o 245
ca bon sou ce used. Dependence o TOC leaching and a ia ions in NO
3-
-N concen a ion ha e also been 246
epo ed by Zhao e al. (2018). In o de o con ol he ca bon con en due o leaching, i is ecommended o 247
conside pos -bio eac o s ea men uni s (e.g. cons uc ed we land, sand il e ) o eci cula ing he s a -up 248
e luen back o he bio eac o (Schippe e al., 2010). 249
The SO
42-
concen a ions we e on a e age highe in he ou low han in he in low wa e s o BR1 and BR2, 250
indica ing leaching o p oduc ion o SO
42-
(Fig. 4). This esul ed in cumula i e leaching/p oduc ion o 165 g 251
and 474 g SO
42-
in BR1 and BR2, espec i ely, o he whole s udy pe iod. In con as , SO
42-
we e on a e age 252
lowe in ou low han in in low wa e s o BR3 and BR4 (Fig. 4), indica ing SO
42-
educ ion/ emo al. 253
Cumula i e SO
42-
emo al o 350 g and 546 g was obse ed in BR3 and BR4, espec i ely, o he whole 254
s udy pe iod. SO
42-
leaching/ emo al inc eased he SO
42-
concen a ion in he ou low by up o 20% 255
compa ed wi h he cumula i e in low SO
42-
o 2.6 kg. Sulpha e leaching/p oduc ion indica ed he po en ial o 256
in e nal sulphu cycling in bio eac o s wi h incomple e N emo al. BR1 and BR2 had incomple e ni a e 257
emo al du ing he s udy pe iod due o sulphide e-oxida ion o sulpha e by sulphu oxidizing bac e ia 258
(SOB), which can use oxygen o ni a e as elec on accep o (Faulwe e e al., 2009) (Fig. S1 and Fig. 3). 259
Sulpha e p oduc ion was obse ed p e iously by Lepine e el. (2016) o a woodchip bio eac o wi h 260
incomple e N emo al. Howe e , highe ni a e emo al in BR3 and BR4 combined wi h hei educed 261
condi ions (Fig.4) a o ed sulpha e educ ion. 262
14
Fig. 4. Sulpha e educ ion/ emo al (+ alues) and leaching/p oduc ion (- alues) in bio eac o s BR1-BR4 263
o e ime a di e en edox po en ial alues (Eh) in in low and ou low o each bio eac o . 264
Redox po en ial was on a e age +340, +354, +312 and +181 mV in BR1, BR2, BR3 and BR4, espec i ely 265
(Fig. 4), indica ing mo e oxidising condi ions in BR1-BR3 and mo e educing condi ions in BR 4. I is well-266
known ha deni i ica ion and mic obial sulpha e emo al cause decline in edox po en ial and ise in pH 267
(Jog and Pa y., 2006). In BR4, o he en i e s udy pe iod when ou le Eh educed om 412 o 116 mV, he 268
pH ended o inc ease abou 2.2 pH uni s ( om 4.6-6.82) (Fig. S 5). Simila ly, in BR1-3 by dec easing he 269
ou le edox po en ial, he pH inc eased 0.89,1.65 and 1.4 pH uni s, espec i ely
.
270
In low wa e pH was a he s able h oughou he expe imen (6.5-7.5) (Fig. 5). Ou low pH o bio eac o s 271
du ing s a -up was 6, 4.3, 5.2 and 3.8 in BR1 BR2, BR3 and BR4, espec i ely. I was hus lowe han 272
in low pH in he ea ly s ages o he expe imen , mos likely as a esul o elease o o ganic acids om he 273
packed ma e ials (Fig. 5). All bio eac o s showed lowe alkalini y in ou low han in in low du ing he s a -274
up pe iod (Fig. 5). A e 2-5 weeks, alkalini y p oduc ion was obse ed in all bio eac o s. 275
15
3.2 Fac o s a ec ing ni a e emo al in woodchip bio eac o s
276
The esul s o one-way ANOVA showed ha NO
3-
emo al a es o whole s udy pe iod did no di e 277
signi ican ly be ween BR1, BR2 and BR 3 (p=0.75), while ni a e emo al in BR4 was highe (Fig. 2d-2h). 278
In he i s h ee mon hs o he expe imen , when in low NO
3-
-N concen a ion a ied be ween 15 and 52 mg 279
L
-1
, all bio eac o s showed simila emo al a es (Fig. 2). A e ha , he bio eac o s esponded di e en ly o 280
inc easing NO
3-
-N in low concen a ions, e.g. he emo al a e declined in BR1-BR3 bu inc eased in BR4 281
(Fig. 2). BR4 eached i s maximum emo al a e o 38 g NO
3-
-N m
-3
d
-1
a he highes NO
3-
-N in low 282
concen a ion (70 mg L
-1
; days 152-184), whe eas BR1, BR2 and BR3 had a emo al a e o 9, 13 and 12 g 283
NO
3-
-N m
-3
d
-1
, espec i ely (Fig. 2). Those di e ences pe sis ed un il day 250, a e which all eac o s again 284
had simila s able emo al a es o a ound 15 g NO
3-
-N m
-3
d
-1
un il he end o he expe imen . Simila ly o 285
emo al a e, he NO
3-
emo al e iciency in BR1-BR3 showed luc ua ions h oughou he s udy pe iod (Fig. 286
2e and 2g). Howe e , BR4 eached s able emo al e iciency o 93% a e a pe iod o luc ua ion a s a -up 287
(Fig. 2h). 288
The wide ange o NO
3-
emo al a es (3-38 g NO
3-
-N m
-3
d
-1
) eco ded in all bio eac o s ollowed he NO
3-
-289
N in low concen a ion luc ua ions. High emo al a e in all bio eac o s occu ed when he in low had high 290
Fig.
5
.
Alkalini y p oduc ion (+ alues) and in low and ou low pH in bio eac o s (BR1-BR4).
16
NO
3-
-N concen a ions. This is consis en wi h p e ious indings ha in low concen a ions con ol emo al 291
a e (e.g. Schippe e al., 2010; Addy e al., 2016). 292
In he p esen s udy, NO
3-
emo al a e in BR4 inc eased signi ican ly wi h inc easing NO
3-
-N in low 293
concen a ion du ing he en i e s udy pe iod (R
2
= 0.93; emo al a e = 0.6 × in luen ni a e concen a ion - 294
1.85) (Fig. 6). This eg ession illus a ed he ac ual ela ionship be ween in low NO
3-
-N concen a ion and 295
emo al a e by excluding NO
3-
-N limi ed e en s (NO
3-
-N concen a ion <0.5 mg L
-1
) (Addy e al., 2016). 296
Likewise, bio eac o s BR1-BR3 showed a simila esponse o NO
3-
-N when days 152-212, wi h high NO
3-
-N 297
concen a ion (55-70 mg L
-1
), we e excluded om he da a (Fig. 6). The sha ply decline in NO
3-
-N emo al 298
du ing days 152-212 was caused due o exceeding he maximum deni i ica ion capaci y in hose bio eac o s. 299
This indica es ha NO
3-
emo al in BR1-BR3 was con olled by an independen pa ame e a high NO
3-
-N 300
concen a ions. The elease a e o deg adable ca bon om he packed media p esumably con olled NO
3-
301
emo al in his concen a ion ange (>52 mg L
-1
) (Schippe e al., 2010). Hence, he ype o ca bon sou ce 302
used in deni i ying bio eac o s can con ol NO
3-
emo al, by p o iding mo e ca bon a ailabili y and 303
di e en mic obial composi ion (Xu e al., 2018; Tangsi e al., 2017). Obse ed DOC in he bio eac o s 304
showed ha ca bon was much mo e eadily eleased om po a o esidues han om any o he o he ca bon 305
sou ces es ed (Table S1). The easily soluble ca bon in po a o esidues esul ed in apid o ma ion o a 306
complex mic obial communi y s uc u e wi h s ong adap i e g ow h o he new en i onmen (Zhao e al., 307
2018). 308
309
17
The maximum NO
3-
emo al a es obse ed in his s udy we e g ea e han hose p e iously epo ed (22 g 310
NO
3-
-N
m
-3
d
-1
)
(Da id e al., 2015; Schippe e al., 2010). This could be due o a combina ion o op imal 311
ac o s: su icien HRT (Lepine e al., 2016; Tangsi e al., 2017) as a esul o dis ibu ed upwa d low 312
(sec ion 2.2) combined wi h high NO
3-
in low concen a ion (Schippe e al., 2010), he o ganic C 313
compounds used (Gibe e al., 2008) and wa e empe a u e (Addy e al., 2016), he e 15.5 ± 1 °C (mean ± 314
SD). A emo al a e o >39 g NO
3-
m
-3
d
-1
epo ed by Lepine e al. (2016) o compa able wa e quali y was 315
associa ed wi h high COD:NO
3-
a io (0.86-1.66) in ea ed was ewa e . This a io can p o ide 42% COD 316
equi ed o deni i ica ion. The COD:NO
3-
a io has been epo ed o be a signi ican pa ame e a ec ing 317
deni i ica ion in bio eac o s (Ja a i e al., 2015). Howe e , in he p esen s udy in low COD p o ided less 318
han 8% o he C/N equi ed o comple e NO
3-
educ ion (Na kis e al., 1979). Hence, he epo ed NO
3-
319
emo al a es in his s udy ep esen he ne alues wi hou a con ibu ion om in low COD. Enhancing 320
ni a e emo al e iciency wi h di e en ca bon subs a es has been in es iga ed p e iously (Gebe e al., 321
2008; Schippe e al., 2010; Hashemi e al., 2011). Hashemi e al., (2011) imp o ed ni a e emo al o 36% 322
in wood bio eac o o 65%, 56 % and 77 % by u ilizing ba ley s aw, ice husk and da e palm lea , 323
espec i ely. Gebe e al., (2008) epo ed so wood (b anches and ba k wi h small amoun s o lea es om a 324
a ie y o ees) as op pe o ming subs a e in deni i ica ion e iciency (>98%) wi h deni i ica ion a e o ~ 325
Fig.
6
.
Ni a e emo al a e e sus ni a e in luen loading in BR1-
4 o he s udy pe iod o 346 days.

18
17 g NO
3-
-N m
-3
d
-1
. Howe e , o he in es iga ed ma e ials such as mix u e o wood chips, sh edded ba k and 326
opsoil, compos (ob ained om he biological decomposi ion o o ganic was es – wood immings, lea es, 327
o en ege ables and ood sc aps) and willow woodchips iden i ied as unsui able ca bon sou ces (see Gebe 328
e al., 2008). Wa neke e al., (2011) epo ed ni a e emo al o ~ 6.5, 6.2 and 3.5 g NO
3-
-N m
-3
d
-1
o whea 329
s aw, maize and g een was e ma e ials, espec i ely compa e o he emo al a e o 1.3 g NO
3-
-N m
-3
d
-1
in 330
so wood (pine) bio eac o o 2- old lowe ni a e inle concen a ion han used in his s udy. Howe e , 331
addi ional po a o esidue o woodchip bio eac o inc eased 13% o ni a e emo al o 38 g NO
3-
-N m
-3
d
-1
332
which is ema kably highe han epo ed emo al abo e. 333
3.3 Mic obial communi y composi ion and p ocess po en ial in he bio eac o s
334
A o al o
9261
quali y- il e ed sequences pe lib a y we e ob ained om wa e and solid samples om he 335
ou bio eac o s (Table 3). Lib a y co e age was ≥94% in all cases, indica ing ha he sequencing dep h was 336
su icien . The numbe o obse ed and Chao 1-es ima ed OTUs was signi ican ly lowe (p<0.001) in il e ed 337
wa e and solid ma e ial om BR4 han in co esponding samples om BR1-BR3. The Shannon di e si y 338
index was also signi ican ly lowe (p<0.001) in BR4 (4.5) han in BR1-BR3. 339
The mic obial communi y in BR4 di e ed s ongly om he mic obial communi y in BR1-BR3 (Figs. S 2A). 340
Smalle di e ences we e de ec ed be ween he mic obial communi ies in BR1-BR3 and be ween wa e and 341
solid samples om all bio eac o s (Figs. S2 B and C). In solid ma e ial, di e ences we e obse ed be ween 342
mic obial communi ies in zone 3 (i.e. op-laye woodchip) and in zone 2 in BR1, BR2 and BR4 (con aining 343
biocha , Sphagnum sp. moss and po a o esidues, espec i ely) bu no BR3 (con aining woodchips) (Fig. 1). 344
In wa e , he di e ences we e much less p onounced (Figs. S2 B and C). 345
Table 3. P oka yo ic di e si y in bio eac o s BR1-BR4. Numbe s o sequences a e aken om he o iginal 346
OTU ables, while all o he di e si y indica o s a e based on OTU ables a i ied a a dep h o 4098 347
sequences. A e age alues o 1-2 eplica es pe sampling poin a e shown. Zone 2 and zone 3 e e o he 348
ca bon sou ce ma e ial es ed and he op-laye woodchip, espec i ely, as indica ed in Fig. 1 349
No. o No. o Co e age OTUs OTUs Shannon
19
sequences samples (%) ichness
(obse ed)
ichness
(es ima ed)
a
BR 1:
Woodchip/
Biocha
Wa e Zone 2 8 550 2 95 441 802 4.64
Zone 3 7 310 2 95 468 761 4.68
Solid Zone 2 6 844 2 94 496 827 4.77
Zone 3 4 935 2 95 398 739 4.36
BR 2:
Woodchip/
Sphagnum
Wa e Zone 2 0
Zone 3 7 500 1 95 450 821 4.67
Solid Zone 2 7 358 1 96 383 697 4.42
Zone 3 6 711 2 96 354 674 4.2
BR 3:
Woodchip/
woodchip
Wa e Zone 2 8 198 2 95 433 749 4.53
Zone 3 8 304 2 94 480 854 4.72
Solid Zone 2 6 942 2 96 378 713 4.29
Zone 3 6 956 1 95 389 897 4.26
BR 4:
(Woodchip/
po a o)
Wa e
Zone 2 9 261 2 96 303 583 3.61
Zone 3 8 148 2 96 337 605 3.78
Solid Zone 2 9 256 2 97 287 505 3.67
Zone 3 8 359 2 96 296 578 3.39
a
OTUs ichness es ima ed by Chao1.
350
Only bac e ial sequences (no a chaeal sequences) we e de ec ed in he bio eac o s. In BR1-BR3, he 351
mic obial communi y was domina ed by P o eobac e ia, Bac e oide es and Ve ucomic obia (Fig. 7). 352
Wi hin he P o eobac e ia, Be ap o eobac e ia we e mos abundan (24-40% ela i e abundance), ollowed 353
by Gammap o eobac e ia (7-26%) and Alphap o eobac e ia (11-28%). In BR4, he mic obial communi y 354
was domina ed by Epsilonp o eobac e ia (15-36%), Bac e oide es (16-29%) and Fi micu es (17-34%) (Fig. 355
7). Amongs he mos abundan gene a, Uliginosibac e ium (up o 11% ela i e abundance), Sul u ospi illum 356
(up o 29%), P e o ella (up o 19%) and Lac obacillus (up o 18%) we e almos exclusi ely de ec ed in BR4, 357
while Rhodobac e (up o 4%), Sphingobium (up o 4%), Rhodo e ax (up o 5%), Pseudomonas (up o 13%), 358
The momonas (up o 6%) and Lu eolibac e (up o 10%) we e almos exclusi ely de ec ed in BR1-BR3 (Fig. 359
20
S3). The gene a Lac obacillus, P e o ella and Sul u ispi illum include known e men e s, some o which can 360
also educe ni a e o ammonium (e.g. K use e al., 2018; Sal e i e al., 2012). The gene a Rhodobac e , 361
Rhodo e ax, Pseudomonas and The momonas include known deni i ie s (e.g. Finne an e al., 2003; 362
Me gae e al., 2003). 363
Fig. 7. Composi ion o he mic obial communi y based on sequence analysis o bac e ial and a chaeal 16S 364
RNA genes om (A) solid ma e ial and (B) wa e samples om woodchip bio eac o s wi h a zone 365
con aining biocha (BR1), Sphagnum sp. moss (BR2), woodchip (BR3) and po a o esidues (BR4). A e age 366
ela i e abundances o 1-2 eplica es pe sample a e shown. Samples we e aken om he op-laye 367
woodchip (zone 3) and he ca bon sou ce ma e ial (zone 2). 368
Func ional p o iles o he bac e ial communi ies we e p edic ed based on 16S RNA gene sequences using 369
PICRUS . I p o ed possible o use a ound 31% o all OTUs and 83% (76-90%) o all sequences o 370
unc ional p edic ion. O e all unc ional p o iles o mic obiological communi ies we e a he simila in he 371
di e en bio eac o s. Selec ed unc ions ela ed o he ni ogen cycle we e assessed in mo e de ail (Fig. 8). 372
21
Func ions ela ed o deni i ica ion (Na G, NapA, Ni K, No B, No C, NosZ) and DNRA (Na G, NapA, 373
N A) we e p edic ed, while unc ions speci ic o ni i ica ion (AmoA, AmoB, AmoC) we e no p edic ed. 374
The memb ane-bound ni a e educ ase Na G was p edic ed in simila ela i e abundance in all bio eac o s, 375
while highe ela i e abundance o he pe iplasmic ni a e educ ase NapA was p edic ed in BR4 han in 376
BR1-BR3 (Fig. 7). The deni i ica ion-associa ed unc ions Ni K, No B, No C and NosZ we e p edic ed wi h 377
highe ela i e abundances o BR1-BR3 han o BR4, while he ni i e educ ase N A (which ca alyses he 378
educ ion o ni i e o ammonia in DNRA) was mo e equen ly p edic ed o BR4 (Fig. 8). This indica es 379
ha bio eac o s BR1-BR3 had highe p edic ed po en ial o deni i ica ion, while he bio eac o wi h po a o 380
esidues (BR4) had highe p edic ed po en ial o DNRA. The ni i e educ ase Ni K may also be p esen in 381
ni i ying o ganisms. Howe e , he con ibu ion o ni i ie s such as Ni ospi a sp. o Ni obac e sp. o Ni K 382
was only 0.15%. 383
Fig. 8. Rela i e abundance o p edic ed ni ogen cycle- ela ed genes in unc ional p o iles o (A) solid 384
ma e ial and (B) wa e samples om woodchip bio eac o s wi h a zone con aining biocha (BR1), Sphagnum 385
sp. moss (BR2), woodchip (BR3) and po a o esidues (BR4). Func ional p o iles we e p edic ed based on 386
16S RNA gene sequences using PICRUS . A e age ela i e abundances o 1-2 eplica es pe sample a e 387
shown. 388
28
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- Woodchip bio eac o s emo ed 31-38 g NO
3-
-N m
-3
d
-1
om in ensi e aquacul u e e luen
- Addi ional po a o esidues o woodchip ma e ial inc eased 13 % o ni a e emo al a e
- The po a o esidue bio eac o hos ed a dis inc ly di e en mic obial communi y
Decla a ion o in e es s
☒
The au ho s decla e ha hey ha e no known compe ing inancial in e es s o pe sonal ela ionships
ha could ha e appea ed o in luence he wo k epo ed in his pape .
☐The au ho s decla e he ollowing inancial in e es s/pe sonal ela ionships which may be conside ed
as po en ial compe ing in e es s: