Bioresource Technology 311 (2020) 123500

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Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech

Humus formation driven by ammonia-oxidizing bacteria during mixed T

materials composting
⁎
Junqiu Wua, Zimin Weia, Zechen Zhua, Yue Zhaoa, , Liming Jiab, Pin Lvc
a
College of Life Science, Northeast Agricultural University, Harbin 150030, China
b
Heilongjiang Province Environmental Monitoring Centre, Harbin 150056, China
c
Institute of Natural Resources and Ecology, Heilongjiang Academy of Sciences, Harbin 150030, China

G R A P H I C A L A B S T R A C T

A R T I C LE I N FO A B S T R A C T

Keywords: The aim of this study was to identify the effects of ammonia-oxidizing bacteria (AOB) inoculation on humus
Ammonia-oxidizing bacteria formation. Both nitrogen conversion and humus formation were considered as the main processes, because
Nitrogen transformation NH4+-N-like compounds not only substrates of nitrification, but also precursors of humus. During composting,
Humus formation the inoculation of AOB indeed increased humus concentration by fixing NH3 emission as NH4+-N, but it has also
Composting
promoted nitrogen transformation. While the main reason was the changed bacteria community structure caused
by inoculating AOB. Moreover, the relationship between bacteria and nitrogen transformation and humus for-
mation has become closer. And bacteria were more likely to synthesize humus. Therefore, it is conjectured that
AOB inoculation could not only provide NH4+-N for humus formation, but also enhance the anabolism of mi-
croorganisms. This suppose has been confirmed by structural equation model in this study. Therefore, AOB
inoculation has a driving effect on promoting humus formation.

1. Introduction
The by-products produced in composting process, such as enzyme,
flora and humus, have strong environmental benefits (Zang et al.,
2020). Especially humus has the functions of repairing soil, increasing
crop yield, absorbing pollution and so on (Awasthi et al., 2019; Zhao
et al., 2019; Zhu et al., 2020; Wei et al., 2020). Humus is formed by
different polymerization pathways of the precursors which includes
polyphenols, quinonyl compounds, reducing sugars, saccharides, and
nitrogenous compounds (Cui et al., 2020; Wu et al., 2017, 2020a).

⁎
Corresponding author.
E-mail address: zhaoyue@neau.edu.cn (Y. Zhao).

https://doi.org/10.1016/j.biortech.2020.123500
Received 28 March 2020; Received in revised form 4 May 2020; Accepted 5 May 2020
Available online 07 May 2020
0960-8524/ © 2020 Elsevier Ltd. All rights reserved.
J. Wu, et al.
Fig. 1. Relationship between humus formation and nitrogen conversion during composting with AOB inoculation.
However, no matter which pathway, the formation of humus is finally
achieved by the condensation of nitrogenous compounds with other
precursors (Tan, 2014). Meanwhile, the ammonia nitrogen-like (NH4+-
N) compounds are the most prominent precursors. Even most of present
studies related to humus formation aim to improve the precursors
concentration (Qi et al., 2020; Shi et al., 2020; Wu et al., 2017, 2020a),
there are few researches focused on promoting NH4+-N.
During composting, NH4+-N will be transformed into NO2−-N by
ammonia-oxidizing bacteria (AOB), which might conductive to redu-
cing NH3 emission, decreasing organic nitrogen loss (Zeng et al., 2018;
Wang et al., 2018; Chen et al., 2019; An et al., 2020). Previous study of
Zhang et al. (2016) had confirmed that NO2−-N concentration was
increased with decreased NH3 emission and NH4+-N concentration by
inoculating enriched AOB. In addition, NH3 recycling, biochar addition
can also alter bacteria diversity to promote nitrogen transformation
(Zhu et al., 2019; Wang et al., 2020). Therefore, bacteria community
plays an important role in nitrogen transformation. Thus, with the
strong interference of AOB inoculation, the relationship between ni-
trogen transformation and humus formation deserves further study.
Accordingly, the purpose of this study is to explore whether microbial
driven nitrogen retention also has positive effects on humus formation.
The relationships between nitrification of nitrogen and humus forma-
tion after AOB inoculation was intensive studied by detecting 1) re-
lationships between cascade effects of nitrogen conversion and humus
formation; 2) effects of distribution of bacterial community on humus
formation; 3) driving effects of AOB on humus formation.
2. Materials and method
2.1. Composting text and humus extraction
Mixed materials (25% chicken manure + 75% rice straw) were
composted in the compost reactor with temperature control system (Lu
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Bioresource Technology 311 (2020) 123500
et al., 2018). Composting pile with 5% (1 × 106 CFU/mL) AOB in-
oculant was set as treatment group (TM), and the other was check
control (CK). The detailed information about composting text and AOB
inoculant obtain have been elaborated by Zhang et al. (2016).
The extraction and separation methods of humus were according to
Wu et al. (2020a). Briefly, 1 g sample was extracted by 10 mL mix
solution of 0.1 M Na4P2O7·10H2O and NaOH. Thereafter, the miscible
liquids were shaken at 25 °C for 24 h and obtained the solution with
11,000 rpm for 15 min. Humus was achieved after the solution was
filtered with 0.45 μm Millipore membrane. Fulvic acids (FA) as the
fraction of humus was separated by adjusting humus solution to pH 1.5
by 6 M HCl. The acidic solution was stood at 4 °C for 12 h, which then
was centrifugated with 11,000 rpm for 15 min, and the supernatant is
FA, while the precipitate is humic acids (HA). The HA was also needed
to be washed by 0.1 M HCl and deionized water alternatively for twice.
Finally, the precipitate was dissolved with 0.05 M NaHCO3. The con-
centration of humus, HA and FA was determined by SHIMADZU TOC-
Vcph analyzer. All the processes were repeated for triplicate and the
mean value was calculated for each case.
Parameters of NH4+-N, NO3−-N and NO2−-N were determined ac-
cording to the method of Zhang et al. (2016) and Wang et al. (2015). In
addition, DNA extraction and the analysis of PCR-DGGE have also been
reported by Zhang et al. (2016). Therefore, no more repetitive de-
scription is made herein.
2.2. Data analysis
Humus, FA and HA concentration analysis were conducted by
OriginPro 2016. Pearson analysis was conducted by IBM SPSS Statistics
22, which was utilized to detect the relationship between nitrogen
transformation and humus formation. Canoco for Windows (Version
5.0) was used to analysis the distribution characteristics of bacterial
community after AOB inoculation. Structural equation model (SEM)
J. Wu, et al. Bioresource Technology 311 (2020) 123500

Fig. 2. Effects of bacteria community distribution on the humus formation and nitrogen conversion (a: bacteria community distribution; b: CK; c: TM).

Fig. 3. Structural equation model confirming the driving effects of ammonia-rich oxidizing bacteria on humus formation during composting. NMDS1 and NMDS2
represent the data derived from the non-metric multidimensional scaling of bacteria community. The path coefficients are adjacent to the arrows, the width of the
arrows represent the strength of the path.

3
J. Wu, et al.
was constructed to confirm the roles of nitrogen component in humus
formation by IBM SPSS AMOS 23.0 (Zhao et al., 2020).
3. Results and discussion
3.1. Cascade effects of nitrogen conversion on humus formation
AOB was initially inoculated to remove pollution, but it was found
that AOB could also improve the humus concentration during com-
posting (Zhang et al., 2016). To analysis the effects of nitrogen con-
version on humus concentration changing is helpful for identifying how
does the AOB inoculation affect humus formation. It shows that the two
processes are substrate competition (Fig. 1). During composting, humus
gradually took HA as the main fraction (Fig. 1d), and the NH4+-N was
also the important precursor of it (Fig. 1a). On the other hand, NH4+-N
was converted to NO2−-N and NO3−-N by AOB (Fig. 1b and c). How-
ever, results presented that the concentration of humus and its aromatic
fraction HA was increased by inoculating AOB, while the NH3 emission
was decreased either during composting (Zhang et al., 2016). Thus, the
decreased NH3 emission might provide precursors for humus formation,
such as the NH4+-N compounds. Previous study of Zhang et al. (2016)
has shown that NH4+-N and NO2−-N concentration were significantly
decreased and increased (p < 0.05) with AOB inoculation. Meanwhile,
NH4+-N as the precursor of humus formation and nitrogen conversion
also significantly promoted humus and NO2−-N production (p < 0.05)
(Fig. 1a, b). Therefore, AOB inoculation was contributed to humus
concentration increasing, even there was substrate competition be-
tween the above two processes. However, it is the chemometric analysis
presented the promotion of humus formation might be due to the
fixation of nitrogen by AOB. In view of the primary role of AOB is ni-
trogen conversion, it is necessary to analyze how does AOB increase the
humus concentration and promote nitrogen transformation as the same
time.
3.2. Effects of bacterial community distribution on humus formation
The inoculation of AOB significantly changed the distribution of
bacteria community (Fig. 2a). Comparing with 0 day, the bacteria
gathered closely in the process of composting. But the distribution
difference between CK and TM is still large. Thus, whether AOB affects
humus formation by reshaping the structure of bacterial community
need further study. While the effects of this result on humus formation
and nitrogen conversion will be analyzed by RDA (Fig. 1b and c). There
was significant correlation between bacteria bands and substance
transformation in RDA (p < 0.05). Comparing CK with TM, the in-
oculation of AOB significantly increased the interaction of bacteria with
the above two processes. As bands 1, 3, 7, 9 and 11 were the inoculated
AOB, they were closely related to nitrogen transformation in CK.
Nevertheless, bands 1 and 11 presented insignificantly correlation with
nitrogen conversion in TM composting. The reason might be that the
extremely significant effects of band 9 covered bands 1 and 11, or the
bacteria represented by bands 1 and 11 showed weak effects on ni-
trogen transformation. In addition, some function of indigenous bac-
teria has also been changed by AOB inoculation. For example, bands 21
and 22 significantly positive affected FA formation in CK. While they
significantly negative affected FA production in TM, which might pre-
sent biodegradation effects on FA (Wang et al., 2018). It indicated that
AOB inoculation changed the utilization direction of bacteria: de-
gradation or synthesis of organic matter. On the other hand, it is also
because that the number of bacteria which closely positive related with
humus formation were increased, such as bands 4, 8, 16, 28 and 29 in
TM. NH4+-N, NO2−-N and NO3−-N related bacteria also showed the
same phenomenon in TM. Accordingly, AOB inoculation might be
beneficial to the microbial synthesis of target products. While the role
of microorganisms in the formation of humus cannot be ignored (Wu
et al., 2017b, 2019; Yu et al., 2019). Therefore, the combination of
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Bioresource Technology 311 (2020) 123500
chemometrics and microbiological analysis showed that AOB inocula-
tion might not only provide NH4+-N for humus formation, but also
enhance the synthesis of humus by microorganisms.
3.3. Driving effects of ammonia-rich oxidizing bacteria on humus formation
The SEM has been conducted to further reveal the relationship
among nitrogen conversion, bacteria community diversity and humus
formation (Wu et al., 2020b). Moreover, it also proved that the AOB
inoculation promoted humus formation from two aspects: providing
substrate and promoting microbial synthesis. Comparing CK with TM,
bacteria as the leading factors were driving the effects of nitrogen
conversion on humus formation (Fig. 3). The diversity of microorgan-
isms involved in these two processes increased significantly. This result
was consistent with RDA (Fig. 2b, c). In TM, NMDS2 represented bac-
teria significantly promoted humus formation, which also obviously
advanced nitrogen conversion (p < 0.05), especially in forming NO2−-
N. In addition, enriched bacteria diversity weakened the relationship
between NMDS1 and ammonia NH4+-N, but it was compensated by
NMDS2. On the other hand, NMDS1 also significantly promoted NO3−-
N formation (p < 0.05) in TM. Therefore, the inoculation of AOB both
promoted nitrogen conversion and humus formation by mobilizing
bacteria diversity. Comprehensively speaking, AOB could not only re-
duce nitrogen loss, but also significantly improve humus formation.
4. Conclusion
This study revealed the driven property of AOB inoculation on both
nitrogen conversion and humus formation by altering the function of
bacteria community. Nitrification of ammonia nitrogen promoted by
AOB which also provide precursors for humus formation by fixing NH3.
Meanwhile, the bacteria in CK was contributed to organic matter de-
gradation, while it tended to synthetic humus in TM. Therefore, in-
oculation of AOB in composting has dual functions of nitrogen fixation
and humus production improvement. During composting, AOB can be
considered as the main factors to provide precursors for humus for-
mation.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
Acknowledgement
This work is funded by the National Natural Science Foundation of
China, No. 51878132; National Key Research and Development Project,
No. 2019YFC1906403; National Natural Science Foundation of China,
No. 51978131, No. 51778116.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.biortech.2020.123500.
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