Ecological Modelling 351 (2017) 6376

Contents lists available at ScienceDirect

Ecological Modelling
journal homepage: www.elsevier.com/locate/ecolmodel

Inuence of agricultural activities in the structure and metabolic

functionality of paramo soil samples in Colombia studied using a
metagenomics analysis in dynamic state
Astrid Catalina Alvarez-Yela a , Mara Camila Alvarez-Silva a , Silvia Restrepo b ,
Johana Husserl c , Mara Mercedes Zambrano d , Giovanna Danies b , Jorge M. Gmez a ,
Andrs Fernando Gonzlez Barrios a,
a
Department of Chemical Engineering, Universidad de los Andes, Bogot, Colombia
b
Department of Biological Sciences, Universidad de los Andes, Bogot, Colombia
c
Department of Civil and Environmental Engineering, Universidad de los Andes, Bogot, Colombia
d
Molecular Genetics, Corporacin Corpogen, Bogot, Colombia

a r t i c l e i n f o a b s t r a c t

Article history: Soil is one of the most variable and complex habitats on earth where microbial communities and their
Received 27 September 2016 metabolic capabilities are greatly affected by agricultural activities. Nevertheless, the impact of these
Received in revised form 9 February 2017 activities has been poorly studied and more detailed analyses are required in order to understand soil
Accepted 10 February 2017
ecosystems at both a structural and metabolic level. Here, we conducted a detailed metagenomics analysis
to establish the effect of anthropogenic interventions on microbial communities and on the biogeochem-
Keywords:
ical cycle dynamics in soil. Our results suggest that the agricultural activities lead to shifts in the structure
Metagenomics
of microbial communities characterized by an increase in ammonia-oxidizing archaea, bacteria with copi-
Metabolic networks
Dynamic ux balance analysis (DFBA)
otrophic features, and ascomycete fungi. The most important biogeochemical processes were related to
Biogeochemical cycles the carbon and nitrogen cycles. These shifts in the composition of microbial communities led to a loss
Metagenomic modelling in the metabolic functional diversity, which was reected by the lower metabolic activity found. On the
other hand, in the non-intervened soil, an increased metabolic activity was observed. The biogeochemical
cycles behaved in an interdependent and synergistic way. We were able to elucidate how soil ecosystems
dynamics is inuenced by agricultural activities in a paramo ecosystem in Colombia.
2017 Elsevier B.V. All rights reserved.

1. Introduction Microbial communities and their metabolic capabilities are

Soil is one of the most variable and complex habitats on
earth. One gram of soil can contain up to 10 billion microorgan-
isms of thousands of different species (Rossell-Mora and Amann,
2001). Thus, soil ecosystems represent a signicant fraction of the
total biomass on earth. The distribution and community struc-
ture of these microorganisms contribute to the heterogeneity of
the soil. Furthermore, they inuence its functional capabilities at
a metabolic level (Ranjard and Richaume, 2001), given that micro-
bial growth shapes the chemical and biological properties of soil
(Lozupone and Knight, 2007).
Corresponding author at: Department of Chemical Engineering, Universidad de
los Andes, Carrera 1A # 18A 12, Bogot, Colombia.
E-mail address: andgonza@uniandes.edu.co (A.F. Gonzlez Barrios).
greatly affected by external stimuli such as mining, oil spills, pollu-
tion and agriculture, among others (Hollister et al., 2010; Hughes,
2003). Agricultural activities are of great relevance because of their
economic importance and because of their impact on soil (Hollister
et al., 2010; Karlsson, 2012). Detailed metagenomic analyses are
required to understand the effects of agricultural interventions on
soil ecosystem dynamics at both structural and metabolic levels.
At a structural level, taxonomic analyses allow the estimation of
species diversity and distribution (Thomas et al., 2012; Delmont
et al., 2011). At a metabolic level, metabolic network reconstruction
and modelling provide information on the biochemical activities
of microbial communities (Durot et al., 2009). Furthermore, the
relation between the microbial community and the biogeochemi-
cal cycles can be established as the metabolic capacity of different
taxa regulate the cycling of nitrogen, carbon, and sulfur in the soil
(Sylvia et al., 2005).

http://dx.doi.org/10.1016/j.ecolmodel.2017.02.010
0304-3800/ 2017 Elsevier B.V. All rights reserved.
64 A.C. Alvarez-Yela et al. / Ecological Modelling 351 (2017) 6376
Metabolic reconstructions lead to models or curated networks of
metabolic reactions which capture the inter-conversion of metabo-
lites through chemical transformations catalyzed by enzymes
(Price et al., 2004). The obtained models can be simulated and
analyzed using a variety of frameworks among which topological
models, kinetic, stochastic, and constraints-based methods are the
most common (Reed and Palsson, 2003). The ux balance analysis
(FBA) is a constraints-based method that is dened by the stoi-
chiometry of reactions and predicts metabolites uxes through
the model in order to optimize an objective function (Varma and
Palsson, 1994). The limitation of this method is the inability to pre-
dict cellular concentrations over time, which has been resolved
with an extension of the FBA called dynamic ux balance analysis
(DFBA) (Mahadevan et al., 2002). This analysis incorporates biolog-
ical and thermodynamic constraints to predict the concentration
of metabolites in the period of time evaluated (Mahadevan et al.,
2002). The DFBA has been of particular interest in ecology due to the
changes that occur in ecosystems over time and space. The dynamic
optimization approach (DOA) of the DFBA is evaluated over the
course of the simulation to predict uxes and the concentration
proles of metabolites. This is done by using a set of differential
equations to represent the mass balance over each metabolite and
to formulate an optimization problem over a period of time. The
problem can then be solved by parameterizing the dynamic equa-
tions with methods of orthogonal collocation on nite elements
(Systems, 1987).
Nowadays, a large number of metabolic reconstructions have
been modeled for isolated genomes (Forster et al., 2003; Duarte
and Herrg, 2004). Metabolic reconstructions of Escherichia coli
and Saccharomyces cerevisiae have been analyzed using a DFBA
(Antoniewicz, 2013). The rst dynamic model formulated for E. coli
was published in 2002 (Mahadevan et al., 2002) and for S. cerevisiae
in 2003 (Jouhten et al., 2012). Furthermore, several approximations
have been used to analyze microbial communities using com-
bined reconstructions of related microorganisms (Stolyar et al.,
2007; Wintermute and Silver, 2010; Zhuang et al., 2011). Scheffer-
somyces stipitis and S. cerevisiae have been modeled as co-culture
to determine their optimum dynamic ethanol production (Hanly
and Henson, 2013). The competitive relationship between Geobac-
ter sulfurreducens and Rhodoferax ferrireducens was also studied to
improve uranium bioremediation (Zhuang et al., 2011; Peter et al.,
2004).
In the eld of metagenomics, the need to model mixed and com-
plex systems is evident. To the best of our knowledge, the dynamic
implementation of metagenomic models has not yet been done.
Most of the metabolic reconstructions only focus on single microor-
ganisms and ignore interactions within the microbial community
and the environment. Some efforts have been directed towards
modeling these interactions as is the case of human metabolic
reconstruction and hostmicrobe relations which inuence health
or disease conditions in humans but are limited to individual
pathogens (Thiele et al., 2013; Bordbar et al., 2010).
In order to perform a metabolic modelling at the metage-
nomic level we have to consider that metagenomic data provide an
overview of the metabolic state of microbial communities under
specic conditions and that more complex interactions between
individual species and the environment itself have to be incor-
porated as biological restrictions to the models. Despite being
relatively new, this approach can identify metabolic characteristics
from sequence data and can help us to recognize global features
of the systems, until experimental data can be used to infer all
metabolite concentrations, social and metabolic driven interactions
and the active metabolism of single species in order to obtain opti-
mized models (Hanemaaijer et al., 2015; Myrold et al., 2014).
In this study, we present a different approach in which
soil microbial ecosystems are represented as dynamic multi-
organisms. Modeling the metabolic behavior at this metagenomics
scale is important to understand the dynamics of an ecosystem and
ultimately to describe the structural and functional characteristics
of microbial communities within that ecosystem. More specically,
the goal of our study was to compare the functional capacity of
a soil without anthropogenic intervention versus one exposed to
agricultural activity (potato elds) in a paramo soil ecosystem. To
accomplish this, we evaluated the dynamic behavior of metabolic
uxes and metabolites of these two soil ecosystems and studied the
microbial community structure, functional abilities, and dynamism
of biogeochemical cycles through taxonomic analyses, metabolic
network reconstructions, and through the evaluation of the models
using DFBA.
2. Materials and methods
2.1. Sample collection, analysis and processing
I. Metagenomic sequence data of two different soil samples
was provided by the Centro Colombiano de Genmica y Bioin-
formtica de Ambientes Extremos (GEBIX) which collected the
samples at the Parque Nacional Natural Los Nevados, located
in the South American Andes mountains, Colombia. The non-
intervened sample corresponded to paramo rhizosphere soil (N
4 44 50.1 W75 26 40.3 ) located at 3612 m above sea level and
with a vegetation composed of Cortaderia selloana, Pernettya
prostata, Buddleja sp, Lunipus albus y Dentropanax sp. Paramos con-
stitute grasslands above the forest tree-line (at elevations of c.
28004700 m) with a high number of endemic species (Madrinn
et al., 2013). The intervened sample corresponded to rhizosphere
soil (N 4 44 45.5 W75 26 39.1 ) of a potato (Solanum tuberosum)
crop located at 3612 m above sea level. This eld was under
conventional farming practices including the application of fertil-
izers, pesticides, synthetic chemicals, and mechanical hand-work
(Cann-Cortzar et al., 2012). The genetic material extracted was
sequenced by Macrogen (Korea) using the Illumina plataform
HiSeq2000 with paired-end reads at 30X depth sequencing.
Sequences were ltered and trimmed based on length and qual-
ity using the FASTX-Toolkit package (Gordon and Hannon, 2010).
Subsequently, sequences were assembled using the CLC assembler
(Frey et al., 2014), using the default parameters and taking the
paired reads with a separation between the end of the rst read
and the beginning of the second read between 150 and 250 base
pairs (bp). The obtained datasets were then used for the subsequent
taxonomic analysis and for the metabolic reconstructions.
Contigs were analyzed using the gene predictor Glimmer MG
(Gene Locator and Interpolated Markov Modeler Metagenomics)
(Delcher et al., 1999) to identify genes from environmental DNA
sequences. This algorithm selects the best of all possible combina-
tions for predicting a gene based on an interpolated Markov model
(IMM) and identies coding sequences with a sensitivity threshold
of 99%.
2.2. Taxonomic analysis
The taxonomic identication of microorganisms in both non-
intervened and intervened soil samples was performed using the
Metagenome Analyzer platform (MEGAN) (Huson et al., 2007) using
default parameters. This platform, uses the Basic Local Alignment
Search Tool (BLAST) to compare the contigs obtained after sequence
assembly to databases such as that in the National Center for
Biotechnology Information (NCBI) (Gubry-Rangin et al., 2010).
 A.C. Alvarez-Yela et al. / Ecological Modelling 351 (2017) 6376
2.3. Functional annotation and metabolic characterization
Functional annotation is the process where an enzymatic activ-
ity is linked to a predicted gene, according to a homology search
using different databases where functional domains are identi-
ed and similarities are scored in order to get the best hit. This
search is performed by BLAST and can deliver reliable mapping
if small e-values are set to retrieve functional characterization
assignments (Conesa et al., 2005). Some assignations of enzymic
activity are susceptible to be overestimated because an enzyme can
be predicted even if not all domains are present. However, these
estimates are improved during the curation stage of metabolic
reconstructions in which bibliographic data and information from
other databases will give support to the results obtained by Blast.
We performed the annotation using KAAS (KEGG Automatic Anno-
tation Server) (Moriya et al., 2007) and Metapathways (Konwar
et al., 2013) with e-values of 10-6 for BLAST research. Databases
such as Kyoto Encyclopedia of Genes and Genomes (KEGG), COG,
Metacyc and Refseq were used to run Metapathways. The anno-
tation with KAAS was performed using the KEGG database, taking
eukaryotes and prokaryotes as representative data and using the
assignment method BBH (bi-directional best hit).
Metapathways was implemented using a cluster with 24 cores,
128 GB RAM, and a running time of around one week. The KAAS
was implemented on line with a computational time of around ve
hours per dataset. Metabolic genes identied were classied based
on the Enzyme Commission (EC) categories and, subsequently, the
enzymes were mapped against the KEGG (Ogata et al., 1999) and
BiGG (Schellenberger et al., 2010) databases to identify the cat-
alyzed reactions. Finally, biochemical pathways associated with the
reactions were identied using the Search Pathway tool of KEGG
and the respective cell metabolism was assigned to each reaction.
Metabolic functional proles were obtained according to the KEGG
metabolic modules. Based on the set of reactions identied in the
annotation process, a metabolic network reconstruction was per-
formed.
2.4. Curation of metabolic reconstructions
Curation of the network reconstructions was performed using
the Gapnd/Gapll program to restore connectivity of metabolites
through the metabolic reactions (Satish Kumar et al., 2007) by i)
the addition of reactions from an external database; ii) the evalu-
ation of the reversibility of the reactions belonging to the original
model; and iii) by the introduction of transport reactions. The algo-
rithm optimizes the minimum number of reactions that must be
introduced to resolve pathologies. METANETX was used to create
the external database of balanced reactions because it integrates
data from different sources and takes into consideration protons
and water molecules that are often omitted in other biochemical
databases (Ganter et al., 2013). We downloaded the database and
extracted the subset of reactions associated to the E.C. numbers
identied at the annotation step. Additionally, metabolic network
reconstructions were evaluated in order to verify biochemical pro-
cesses carried out by specic species that were taxonomically
identied.
Curation was implemented in GAMS 24.1.3 (Bussieck and
Meeraus, 2004) in an Intel Core2 Duo computer with 4GB RAM.
Working times were about 1 min to 5 h for gapnd and gaplling,
respectively.
Adittionally, information related to mass, charge, and formula
for metabolites was incorporated assuming an intracellular pH
of 7.2, generic reactions that contain generic metabolites were
removed, directionality and reversibility of the reactions were cal-
culated.
65
2.5. Thermodynamic analyses
Directionality and reversibility of the reactions were deter-
mined with GCM (Jankowski et al., 2008) based on the groups
contribution method (Mavrovouniotis, 1990). In this method, the
standard energy of formation of a molecule is calculated from the
contribution of their constituent molecules. Estimations were car-
ried out at pH 7. The data for some reactions was completed using
MetaCyc database reports (Caspi et al., 2014) and literature. Reac-
tions with energy changes equal to or close to zero were considered
reversible because energy shifts are sensitive to small changes in
the concentration of participating metabolites (Jankowski et al.,
2008). We considered the standard deviation reported for the group
contribution method (Fleming et al., 2009) and a range between
1.4 to 1.4 K cal mol1 was established for the reactions with dou-
ble directionality. The obtained data were taken into account to
conduct the topological network analysis.
2.6. Topological analysis of metabolic networks
In order to understand the information obtained from the
metabolic reconstructions a topological analysis was done using
the Cytoscape platform (Doncheva et al., 2012). We used the Net-
work Analyzer plugin for detailed topology parameter analysis.
Clustering coefcients, network diameters, numbers of neighbors,
the shortest paths, and grades were compared to determine the
connectivity between nodes and network redundancy. Topology
was assessed taking into account the thermodynamic constraints.
2.7. Principal component analysis
The main features of the metabolic networks were estimated
by conducting a principal component analysis (PCA). The PCA is a
dimensional reduction which captures the most important infor-
mation of a dataset and expresses it as a set of orthogonal variables
called principal components (Abdi and Williams, 2010). Each prin-
cipal component is associated with an eigenvalue representing
the amount of variability attributed to that component. Results
are represented as dots within a pattern map showing similarity
of observations and variables. Analysis was performed using the
stoichiometric coefcients matrix and the PCA package of Orange
(Demsar et al., 2013).
2.8. Dynamic modeling of metabolic networks
The orthogonal collocation method on nite elements was
implemented to get a faster and more accurate solution to the
optimization problem at a dynamic state and to determine the pro-
les over time for metabolic uxes and metabolite concentrations.
The DFBA optimization problem was formulated using the DOA
(Systems, 1987). Metabolic networks for the evaluated soil sam-
ples were described as a set of ordinary differential equations (ODE)
for all metabolites, which correspond to the mass balance over the
reactions in which they participate:
dy
= Sprod vn prod Scons vncons = S v (1)
dt
where y represents the metabolites concentrations, vn prod rep-
resents the uxes for production reactions [mmol h1 g1 ], vncons
represents the uxes that consume the metabolite [mmol h1 g1 ],
and Sprod and Scons represent stoichiometric coefcients for pro-
duction and assimilation reactions, respectively. Time was divided
into 50 nite elements i and functions were approximated as com-
66 A.C. Alvarez-Yela et al. / Ecological Modelling 351 (2017) 6376

Table 1
Reactions of biogeochemical cycles within objective functions evaluated with the dynamic ux balance analysis for soil ecosystems at the Parque Nacional Natural Los Nevados.

Metabolic pathway Denition

Carbon metabolism Methane + oxygen + NADH + H(+) Methanol + NAD+ + H2 O

d-ribulose 1,5-bisphosphate + CO2 + H2 O 2,3-Phospho-d-glycerol
ATP + Pyruvate ADP + Phosphoenolpyruvate
ATP + Pyruvate + H2 O AMP + Phosphoenolpyruvate + orthophosphate
ATP + Pyruvate + orthophosphate AMP + Phosphoenolpyruvate + Difosfato
ATP + Acetate ADP + Acetyl phosphate
(S)-Malate + NAD+ Oxaloacetate + NADH + H(+)
O-Acetyl-l-serine + Hydrogen sulde l-Cysteine + Acetate

Nitrogen 6 H(+) + 1 nitrite + 6 ferrocytochrome c 6 ferricytochrome c + 1

metabolism NH4 (+) + 2 H2 O
7 H(+) + 1 nitrito + 6 Ferredoxin 1 NH4 (+) + 2 H2 O + 6 Aceptor
8 H(+) + 8 Ferredoxin + 16 H2 O + 16 ATP + 1 dinitrogen 2 NH4 (+) + 1
dihydrogen + 8 A + 16 ADP + 16 phosphate
2 H(+) + 2 ferrocytochrome c + 1 dinitrogen oxide 2 ferricytochrome
c + 1 H2 O + 1 dinitrogen
7 H(+) + 1 nitrite + 6 ferrocytochrome c 6 oxidade cytochrome C + 1
NH4 (+) + 2 H2 O

Sulfur metabolism 3 H2 O + 3 NADP(+) + 1 Hydrogen sulde 1 H(+) + 1 sulphite + 3 NADPH

1 sulphite + 1 AMP + 1 FAD 1 adenosine 5-phosphosulfate + 1 FADH2
Thiosulfate + 5 H2 O + 8 ferricytochrome c 2 sulphite + 8 ferrocytochrome
c + 10 H(+)

Fig. 1. Workow for the metagenomics analysis done on the non-intervened and agriculturally intervened soil samples collected at Parque Nacional Natural los Nevados.
 A.C. Alvarez-Yela et al. / Ecological Modelling 351 (2017) 6376 67

binations of Lagrange polynomials using the collocation orthogonal Table 2

Taxonomic distribution in soil ecosystems at the Parque Nacional Natural Los
method. Polynomials were calculated at discrete points j as follows:
Nevados.
k Domain Non-intervened soil (% reads) Intervened soil (% reads)
y (t) = lj (t) yi,j (2)
j=0 Bacteria 98.50 97.29
Archaea 0.16 0.45
Eukarya 1.30 2.22

k
t tk t t0 t tj1 t tj+1 t tk
Viruses 0.04 0.04
lj (t) = = ... ...
tj tk tj to tj tj1 tj tj+1 tj tk
k=0, =
/ j
literature and allowed us to evaluate the ux through the system
Where yi,j is the value of the variable in the j-th point of the i-th nite (Van Hees et al., 2005; Rling et al., 2007).
element. A step size h = 0.1 h was taken. Legendre polynomials of Values for upper and lower bonds for the uxes were 100
order four were chosen as the collocation points to nd a solution and 100 mmol g1 h1 for reversible reactions and 0 and 100 for
and these were redened for the step size. Derivatives for metabo- direct reactions. Concentrations of 0, 5, and 10 mmol g1 h1 were
lites concentrations by orthogonal collocation approximation were evaluated as starting values for uxes in the optimization prob-
dened as: lem. Eight different conditions for the objective function were
dy (t) k dlj (tk ) assessed, selecting reactions from Table 1; these reactions have
= yi,j (3) been reported as representative for the carbon, nitrogen, and sul-
dt j=0 d
fur cycles (Sylvia et al., 2005). Responses were evaluated for linear
Thus, the system became a nonlinear programming problem combinations of one, three, and 16 reactions, and are described in
(NLP) where concentrations are equivalent to their approxima- detail in S2 Table. Proles of uxes for the 51 reactions were evalu-
tions at the collocation points. The continuity condition for the ated. Among these, 19 corresponded to the carbon cycle, 18 to the
concentration of the metabolites between each nite element was nitrogen cycle, and 14 to the sulfur cycle.
established as: The evaluated reactions were chosen based on their impor-
k tance in each cycle, as they are crucial for the transformation of
yi+1,0 = lj (1) yi,j i = 1, . . .., N 1 (4) compounds in ecosystems. For carbon, the reactions were those
j=0
involved in carbon xation processes (pentose phosphate cycle,
dicarboxylic acid cycle, photosynthesis), methanogenesis, glycol-
Finally, the residual between the orthogonal collocation
ysis, the citric acid cycle, glyoxylate cycle, nucleotide, and amino
approach and the rate of change for the concentrations over time
acid metabolism. For nitrogen, the reactions evaluated were those
was calculated to establish the optimization problem as follows:
 involved in processes of nitrogen xation, nitrication, denitri-
Max Z(t),v(t) w.vi (5) cation, and ammonication. Finally, for the sulfur cycle the
reactions selected were those involved in sulfate and sulfur reduc-
subject to: tion processes, thiosulfate oxidation, and oxidation of other sulfur
compounds.

k
dlj (tk ) The proles for the intracellular metabolites showed two main
Residual = yi,j S.vi = 0
d trends: one stage of production and subsequent depletion or
j=0
exhaustion since the initial time. Therefore, intracellular metabo-
lites were evaluated by determining the presence and absence
vlo v vup of maximum concentrations over time. For extracellular metabo-
lites the consumption rates were evaluated. The dynamics of the
tf t0
ti = t0 + i i = 0 N metabolic uxes was assessed by measuring the interquartile range
N (IR), plotting frequency diagrams, and boxplot graphs in order to
  evaluate the dispersion and the uctuation in their behavior. A
y 0t t0 , tf summary of the methodology used in this study is shown in Fig. 1.

y (t0 ) = y0
Where w represents the weight function for reactions involved
in biogeochemical cycles. The variables vlo and vup correspond to
the lower and upper limits of uxes [mmol h1 g1 ], respectively.
The variable y0 represents the initial concentration of the metabo-
lites [mmol g1 ]. The algorithm was implemented in GAMS 24.1.3
(Bussieck and Meeraus, 2004), using an Intel Core2 Duo computer
with 4GB RAM, and it was resolved with CONOPT in about 20 min. A
total of 4357 and 4098 uxes were identied in the non-intervened
and in the agriculturally intervened soil, respectively, with 217,750
and 204,800 degrees of freedom, respectively.
Due to the difculty of nding experimental values that rep-
resent the real conditions of the soils under study, and in any
metagenomic study (Hanemaaijer et al., 2015), we evaluated dif-
ferent values which could initialize the system and let us evaluate
the response over time. Concentrations of 0.05 mmol g1 for intra-
cellular metabolites and 5 mmol g1 for extracellular metabolites
were established after several tests on the GAMS platform and bib-
liographic review. The values were within the ranges suggested by
3. Results
In total, 205,850,654 and 202,853,882 reads for the non-
intervened and for the agriculturally intervened soil samples,
respectively, were obtained after sequencing. Reads had an aver-
age length of 102 base pairs (bp) and were distributed into 22 data
sets related to the forward and reverse reads. This was reduced
to 153,838,374 reads of an average length of 91 bp after cleaning.
Finally, the assembly step produced 2,056,260 and 1,649,203 con-
tigs for the non-intervened and for the intervened soil, respectively,
with an average length of 446.82 bp. These contigs were used for
taxonomic analysis and metabolic reconstruction.
3.1. Effects of agricultural activities on the taxonomic distribution
in soil
Only 11 and 13% of the reads were assigned to taxonomic cat-
egories for the non-intervened and intervened soils, respectively.
Table 2 shows the taxonomic distribution between the domains of
68 A.C. Alvarez-Yela et al. / Ecological Modelling 351 (2017) 6376

A. B.

C.

Fig. 2. Taxonomic distribution of bacteria in the non-intervened and intervened soil samples. Phyla represented by 5% or more of the assigned reads (2A), phyla represented by
less than 5% of the data (2B), and bacterial orders within the main bacterial phyla involved in the carbon, nitrogen, and sulfur cycles. , , , -Proteobacteria, A: Acidobacteria,
T: Actinobacteria, B: Bacteroidetes, N: Nitrospirae (2C).

life and viruses under both conditions tested. There is a slightly
higher proportion of Archaea and Eukaryotes in the intervened
soil. Differences related to Archaea communities comprise mainly
ammonia-oxidizing archaea related to the Crenarchaeota and Thau-
marchaeota phyla, which are represented by the Thermoprotei and
Nitrosopumilus classes, respectively, and which were only identied
in the intervened soil.
In the case of bacteria, a prevalence of Proteobacteria, Bac-
teroidetes, Acidobacteria, and Actinobacteria was found in the two
ecosystems studied (Fig. 2A, B). In the non-intervened soil, a greater
proportion of oligotrophic microorganisms such as Acidobacteria
was found. However, a greater abundance of copiotrophic microor-
ganisms such as Proteobacteria, Bacteroidetes, and Firmicutes was
observed in the intervened soil.
Microorganisms mentioned above have an important inuence
on the behavior of biogeochemical cycles in soil ecosystems. They
are involved in transport and regulation of carbon, nitrogen, and
sulfur compounds. The distribution of the main bacterial orders
within these phyla, found in the non-intervened and agricultur-
ally intervened soils studied, as well as their association with the
biogeochemical cycles are shown in Fig. 2C.
Fig. 3 summarizes the taxonomic distribution within the domain
Eukarya for both ecosystems studied. Fungi (and in particular
Ascomycota and Basidiomycota) were the most abundant eukary-
otic organisms found in the soils studied followed by the kingdoms
Metazoa, Viridiplantae, and Amoebozoa, respectively. Overall,
ascomycetes were more abundant than basidiomycetes. A higher
number of ascomycetes and a lower number of basidiomycetes
were observed in the agriculturally intervened as compared to the
non-intervened soil studied (data not shown).
Finally, we compared the proportion of viruses reported in the
analysis, which constituted 0.04% of the microorganisms found in
both ecosystems. They were mostly represented by dsDNA viruses.
Despite their low representation with respect to other domains
of life, they may be acting as natural regulators due to their high
pathogenicity and reproduction rates.
 A.C. Alvarez-Yela et al. / Ecological Modelling 351 (2017) 6376 69

50 nine, aspartate, and glutamate metabolisms, valine and isoleucine

Non-intervened soil metabolisms, and fatty acid biosynthesis prevailed in both ecosys-
Intervened soil tems (data not shown).
40 As a general appreciation, the metabolic functionality was simi-
Abundance percentage

30
20
10
0 dation of aromatic compounds.
i
ng ta e
Fu lan
ip
V i ri d
Fig. 3. Taxonomic assignment of individuals within the Eukaryotic domain present
in the non-intervened and in the agriculturally intervened soil samples.
3.2. Functional annotation and metabolic characterization of the
intervened and non-intervened soils
Genomic annotation of sequences is the rst and one of the
most important steps in a metabolic reconstruction. In this step,
genes are identied and subsequently categorized into metabolic
processes. After the annotation process, we obtained a total of
1617 reactions and 2334 enzymes for the non-intervened soil
and 1484 reactions and 2081 enzymes for the agriculturally inter-
vened soil. The relation of these reactions to different metabolic
pathways is shown in Fig. 4. In both, the non-intervened and
the intervened soils, most metabolic activities involved carbo-
hydrate, amino acid, and lipid metabolisms. Essential metabolic
functions related to these metabolisms like pyruvate metabolism,
glycolysis/gluconeogenesis, citric acid cycle, starch, sucrose, ala-
lar for the non-intervened and the intervened soil samples studied.
A total of 1996 reactions were predicted for both ecosystems
studied, representing 86% and 96% of the reactions for the non-
intervened and the intervened soil, respectively. The remaining
percentage of metabolic activity for the soil without interven-
tion were due to activities involved in carotenoid biosynthesis and
arginine, proline, purine, porphyrins, and chlorophyll metabolism.
Meanwhile, for the agriculturally intervened soil, the remaining
4% of the metabolic activities were involved in the synthesis of
secondary metabolites, inositol phosphate metabolism, and degra-
oa To test if differences in environmental conditions affect spe-
zo
a
ta z bo cic metabolic occurrences, we analyzed the richness (number) and
Me mo
e
A evenness (relative abundance of a particular metabolic process in a
sample) of specic metabolic processes. Fig. 5 shows the percentage
of reactions assigned into the functional categories, taking as refer-
ence all reactions in the KEGG database. This allowed us to evaluate
the abundance factor of diversity. Richness of metabolic processes
was similar for both ecosystems. However, a slightly greater even-
ness of metabolic processes was observed in the non-intervened
soil studied, with a diversity index of 1.537 versus a 1.529 for the
intervened soil. Differences of metabolic processes related to ter-
penoids and polyketides metabolism, amino acids, nucleotides, and
energy metabolism were observed.
Reactions that were obtained after the annotation process
were translated to the METANETX database notation to unify the
language for curation. Gaps in some important reactions were
identied in both reconstructions. The non-produced and non-
consumed metabolites identied after gapnd were evaluated to
verify their association to the main metabolic pathways involved
in carbon, nitrogen, and sulfur cycles. Specic problems were found
for metabolites within reactions related to glycolysis, methano-

Non-intervened soil
25 Intervened soil

20
Reactions (%)

15

10

0
es ds ds rs ic s gy es de ds es ns
d rat o aci Lipi facto biot Ener eotid yketi o aci bolit lyca
h y in co eno cl ol in ta G
rbo Am nd X Nu nd p r am y me
Ca a a e r
in s id Oth onda
i tam e no ec
V r p rs
Te h e
Ot
Fig. 4. Metabolic functions present in soil samples.
70 A.C. Alvarez-Yela et al. / Ecological Modelling 351 (2017) 6376
60
Reactions (%) 40
20
0
y s
erg tide ate cid to can cid Lipid etide tioco olite
En cleo hydr ino a cofac Gly ino a
Nu arbo Am and
C s
am
in
Vit
Fig. 5. Metabolic functions in the non-intervened and the agriculturally intervened soil samples. The total number of reactions reported in the KEGG database were taken as
a reference to calculated the percentages.
Table 3
Summary of the data used for the metabolic reconstructions of non-intervened and
agriculturally intervened soils samples.
Reconstruction data Non-intervened soil
Reactions 4357
Exchange reactions 1109
Metabolites 2784
Extracellular metabolites 427
genesis, nitrication, and denitrication, sulfate reduction, and
thiosulfate oxidation. Some reactions within these processes were
not identied after the annotation step but they were incorpo-
rated after the evaluation of metabolic reconstructions reported
in METANETX for organisms considered within our taxonomic
prole. Some examples of these organisms are Agrobacterium
tumefaciens, Pseudomonas putida, Desulfovibrio vulgaris, Geobacter
metallireducens, Rhodospirillum rubrum, Bacillus subtilis, Nitrosop-
umilus maritimus, Xanthomonas campestris, etc.
After the curation process, a total of 2215 and 2073 reactions
were added to the set of reactions for the non-intervened and the
agriculturally intervened soils, respectively. Half of these reactions
corresponded to exchange reactions between the cytosol and the
extracellular environment, while the other half of the aggregate
reactions were related to different metabolic processes. A total of
1732 reactions were introduced to both sets of data, which repre-
sented 78% and 82% of the reactions in the non-intervened and
in the agriculturally intervened soils, respectively. Some of the
differences may be attributed to the biosynthesis of secondary
metabolites, and metabolism of essential amino acids, among oth-
ers (data not shown).
Table 3 shows the nal results of the metabolic reconstructions
for both the non-intervened and intervened soil ecosystems from
which the network analysis and dynamic modeling was performed.
Complete information for each ecosystem is reported in the Sup-
Non-intervened soil
Intervened soil
s s rs s s s s s s
ly k b i ta b
ra
m po e no y m e
h e o id, X ar
Ot rp e
n
on
d
Te s ec
r
he
Ot
porting informations II and III. Results show a higher metabolic
diversity in the non-intervened soil.
Intervened soil
4098 3.3. Slight differences at topological level imply a more complex
1084 network for non-intervened soil
2608
428
A total of 2856 and 2732 reversible reactions were identi-
ed for the non-intervened and the agriculturally intervened soil,
respectively. This information was used in the curation of the recon-
struction and for the analysis of the structural properties of the
networks at a metagenomic scale. We performed a topological
and PCA analysis for the metabolic components of the networks
constructed using metagenomic data to identify their principal fea-
tures. Our results suggest that these networks behave in a way
similar to the networks at a genomic level. Yet, some discrepancies
were observed and will be discussed here.
Networks were represented as metabolitemetabolite graphs
(Fig. 6). Statistical analyses of the network connectivity allowed
us to conclude that the constructed networks were scale-free
networks. Unlike random networks, these are extremely hetero-
geneous and a few highly connected nodes that connect other less
connected nodes dominate their topology.
We evaluated the node degree, dened as the number of links
connected to each metabolite; the node degree represents the
number of reactions that produce this metabolite (in degree) and
the number of reactions consuming that metabolite (out degree)
(Doncheva et al., 2012). The distributions show a behavior that
follows the power law with a in = 0.978 and a out = 0.991 for
the non-intervened soil and a in = 0.981 and a out = 0.985 for the
agriculturally intervened soil. These distributions also showed the
metabolites that were most highly connected within the networks.
These highly connected metabolites are shown in Table 4 along
with the degree values of some metabolites involved in biogeo-
chemical cycles. In both ecosystems the main metabolites included
exchange energy molecules (ATP, NADPH, H (+), diphosphate, etc.)
 A.C. Alvarez-Yela et al. / Ecological Modelling 351 (2017) 6376 71

Fig. 6. Metabolic networks for the non-intervened (left) and the agriculturally intervened (right) soil samples. Intracellular metabolites are represented in green and
extracellular metabolites are represented in pink. (For interpretation of the references to colour in this gure legend, the reader is referred to the web version of this article.)

Table 4
Distribution of metabolites in the metabolic networks for non-intervened and intervened soil ecosystems at Parque Nacional Natural Los Nevados.

Non-intervened soil Intervened soil

Name Metabolite Degree Name Metabolite Degree

MNXM1[c] H+ 5419 MNXM1[c] H+ 4899

MNXM2[c] H2 O 4736 MNXM2[c] H2 O 4270
MNXM3[c] ATP 1786 MNXM3[c] ATP 1584
MNXM4[c] Oxygen 1276 MNXM9[c] Orthophosphate 1270
MNXM9[c] Orthophosphate 1264 MNXM4[c] Oxygen 1164
MNXM7[c] ADP 1207 MNXM7[c] ADP 1158
MNXM8[c] NAD+ 1139 MNXM8[c] NAD+ 1078
MNXM5[c] NADP+ 876 MNXM13[c] CO2 876
MNXM11[c] Diphosphate 866 MNXM10[c] NADH 802
MNXM10[c] NADH 845 MNXM1[e] H+ extracellular 774
MNXM1[e] H+ extracellular 785 MNXM5[c] NADP+ 739
MNXM13[c] CO2 773 MNXM11[c] Diphosphate 738
MNXM15[c] NH3 693 MNXM15[c] NH3 704
MNXM6[c] NADPH 664 MNXM6[c] NADPH 557
MNXM89557[c] l-glutamate 536 MNXM89557[c] l-glutamate 520
MNXM35[c] Aceptor 460 MNXM12[c] CoA 494
MNXM27[c] Sodium 438 MNXM27[c] Sodium 491
MNXM27[e] Sodium extracellular 433 MNXM27[e] Sodium extracellular 480
MNXM14[c] AMP 426 MNXM35[c] Aceptor 468
MNXM16[c] S-adenosylmethionine 398 MNXM23[c] Pyruvate 384
MNXM23[c] Pyruvate 373 MNXM26[c] Acetate 178
MNXM26[c] Acetate 160 MNXM169[c] Ferredoxin 140
MNXM41[c] d-Glucose 149 MNXM107[c] Nitrite 118
MNXM105630[c] Sulte 140 MNXM41[c] d-Glucose 115
MNXM169[c] Ferredoxin 122 MNXM105630[c] Sulte 99
MNXM107[c] Nitrite 113 MNXM89582[c] H2 S 73
MNXM207[c] Nitrate 94 MNXM207[c] Nitrate 66
MNXM89582[c] H2 S 79 MNXM280[c] Acetyl phosphate 32
MNXM280[c] Acetyl phosphate 49

and compounds such as l-glutamate and NH3 that are involved in Table 5
Parameters that dened the topology of the metabolic networks constructed for the
key metabolic processes of all microorganisms such as cell growth
non-intervened and intervened soil ecosystems studied.
and formation of macromolecular compounds.
A comparison of other topological parameters is shown in Parameter Non-intervened soil Intervened soil
Table 5. Among these, the diameter of the networks, dened as Clustering coefcient 0.218 0.216
the maximum distance between a pair of nodes (Doncheva et al., Diameter 9 8
2012), was slightly higher in the soil without intervention. Due to Shortest path 6,015,159 (77%) 5,488,761 (74%)
Average number of neighbors 7.589 7.419
the fact that for metabolic networks the distances correspond to
Number of nodes 2.788 2.715
links between different biochemical reactions, it can be inferred
72 A.C. Alvarez-Yela et al. / Ecological Modelling 351 (2017) 6376
that the metabolic behavior of the soil without intervention is
slightly more complex in terms of biochemical process because it
has more metabolites reacting through more reactions catalyzed by
enzymes. The clustering coefcient which estimates the relation-
ship between individual connections of nodes with their neighbors
and all possible connections between them, indicated the level
of partnership within each system. This coefcient takes values
between 0 and 1 as both low and high groupings are observed,
respectively (Doncheva et al., 2012). In both systems, similar val-
ues were found with a slightly higher value for the non-intervened
soil, which could indicate a higher grouping in this sample. The
shortest path, which describes the expected minimum number of
connections between two nodes (Doncheva et al., 2012), was lower
for the intervened soil, which could imply a faster shift on metabolic
responses for this ecosystem.
A principal components analysis was performed to quantita-
tively describe the relationship between components within the
networks. The principal components were similar for the two
reconstructions made. Metabolites such as H+, H2 O, ATP, NADP+,
orthophosphate, ADP, NAD+, extracellular H+, NADH, and NH3 are
the fundamental metabolites in the networks because they are
the major contributors to the overall metabolic dynamic behavior
within the biological systems. Results showed similarity with the
description of the most interconnected metabolites at a topologi-
cal level. These metabolites are involved in the most representative
reactions for carbon, nitrogen, and sulfur cycles, which will be dis-
cussed in the dynamic modeling (see below) and which conrms
the dependence of these ecosystems on the biogeochemical pro-
cesses. The graphs of these analyses are presented in Figs. S1 and S2
. Additionally, we found that 99% percent of the variance was repre-
sented by 2170 metabolites from a total of 2784 metabolites in the
non-intervened soil. In the agriculturally intervened soil 99% of the
variance was represented by 1780 of a total of 2608 metabolites.
3.4. Synergistic metabolic behavior is lost after agriculture
activities
The metabolic ux proles and metabolites showed some differ-
ences between the non-intervened and intervened soils. In general,
uxes for the non-intervened soil had higher dispersion and uctu-
ations in their values. Additionally, in this ecosystem the number of
active uxes was greater under the objective functions evaluated.
Comparison of the results under the different objective functions
showed that the optimization of reactions of a specic cycle has an
important effect on other cycles in the non-intervened ecosystem
and that there is a synergistic behavior on the metabolic processes
that take place in this soil. On the contrary, an uncoupled effect
on biogeochemical processes was observed in the agriculturally
intervened soil.
The interquartile range (IR), an indicator of the scattered dis-
tribution of the uxes, showed that many uxes increase their IR
when reactions involved in the nitrogen cycle are optimized. Fur-
thermore, a decrease in the number of active uxes was observed
for carbon and sulfur cycles when reactions related to the carbon
cycle were optimized. When the sulfur cycle reactions were opti-
mized a decrease in the number of active uxes for the nitrogen
cycle was observed. Finally, if all reactions were optimized together,
a decrease in the IR and an activation of uxes was observed for
most of the reactions from all cycles in the non-intervened soil.
A less coupled behavior related to biogeochemical cycles was
found for the agriculturally intervened soil. The IR for reactions
related to the nitrogen cycle changed when nitrogen reactions were
optimized. Furthermore, the number of active uxes for carbon
and sulfur cycles decreased. Only the IR for carbon related uxes
were inuenced when reactions related to the carbon cycle were
optimized. When the sulfur cycle was optimized, the IR for uxes
related to sulfur cycle changed and the number of active uxes
related to the nitrogen cycle was reduced. Finally, when all reac-
tions were optimized together, only the IR of uxes related to the
carbon cycle were affected.
The intracellular metabolite proles were analyzed (Fig. 7).
A greater number of maximum concentration peaks (those that
reached higher values and appeared earlier in time) were found
in metabolites from the non-intervened soil. Concerning the objec-
tive functions evaluated, we found more peaks when independent
reactions were optimized, particularly if these reactions were asso-
ciated with the carbon and nitrogen cycles. A lower number of
peaks occurred when all reactions were optimized together. The
analysis of the extracellular metabolites proles (Fig. 8) showed
higher consumption rates in the agriculturally intervened ecosys-
tem. The highest rates corresponded to metabolites that were
directly related to the nitrogen cycle, followed by the carbon, and
sulfur cycles. It was also noted that in many cases the rates were
similar for both the non-intervened and the agriculturally inter-
vened ecosystems, and no particular pattern was observed under
the objective functions evaluated. Graphs of the IR distances, the
frequency distribution, and the metabolites proles are shown in
Supporting information I.
4. Discussion
4.1. Changes in the microbial communities determine differences
between non-intervened and agriculturally intervened soils
The most representative microbial communities present in the
soil samples evaluated from the paramo ecosystem were identied.
It is important to highlight that most of the microorganisms in the
soil are non-cultivable and might therefore not be identied using
current databases. This most certainly led to a low coverage and
thus, an underestimation of the diversity present in the soil samples
studied (Streit and Schmitz, 2004).
Our results suggest that the agricultural activities lead to a
shift in the structure of microbial communities characterized by an
increase in ammonia-oxidizing archaea, bacteria with copiotrophic
features, and ascomycete fungi as a result of natural selection.
These changes are important due to the role of these microor-
ganisms in nutrient cycling regulation in soils. Carbon acts as the
main source of energy for microorganisms and plants. Nitrogen is
one of the most limiting nutrients for plant growth and has a pro-
found detrimental effect on the ozone layer due to the release of
nitrogen oxides (NO and N2 O). Sulfur, is required for the synthesis
of vitamins, hormones, coenzymes, and structural elements. The
ammonia oxidizers, play a key role in the nitrication process. The
process of oxidizing ammonia to form nitrate, is a major step in
the global nitrogen cycle and the regulation of nitrication rates
(Gubry-Rangin et al., 2010). Archaea oxidizers have been found in
greater proportions than their bacterial counterparts in agricultural
soils (Leininger et al., 2006; Schauss et al., 2009).
Related to Bacteria, microorganisms from the orders Actino-
mycetales and Pseudomonales are involved in cellulose, lignin,
and chitin degradation (Sylvia et al., 2005). Bacteria belonging
to the order Nitrosomonadales are particularly involved in the
nitrication process by the autotrophic conversion of ammo-
nia (NH3 ) to nitrite (NO2 ). Oxidation of the latter to nitrate
is done by bacteria in the order Nitrospirales. Burkholderiales
and Actinomycetales oxidize ammonia to organic nitrogen. Addi-
tionally, some species of Pseudomonales, Burkholderiales, and
Flavobacteriales are involved in denitrication to reduce nitrate
to nitrogen oxide. Microorganism with the ability to x nitro-
gen either as mutual associations with legumes and plants, such
as Rhizobiales, Actinomycetales, and Enterobacteriales, or as free
 A.C. Alvarez-Yela et al. / Ecological Modelling 351 (2017) 6376 73

20 20

18 F.O 1
F.O 2 F.O 1
16 F.O 3 F.O 2
15

Concentration (mmol g-1)

Concentration (mmol g-1)

F.O 4 F.O 3
14 F.O 5 F.O 4
F.O 6 F.O 5
12 F.O 7 F.O 6
F.O 8 F.O 7
10 10 F.O 8

6
5
4

0 0
0.0 0.5 1.0 1.5 2.0 2.5 3.0 0.0 0.5 1.0 1.5 2.0 2.5 3.0

Time (hours) Time (hours)

Fig. 7. Comparison of the maximum concentrations reached by the intracellular metabolites in the non-intervened soil (left) versus the agriculturally intervened soil (right).
The example shown corresponds to nitrite under different objective functions (FO) related to biogeochemical cycles.

20
Non-intervened soil 18 Non-intervened soil
Intervened soil Intervened soil
20
Consumption rate (mmol h-1 g-1)

Consumption rate (mmol h g )

16

-1
-1 14

15 12

10

8
10
6

4
5 2

0
0 1 2 3 4 5 6 7 8
0 1 2 3 4 5 6 7 8
Objective function
Objective function

Fig. 8. Comparison of consumption rates for the extracellular metabolites. The example shown corresponds to the results obtained for CO2 (left) and acetate (right).

living organisms, such as Pseudomonadales, Rhizobiales, and Xan- carbon, nitrogen, and phosphorus (Klaubauf et al., 2010). For exam-
thomonadales, were also found. Additionally, microorganisms ple, Verticillium and Trichoderma degrade chitin of fungi cell walls;
belonging to Acidithiobacillales, Hydrogenophilales, Actinomyc- Penicillium and Fusarium depolymerize structural polysaccharides
etales, Pseudomonadales, and Chromatiales decompose organic such as cellulose and hemicellulose, and some Basidiomycetes are
matter producing soluble organic sulfur compounds. The orders able to break lignin, the main constituent of the plant cell wall.
Desulfovibrionales and Desulfuromonadales are the most promi- With respect to the nitrogen cycle, Ascomycetes such as Aspergillus
nent in the inverse process of reducing sulfate to hydrogen sulde oxidize ammonium or organic nitrogen, which is then assimilated
(Sylvia et al., 2005). by plants (Sylvia et al., 2005). As a result of the soil interven-
The distribution observed for the described orders conrms tion, Ascomycetes constitute the major group of fungi responsible
the increased proportion of Acidobacteria in the non-intervened for waste degradation in agricultural soils (Ma et al., 2013). The
soil. These microorganisms have been classied as oligotrophic increased biomass residues formed by the constant crop renewal is
microorganisms that exhibit slow growth rates and metabolize the primary source of carbon and energy for this group of fungi. Sol-
chitin, lignin, hemicellulose, keratin, and other plant and fungi uble compounds released, such as amino acids, organic acids, and
polymers. They also prevail under low nutrient availability and sugars, are rapidly assimilated by bacteria and fungi in soil (Sylvia
have high afnities for specic substrates (Wessn et al., 2010). et al., 2005).
On the contrary, the abundance of copiotrophic microorganisms Finally, viruses are important because of their role in soil ecol-
was higher in the soil that was agriculturally intervened. These ogy as temporary contaminants after fecal deposition from humans
microorganisms consume preferably labile compounds and organic and animals or as natural members of the microbial community by
compounds rich in carbon, have high nutritional requirements and infecting plants, bacteria, and fungi (Sylvia et al., 2005).
high growth rates when resources are abundant (Sylvia et al., 2005),
which results after the application of fertilizers. This was conrmed 4.2. Shifts in the functional response of ecosystems
by the high ratio (7.7%) of Proteobacteria to Acidobacteria observed
(Fierer et al., 2012). Shifts in the composition of microbial communities has been
Fungi play a central role in most soil ecosystems as important associated with shifts in the physiological and functional response
decomposers because they are involved in the natural cycling of of ecosystems (Torsvik, 2002; Waldrop et al., 2000). Thus, signif-
74 A.C. Alvarez-Yela et al. / Ecological Modelling 351 (2017) 6376
icant variation in specic metabolism occurrences exists under
different environmental conditions (Dinsdale et al., 2008). We con-
rmed that shifts in the composition of microbial communities
have an effect in the physiological and functional response of the
studied ecosystems. In this case, agriculturally intervened soils led
to a loss of diversity in its metabolic functionality. This is impor-
tant because a loss in metabolic capabilities of communities can
decrease the ecosystems resistance under conditions of stress and
disturbance (Fierer et al., 2012; Degens et al., 2001). It can be
presumed that microorganisms in the non-intervened soil have a
higher enzymatic exibility to respond to external stimuli while
microorganisms in agriculturally intervened soils present limited
metabolic capabilities.
It is important to consider that energy metabolism is important
for the regulation of biogeochemical cycles. Energy metabolism
affects oxidative phosphorylation, photosynthesis, carbon x-
ation, methane metabolism, nitrogen metabolism, and sulfur
metabolism. These processes are directly related to the ecologi-
cal roles performed by the microorganism that are favored by the
availability of nutrients after fertilization during agricultural activ-
ities.
4.3. Metagenomic networks are similar to genomic ones but more
complex
The free scale networks are specic to biological systems and
have been described for many genomes where growth phenomena
occur and new nodes preferably attach to nodes already established
(Jeong et al., 2000). This behavior was observed in our networks, is
similar to that described for other metagenomes (Zamora et al.,
2015) and has been conrmed by the degree distribution values
(Doncheva et al., 2012). The difference with genomic networks in
this case is related to the values. For genomes, typical val-
ues range between two and three as assessed by Jeong et al. for
43 organisms in all domains of life (Jeong et al., 2000). However,
metagenomic reconstructions integrate a wide variety of microor-
ganisms with diverse metabolic interactions and therefore a greater
variance is expected in the distribution of links connecting the
nodes. Thus, this results in values smaller than two in metage-
nomics reconstructions (Zhu et al., 2007).
The connectivity between nodes allowed us to identify the most
highly active metabolites within networks. These highly active
metabolites, represent activity centers in biochemical processes
and can be assigned as essential metabolites involved in many dif-
ferent reactions (Zhu et al., 2007). Due to this role, and the fact that
they are involved in most reactions within the central metabolism
of all microorganisms, the metabolites listed in Table 3 also were
identied as the principal components by the PCA. These results
are similar to those found at a genome level and show that the
metabolic behavior is led by the same biological processes involved
in central metabolism (Barrett et al., 2009).
Additionally, we noted that the network for the non-intervened
soil presented greater connectivity between its metabolites. Fur-
thermore, a higher diameter and clustering was observed, which
implies a more complex network with more enzymes and reac-
tions. This reects an increased metabolic capability, which was
also observed after the functional annotation for this ecosystem. By
evaluating the diameters, we observed that in general the genome
networks differ among them because their diameters usually reach
values of three and the clustering is generally high (Jeong et al.,
2000). At a genomic level these are dened as small networks
(small world) (Wagner and Fell, 2001), but at a metagenomics
level the complexity and diversity of biological processes that are
represented increases. Although they are highly conserved in most
organisms inside the microbial communities, biological processes
are also diverse and could extend the range of connections binding
metabolites and representing all possible variations in metabolic
activities that could be found in ecosystems.
Finally, the PCA results allowed us to deduce that the networks
are not redundant because the total variance of the system can-
not be expressed by a small number of principal components.
This implies that the metabolic reactions are not repetitive and, as
expressed before, these features are also found at a genomic level
(Barrett et al., 2009). In general, our results indicate that metage-
nomics networks are more complex and robust than the genomic
ones. The multi-organism approach extends the topological and
structural perspective of the networks for individual microorgan-
isms.
4.4. Agricultural activities greatly affect the dynamic of
biogeochemical cycles
The DFBA allowed us to observe that agricultural activities have
an important effect on the dynamics of biogeochemical cycles. In
general, the features of the non-intervened soil at the taxonomic
and metabolic levels led to a synergistic behavior of the metabolic
processes performed by microbial communities and to a functional
coupling of the carbon, nitrogen, and sulfur cycles.
Results suggest that in both ecosystems studied, the dynam-
ics of the reactions related to the nitrogen cycle have an important
effect on the other cycles. In the non-intervened soil, they inuence
uxes and their uctuation in time. Whereas in the agriculturally
intervened soil, the effect is reduced. The dynamics of reactions
associated with the carbon and sulfur cycles affects other cycles
in the non-intervened soil while the effect is self-regulating in the
agriculturally intervened one. The uxes behave in a uniform way
when all cycles are evaluated together in the non-intervened soil
by reducing their uctuation and with an increase of active uxes.
Meanwhile, for the soil under agricultural activities only the reac-
tions related to the carbon cycle are relevant and present greater
uctuations.
It may be proposed that the increased metabolic capacity in
the non-intervened soil, mainly related to energy metabolism
(described above), allows this ecosystem to have an active
metabolic activity which is variable and uctuates in time, and
that the dynamics of the biogeochemical cycles of carbon, nitro-
gen, and sulfur are highly synergistic. In the intervened soil the
metabolic capabilities are mostly focused on particular processes
of the nitrogen and carbon cycles that occur after fertilization. As
a consequence, the activities related to each cycle have no major
implications for others. Results are consistent with the higher
proportion of nitrogen xation and carbon decomposition microor-
ganisms that could tip metabolic activities towards these particular
processes (Karlsson, 2012; Herridge et al., 2008). Additionally, our
results showed agreement with previous metagenomics studies
in paramo ecosystems where microbial adaptation capability was
suggested to be materialized in the distribution of metabolic ows,
especially related to nitrogen cycle (Zamora et al., 2015).
Additionally, the lower concentrations of intracellular metabo-
lites produced in the non-intervened soil are a result of their
transformation through a higher number of reactions as more path-
ways and biological processes are active. Finally, the observations
of the faster consumption of extracellular metabolites in the inter-
vened soil may be related to the increased metabolic capacity
that focuses on carbon sequestration and nitrication processes
described in agricultural soils (Li et al., 1994; Freibauer et al., 2004).
These activities are directly related to changes in the composition
of microbial communities and metabolic functionality discussed for
this ecosystem.
 A.C. Alvarez-Yela et al. / Ecological Modelling 351 (2017) 6376
5. Conclusions
We performed a detailed metagenomic study of two paramo
ecosystems to understand the impact of agricultural activities on
these soils, at both a structural and metabolic level. Our results
suggest that the agricultural activities lead to shifts in the struc-
ture of microbial communities characterized by an increase in
ammonia-oxidizing archaea, bacteria with copiotrophic features,
and ascomycete fungi as a result of fertilizer application, a high
nutritional quality of soil, and the increased biomass residues.
These shifts in the composition of microbial communities have
an effect in the physiological and functional response of ecosys-
tems. Agricultural activities cause a loss in the microbial diversity
and hinder the metabolic functionality of the soil, thus reducing
its resistance under stress and disturbance conditions. We found
that metagenomics networks are similar to genomic ones but
more complex. The free scale features are conserved but greater
variance in the distribution of links connecting the nodes, higher
diameters, and lower clustering coefcients were found. Addition-
ally, the topological parameter conrmed the higher complexity
of the network for the non-intervened soil with more enzymes
and reactions. Finally, our results suggest that agricultural activ-
ities greatly affect biogeochemical cycles because the dynamics of
the reactions related to carbon, nitrogen, and sulfur changes. In the
non-intervened soil, we found an active metabolic activity which is
variable and uctuating in time and where biogeochemical cycles
are highly synergistic. In the intervened soil, the metabolic capa-
bilities were mostly focused on particular processes of the nitrogen
and carbon cycles that occur after fertilization.
Acknowledgements
This work was funded by the Department of Science, Tech-
nology, and Innovation (COLCIENCIAS)-SENA (project number
6570-392-19990) and was done under the MAVDT contract 76-
2013 for access to genetic resources and the UAESPNN research
permits, DTNO-N-20/2007 and PIDB DTAO 021-10. The authors
declare that there is no conict of interest regarding the publication
of this paper.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.ecolmodel.2017.
02.010.
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