processes

Article
Diazotrophic Behaviour in a Non-Sterile Bioreactor: The Effect
of O2-Availability
Amber Yasemin Shirin de Zoete , Hendrik Gideon Brink * , Joshua Cornelus Beukes,
Ignatius Leopoldus van Rooyen and Willie Nicol

Department of Chemical Engineering, University of Pretoria, Pretoria 0002, South Africa;

aysdezoete@gmail.com (A.Y.S.d.Z.); joshuabeukes@gmail.com (J.C.B.); ignatiuslvr@gmail.com (I.L.v.R.);
willie.nicol@up.ac.za (W.N.)
* Correspondence: deon.brink@up.ac.za

Abstract: The behaviour of a locally isolated diazotrophic consortium was investigated with the
prospect of agricultural applications. A repeatable culture was obtained in a non-sterile bioreactor.
Metagenomic analysis indicated Chryseobacterium ssp. and Flavobacterium ssp. were the dominant
species, making up approximately 50% of the microbial community. The oxygen supply was var-
ied and mass-transfer limited growth was attained under all experimental conditions. Negligible
amounts of aqueous metabolites were formed, indicating a high selectivity towards biomass pro-
duction. High oxygen availability resulted in decreased growth efficiencies i.e., the specific energy
requirements for biomass synthesis. This was attributed to reduced electron transport chain effi-
ciencies and nitrogenase protection mechanisms. Mass and energy balances indicated that sessile
biomass with a high C:N served as a carbon sink. The most efficient growth was measured at an



aeration feed composition of 21% oxygen and 79% nitrogen. The study presents one of the only


known investigations of operational conditions on diazotrophic growth in a non-sterile bioreactor.
Citation: de Zoete, A.Y.S.; Brink, In addition, it provides a strong foundation for the development of a Biological Nitrogen Fixation
H.G.; Beukes, J.C.; van Rooyen, I.L.; process with scaling potential.
Nicol, W. Diazotrophic Behaviour in a
Non-Sterile Bioreactor: The Effect of
Keywords: diazotrophs; bio-fertilizer; nitrogen fixation; ATP down-regulation
O2 -Availability. Processes 2021, 9, 2039.
https://doi.org/10.3390/pr9112039

Academic Editors: Clarisse Brigido

1. Introduction
and Francesca Raganati
Population growth has been catalysed in the past decade by the rapid industrialisation
Received: 7 October 2021 of developing countries and technological advancements. A 200% population increase with
Accepted: 12 November 2021 reference to 2011 is predicted to take place by 2050 [1] . Due to this ever-expanding popu-
Published: 15 November 2021 lation, one of the main future challenges is food security [2]. To accommodate this surge
in food demand, a concomitant exponential increase in food production—and therefore
Publisher’s Note: MDPI stays neutral fertilizer usage—is required [3].
with regard to jurisdictional claims in In the late nineteenth century, a similar rise in food demand occurred which necessi-
published maps and institutional affil- tated industrialised nitrogen fixation. This led to the invention of nitrogen-fixing processes
iations. in the early twentieth century [4]; the Haber-Bosch process (Equation (1)) for synthetic
nitrogen production became the most prevalent in modern agriculture. Currently, 450 mil-
lion tonnes of nitrogen fertilizer are produced through this process each year [5]. The
Haber-Bosch process has two major drawbacks. Firstly, the process is energy-intensive
Copyright: © 2021 by the authors. as it relies on fossil fuels (1–2% of world’s annual energy supply) [5]. At ambient temper-
Licensee MDPI, Basel, Switzerland. atures, the Haber-Bosch process has a spontaneous standard Gibbs free energy change
This article is an open access article of −10.9 kJ/mol H2 . Unfortunately, at these conditions low productivities are observed
distributed under the terms and due to slow reaction rates. Elevated temperatures, up to 773 K, result in faster reaction
conditions of the Creative Commons rates, but this is at a Gibbs free energy cost (∆G o = 23.6 kJ/mol H2 at 773 K). Consequently,
Attribution (CC BY) license (https:// the process requires elevated pressures up to approximately 20 MPa [6] to maintain an
creativecommons.org/licenses/by/ exergonic Gibbs free energy change of −10 kJ/mol H2 at a temperature of 773 K [7].
4.0/).

Processes 2021, 9, 2039. https://doi.org/10.3390/pr9112039 https://www.mdpi.com/journal/processes

Processes 2021, 9, 2039 2 of 15

3H2 ( g) + N2 ( g) ↔ 2NH3 ( g) (1)

The Haber-Bosch process has a significant negative impact on the environment due to
greenhouse gas emissions resulting from these energy requirements [8]. Ammonia produc-
tion is responsible for circa 1.6% of total global carbon-dioxide emissions [6]. Secondly, the
addition of synthetic nitrogen to crops has led to leaching of nitrogen into groundwater,
rivers, lakes, and estuarine zones. This has created an offset in hydrospheric nitrogen which
results in phenomena such as aquatic biodiversity loss and eutrophication [9]. In addition,
the release of nitrogen from synthetic fertilizers as nitrous oxides has a detrimental effect
on the global climate due to its high global warming potential [9].
Prior to industrialisation, biological nitrogen fixation (BNF) was responsible for sup-
plying 58 million tonnes of nitrogen per year [4]. BNF is the process in which nitrogenase-
bearing prokaryotes reduce atmospheric nitrogen to more bio-available forms of nitro-
gen [10]. Many nitrogen-fixers form symbiotic relationships with plants by providing plants
with nitrogen, while consuming plant exudates as their carbon-source [11]. In addition,
these microbial communities release several plant-growth hormones, aid in nutrient cycling,
and combat pathogens [9]. Non-symbiotic or free-living nitrogen fixers, referred to as
diazotrophs, show much promise in aiding the development of sustainable agriculture [9].
Naturally occurring diazotrophs could be used for bio-fertilizer production as they
significantly promote soil fertility and plant growth [9]. Bio-fertilizers are already in use,
however, there is a need for improved formulations for commercialisation. Several desirable
characteristics including: easily adjustable pH and nutrient addition, non-toxic, simple
application, and biodegradable were described by Mahanty et al. [9]. Liquid bio-fertilizers
from diazotrophic cultures could be the solution to this need. Utilizing diazotrophic cultures
in broths would ensure a measurable and controllable process [9].
There is a need to reevaluate current agricultural practices and develop more sustain-
able approaches. Therefore, the development of a green, effective nitrogen-source which
supports the natural nitrogen cycle and minimally disturbs the microbial community is
essential. Bio-fertilizer production utilizing diazotrophs is a promising method of safe-
guarding future food supply, while minimizing or completely negating imbalances with
the natural environment. To this end, the development of a process utilizing controlled
bio-reactor environments is proposed. The use of a microbial consortium, instead of a pure
culture, was identified as a suitable solution for agricultural application. Metabolic coop-
eration, increased biomass, and enhanced catalytic function are a few benefits previously
identified in microbial consortia [12]. In addition, a non-sterile environment allows for
the development of a robust culture, which is resilient to environmental changes. Table 1
maps out the main themes throughout literature relating to diazotrophs. The bibliometric
data shows a disproportionately small amount of research focused on bioreactors and
bio-fertilizers in parallel with diazotrophs.

Table 1. Bibliometric data (4 July 2021).

Scopus Web of Science Science Direct Average

Keywords
# (%) # (%) # (%) (%)
Diazotrophs 1428 100.00 1401 100.00 2074 100.00 100.00
Diazotrophs free-living 552 38.66 141 10.06 858 41.37 30.03
Diazotrophs aerobic 271 18.98 38 2.71 674 32.50 18.06
Diazotrophs batch 80 5.60 10 0.71 265 12.78 6.36
Diazotrophs bio-fertilizer 16 1.12 7 0.50 214 10.32 3.98
Diazotrophs bioreactor 25 1.75 2 0.14 95 4.58 2.16
Diazotrophs chemostat 19 1.33 2 0.14 62 2.99 1.49
Bioreactor 90,865 100.00 41,397 100.00 79,249 100.00 100.00
Bioreactor fertilizer 2381 2.62 189 0.46 5652 7.13 3.40
Bioreactor nitrogen fixation 1207 1.33 47 0.11 3719 4.69 2.04
Processes 2021, 9, 2039 3 of 15

BNF is highly energy-intensive for microbes with a theoretical cost of 16 ATP/N2

fixated and an even higher practical cost. Therefore, process conditions should be optimized
to maintain a minimal energy requirement for nitrogen fixation. Due to the lack of literature
on diazotrophic cultures in bioreactors, a need for diazotrophic behavioural studies in
bioreactors was identified.
This study aimed to investigate the behaviour of a non-sterile diazotrophic consortium
with the prospect of utilising their nitrogen-fixing ability in agricultural applications. The
main objectives of the investigation were: to obtain a repeatable, non-sterile diazotrophic
culture; to study the behaviour of the consortium under various aeration conditions; and
to investigate their energy expenditure.

2. Materials and Methods

2.1. Materials and Reagents
A nitrogen-free, modified Burke’s medium was utilised during laboratory experiments.
The medium consisted of the following: 1 g/L KH2 PO4 ·7H2 O, 0.2 g/L MgSO4 ·7H2 O,
0.1 g/L CaCl·2H2 O, 0.00145 g/L FeSO4 ·7H2 O, 0.0002 g/L Na2 MoO4 ·2H2 O, 0.05 g/L KOH,
and 5 g/L glucose. The pH was controlled through the addition of a 1 M NaOH solution.
All chemicals were purchased from Merck (Midrand, South-Africa). The aeration feed
was made up of varying ratios of oxygen (99.5%) and nitrogen gas (99.5%). Gasses were
purchased from Afrox (Pretoria, South-Africa).

Figure 1. Diagram of the laboratory-scale reactor setup.

2.2. Equipment
All experiments were conducted in a bench-scale bioreactor (Figure 1) with a volume
of ±300 mL. The bioreactor was continuously mixed by a magnetic stirrer at 105 rpm. A
recycle line with a total volume of ±100 mL was also implemented, which was utilized
for aeration. The aeration gas composition was controlled by Brooks mass flow regulators
where nitrogen and oxygen were fed at the desired composition to a 2 L holding vessel
to ensure complete mixing. The aeration gas from the holding vessel was injected into
the recycle line by a peristaltic pump, which produced a Taylor bubble flow for improved
Processes 2021, 9, 2039 4 of 15

gas-to-liquid mass transfer. The recycle line also served as part of a heat exchanger as
approximately 90% of the recycle line (ID 3 mm) was submerged in a 5 L bottle of water
that was heated by a heating plate. The temperature of the water was maintained at 2 ◦ C
above the desired reactor temperature to account for heat losses. An Endress+Hauser
Memosens COS81D oxygen sensor (Johannesburg, South-Africa) was utilised to read and
log dissolved oxygen and to indicate the temperature inside the reactor. The pH was
controlled through proportional control. A DFRobot pH-sensor was utilized in conjunction
with a peristaltic pump for base addition. An overflow system was used for level control.

2.3. Experimental Methods and Analyses

2.3.1. Inoculum Procurement and Development
A soil sample from N-lean soil at 10 mm depth (coordinates: 25.75361° S, 28.229721° E)
was obtained. To extract microorganisms, the soil sample was suspended in distilled
water. The distilled water containing the soil particles was agitated by mixing the particles
thoroughly with a spatula and manually swirling the solutions. Thereafter, the particles
were allowed to settle and the supernatant was decanted. This was used as an initial
inoculum. The bioreactor and adjacent lab space was disinfected using surfactants, distilled
water, ethanol, and VirkonTM prior to each run to minimize the microbes present in the
bioreactor space. The system was intentionally not autoclaved as the focus of the study was
to develop a robust system able to perform under non-sterile operational conditions. The
inoculum was cultured in a 1.5 L vessel at a pH of 6.8 and 25 ◦ C. Aeration with atmospheric
air was supplied to prevent fermentation products. The inoculum was cultured until the
glucose ran out. Then, 10 mL of the culture was placed in fresh medium and left to grow.
This was repeated several times. After several runs, a natural selection had occurred as the
microbial behaviour became relatively repeatable based on measured concentration profiles:
suspended biomass, byproducts, and glucose concentrations. The resulting solution was
stored in 10 mL vials. One vial was utilized as inoculum for each experimental run.

2.3.2. Mass-Transfer Experiments

To determine the volumetric mass transfer coefficient of the aeration in the recycle
line, a mass-transfer experiment was completed in triplicate at 30 ◦ C. First, the system
was purged with nitrogen and allowed to reach a low dissolved oxygen level (<2 mg/L).
Then, the aeration pump (10 rpm) was turned on to replenish the system with oxygen,
the dissolved oxygen was recorded until the saturated dissolved oxygen concentration
was approached. The experiment was done utilizing atmospheric air to increase the
oxygen concentration. The volumetric mass transfer coefficient (kla) in s−1 was obtained
by utilizing Equation (2), where t is the time in s, DO is the dissolved oxygen measured in
mg/L, and DO∗ is the saturated dissolved oxygen in mg/L.

dDO
= kla × ( DO∗ − DO) (2)
dt

2.3.3. Experimental Conditions

The experimental conditions were set at 30 ◦ C, ambient pressure and a pH of 6.8.
These conditions were selected as they were most commonly used in literature [13–16]. The
reactor was covered to be completely dark, in order to prevent algal growth. To investigate
the effect of varying aeration feed compositions, three different compositions were tested:
oxygen-rich, atmospheric air, and oxygen-poor. Each condition was tested in triplicate to
confirm repeatability. The oxygen-rich feed (35% oxygen, 65% nitrogen) was labelled O2 _35,
whereas the oxygen-poor feed (7% oxygen, 93% nitrogen) was labelled O2 _7 to reflect their
respective oxygen concentrations. The atmospheric air (21% oxygen, 79% nitrogen) feed
was labelled O2 _21. The oxygen-rich and oxygen-poor compositions were chosen such that
they lay equal distances (14 percentage point) away from the atmospheric air condition
and consequently the change in oxygen% compared to 21% oxygen or ( 23 of 21%) would be
sufficiently different to ensure marked differences in the operational conditions.
Processes 2021, 9, 2039 5 of 15

2.3.4. Sample Analysis

Samples were taken periodically and in duplicate. Absorbance readings were done
using 3 mL cuvettes at a wavelength of 660 nm in a spectrometer (±0.0001) (Agilent Tech-
nologies, Johannesburg, South-Africa—Cary 60 UV-Vis). The absorbance readings were
related to dry-cell weight (DCW) through the following procedure: an empty sample
vile was weighed after remaining in an oven at 60 ◦ C overnight to allow evaporation of
any liquids; the culture samples were placed in the vile; the samples were centrifuged
and washed three times; then the samples were dried overnight at 60 ◦ C; the viles with
sample were weighed. The difference between the two measurements was taken to deter-
mine the mass of the biomass. This was related to the sample absorbance reading. The
carbon-compound and acid concentrations were measured in the high-pressure liquid
chromatography (HPLC) (±0.00001 g/L) (Agilent Technologies—1260 Infinity). Before
analysis, samples were centrifuged in an Eppendorf MiniSpin 5425 for 90 s at 120 rpm.
After that, they were filtered (0.45 µm). To determine ammonia concentrations, Ammonia
test kits (Spectroquant, Merck, South-Africa) were utilised. The samples were prepared
as per suppliers manual and the resulting solution was analysed in the spectrometer at
690 nm (Agilent Technologies, Johannesburg, South-Africa—Cary 60 UV-Vis).

2.3.5. C:N Determination

The total nitrogen was determined at the end of each run. A DMP Spectroquant total
nitrogen test kit (Spectroquant, Merck, South-Africa) was used which determined the total
nitrogen based on the sum of total Kjehldahl nitrogen, nitrate, and nitrite [17]. Samples
were prepared as per supplier’s instruction (Spectroquant, Merck, South-Africa) which
included digestion (analogous to method EN ISO 11905-1) [17] of biomass-containing
samples at 120 ◦ C for 1 h. An absorbance reading at 340 nm was taken to determine the
total nitrogen present in the samples (analogous to method DIN 38405-9) [17] . This was
done for filtered and unfiltered samples. The difference in readings was indicative of the
nitrogen content of the suspended biomass.
Samples were taken at the end of each run for the total organic carbon analysis. The
samples were centrifuged at 2500 rpm for 5 min and filtered (45 µm) and then diluted
to 30 mL solutions. The solutions were analyzed on a Total Organic Carbon Analyzer
(Shimadzu, Kyoto, Japan). The TOC-V (liquid samples) setting was used to determine the
non-purgeable organic carbon. Nitrogen was used as a carrier gas and sodium persulfate
and phosphoric acid were used as oxidizers.

2.3.6. ngDNA Sequencing

Next-generation DNA sequencing was outsourced to Inqaba Biotec (Pretoria, South-
Africa). A metagenomic analysis of full length 16 s gene amplicons was performed on a
sample from each experimental condition. A two-step PCR was performed on each sample
and 16S (forward and reverse) primers (27F and 1492R) tailed with PacBio universal
sequences were used [18]. To process raw subreads the Circular Consensus Sequences
(CCS) algorithm accessible through the software SMRTlink (v9.0) was utilized to produce
highly accurate reads (>QV40).

2.3.7. Statistical Analysis

To quantify the repeatability of experimental runs under a specific condition, standard
deviations (σ) were calculated according to Equation (3), where Xm is the mean, Xi is the
value of the data point, and n is the number of data points.
p
( Xi − Xm)2
σ= (3)
n−1
In addition, the relative standard deviation (RSD) for each data point was calculated
using Equation (4), where M signifies the mean.
Processes 2021, 9, 2039 6 of 15

σ
RSD = × 100 (4)
M
To quantify the similarity of the culture compositions, the Bary-Curtis dissimilarity
index (BC) was calculated. The BC is a number between 0 and 1, where 0 signifies that
the data sets were identical and 1 signifies there was no overlap. Equation (5) was used to
compute the BC, where A and B are the sum of frequencies for the two data sets and where
W is sum of minimum frequencies among the data sets [19].

2×W
BC = 1 − (5)
A+B

3. Results and Discussion

3.1. Diazotrophic Growth
One of the objectives of this study was to obtain a repeatable culture in a non-sterile
environment. As seen in Figure 2, good repeatability among experimental runs at the
same condition was observed from a bio-reactor performance point of view. This was
confirmed by the average RSD of the growth curve data. The average RSD values were
calculated as 15.7%, 8.1%, and 12.9% for O2 _21, O2 _35, and O2 _7, respectively. It should
be noted that the biomass concentration profiles presented were based on suspended
biomass measurements.
The biomass concentration profiles during all three conditions indicate that different
growth-regimes occurred. The different growth-regimes coincide with the trends in the
dissolved oxygen profiles. An initial lag phase is present to establish the culture, where the
dissolved oxygen is high as there is a low oxygen demand. In this phase, little to no growth
is observed. This phase is followed by a two-part growth phase. Initially, exponential
growth is observed. At this stage, an exponential decrease in dissolved oxygen occurs,
while the biomass is increased. This is followed by mass-transfer limited growth at low
dissolved oxygen concentrations. This is visible in the biomass concentration from its
straight line trends, indicating a constant growth rate. After this, a slight bend in the
biomass concentration profile indicates glucose nears depletion. Once all the glucose is
consumed, growth terminates and a sudden increase dissolved oxygen is observed as the
oxygen demand reduces drastically.
The glucose concentration profiles corresponded well to the biomass concentration
profiles. A significantly shorter lag phase was observed, however, which was followed by
an exponential decrease in glucose. The initial glucose consumption only reflected in the
microbial growth profiles after delay, likely due to biofilm formation. Biofilm formation in
diazotrophs forms part of their oxygen-stress response [20]. Since oxygen-stress is high at
the start of the experimental runs, the initiation of rapid biofilm formation would protect
the consortium and increase its survival chance afterwards. As the mass-transfer limited
growth is reached, a straight line trend is present in the glucose profiles. This indicates a
constant glucose consumption rate. The ratio between glucose uptake rates at condition
O2 _35 and O2 _21 was 1.50 ± 0.10 (mean ± σ) ( rrss 3521 ), whereas the ratio of growth rates
between the two conditions was 1.12 ± 0.09 ( rrxx 21
35
). This shows that condition O2 _35 was
less efficient in glucose consumption compared to condition O2 _21. For condition O2 _7
and O2 _21, a glucose consumption ratio of 1.29 ± 0.18 ( rrss21
7 ) was calculated, whereas the
corresponding ratio of growth rates was 2.71 ± 0.34 ( rrxx21
7 ). This indicates condition O2 _21
utilized significantly more glucose towards biomass formation than condition O2 _7. The
lower efficiency at condition O2 _7 could be attributed to a less efficient metabolism.
Processes 2021, 9, 2039 7 of 15

Figure 2. The concentration profiles for runs at various aeration feed conditions (O2 _21, O2 _35, and O2 _7) are shown. The dissolved
oxygen profiles for each condition are shown in the top row. The middle row shows biomass concentrations, whereas the bottom row
shows glucose concentrations.

The yield of biomass produced to glucose consumed, biomass productivity, mass-

transfer limited growth rate, and glucose consumption rate for each condition are shown in
Table 2. When comparing the results at each condition, significant changes in growth rate,
and glucose consumption rate were observed in the mass-transfer limited growth regime
as a direct effect of the variation in oxygen supply. The change in growth rate is, however,
not proportional to the change in oxygen supply. This is likely due to a change in energy
requirement and biofilm formation. The data from all experimental runs for each condition
was averaged in the x- and y-direction and compared as shown in Figure 3; the error bars
show the standard deviations in both x- and y-directions. From the averaged data, a clear
variation in slope for each condition was observed.
Processes 2021, 9, 2039 8 of 15

Figure 3. The microbial growth data for all experimental runs was averaged in x- and y-direction.
Time-shifted to start their mass-transfer limited growth at the same point in time (vertical line). The
error bars show the standard deviations in the results from the triplicate repeat runs.

Table 2. The effect of aeration feed composition on yield, productivity, growth rate, and glucose
consumption rate. The standard deviation was calculated for each slope and shown in the slope data.

Yield Productivity Growth Rate Glucose Consumption Rate

Run
(g/g) (mg/L·h) (g/L·h) (g/L·h)
O2 _21 0.20 6.03 0.0732 ± 0.003 0.262 ± 0.003
O2 _35 0.19 7.59 0.0820 ± 0.006 0.393 ± 0.031
O2 _7 0.12 2.75 0.0277 ± 0.003 0.207 ± 0.033

From carbon-compound analyses, it was clear that malic acid was the only byproduct
in the liquid for condition O2 _21 and O2 _35. No other fermentation products were pro-
duced at these conditions. Figure 4 shows the malic acid concentration profiles at each
condition. For condition O2 _21, malic acid increased exponentially during the exponential
growth-regime and had an average concentration of 0.07 g/L. The malic acid concentration
decreased when the point of glucose depletion was approached. This indicates malic acid
was utilised as a carbon source by the diazotrophic culture when glucose started depleting.
For condition O2 _35, the rise in malic acid was slightly delayed into the mass-transfer
limited growth-regime. An average final concentration of approximately 0.07 g/L was
obtained. In run O2 _35_3, malic acid consumption was observed. This phenomenon was
absent in run O2 _35_1 and O2 _35_2 as their termination point occurred before malic acid
could start being consumed.
Condition O2 _7 showed malic acid production and consumption occurred in a similar
manner to the other conditions. In run O2 _7_1, however, some carbon was spent on ethanol
and acetic acid formation. A final amount of 0.45 g/L of ethanol and 0.25 g/L of acetic
acid was produced. This could explain lower malic acid concentrations compared to run
O2 _7_2 and O2 _7_3. Trace amounts (<0.05 g/L) of ethanol and acetic acid were found in
run O2 _7_2 and run O2 _7_3.
Processes 2021, 9, 2039 9 of 15

Figure 4. Malic acid concentration profiles for each experimental condition show relatively high
malic acid is attained in each run. Slight decreases in malic acid are visible towards the end of an
experimental run, where glucose is near to depletion.

When analysing the cell-based glucose uptake rate for each condition, a distinct peak
was present during the exponential growth phase (Figure 5). This peak was followed by
a steep drop in glucose uptake rate and a downward trend of the uptake rate during the
mass-transfer limited regime. There was a significant increase in the malic acid around the
glucose transition point. Thus, the malic acid was produced at the glucose uptake peak, as
well as afterwards, during the start of mass-transfer limitation and the exponential growth
phase. To this end, the idea that a glucose overflow is present at the start, generating large
amounts of energy, was proposed. Due to the oxygen limitation, this energy would remain
unused. To facilitate down-regulation of the metabolism, malic acid could be used as a net
zero ATP carbon-sink [21].
Processes 2021, 9, 2039 10 of 15

Figure 5. Glucose uptake rates increase during the lag phase and exponential growth, but stabilize
during mass transfer limited growth. It is hypothesised that malic acid is formed during exponential
growth and the start of mass transfer limited growth to counteract the excess glucose.

There could be various mechanisms at play that affect the energy requirements of
the microbial culture. First of all, diazotrophs contain the nitrogenase enzyme which is
deactivated by oxygen. To avoid nitrogenase deactivation, various protection mechanisms
have been proposed in aerobic diazotrophs. One such mechanisms is increased substrate
usage [20], which reduces the exposure of the nitrogenase enzyme to oxygen by increasing
the consumption of oxygen. Following this argument, the production of malic acid is likely
part of this protection response. In addition, malic acid production is both NADH and ATP
neutral when produced from glucose. Thus, the production of malic acid facilitates the
removal of glucose from the system without generating excess ATP for growth.
Another protection mechanism is biofilm formation [20]. Bacterial communities form
biofilms in order to increase their resistance against environmental stress. According
to Wang et al. [22], extracellular polysaccharides in the biofilm matrix facilitate aeration
management and generate a suitable micro-environment for the microbes. During all
experimental conditions, a light-yellow biofilm formed in varying amounts inside the
recycle line and on the pH and DO probe. Since the absorbance measurements were only
based on suspended biomass [23], biofilm attachment would lead to an underestimation in
yield and productivity. Much of microbial biofilms comprises of extracellular polymeric
substances (EPS), attributing to 50–90% of the biofilm’s total organic carbon [24]. These
EPS could serve as a carbon sink.
Lastly, as discussed by Oelze [10], when oxygen supply is increased past the oxy-
gen demand of the organisms, a mechanism initiates to down-regulates ATP production.
Diazotrophs have the ability to change their electron transport chain (ETC) during ox-
idative phosphorylation. Changing the ETC allows for less protons to move across the
ATP complex for each oxygen consumed, this decreases the chemical gradient which, in
turn, decreases the ATP production. Therefore, increasing the oxygen supply past a certain
turning point would no longer benefit the growth rate.
A mass balance was performed over the mass-transfer limited regime using Equa-
tion (6). Elemental balances of C, H, O and N were performed, and the oxygen consumption
rate and glucose consumption rate were specified from experimental data. The biomass
composition was estimated as CH1.8 O0.5 N0.08 . This was done through total nitrogen
Processes 2021, 9, 2039 11 of 15

analysis, where the mass% of nitrogen in the biomass was converted to its equivalent
mol%. The other elemental ratios were based on the following biomass composition:
CH1.8 O0.5 N0.2 [25].
Since, ammonia excretion was very low in all runs (<20 mg/L), the nitrogen require-
ment for ammonia was omitted in the model. The malic acid (C4 H6 O5 ) production rate
during the mass-transfer limited regime was calculated from the experimental data.
CH2 O + O2 + N2 → CH1.8 O0.5 N0.08 + CH3/2 O5/4 + CO2 + H2 O (6)

The oxygen uptake rate (OUR) in mol/L.h was calculated using Equation (7), where
dDO
dt signifies the rate of change in dissolved oxygen over time.
dDO
OUR = kla × ( DOsat − DO) − (7)
dt

The gas-liquid mass transfer coefficient (kla) was determined to be 0.0027 s−1 . Figure 6
shows the experimental data for the mass transfer coefficient calculation.

Figure 6. The volumetric mass transfer coefficient was found as the slope of the experimental data.
Repeatable data was obtained.

To calculate the overall ATP requirement, Equation (8) was utilised. Where the oxida-
tive phosphorylation efficiency (P/O) was assumed to be 1.5 ATP/O atom. The energy cost
of nitrogen fixation was assumed to be the theoretically reported value of 16 ATP/N2 [26].
The rate of nitrogen fixation (r N2 ) was determined from the mass balance. It was assumed
that oxidative phosphorylation was solely responsible for energy generation.
2 × P/O × rO2 − γ × r X − β × r N2 = 0 (8)

In the above approach, the assumption was made that all biomass had the same
nitrogen content. This resulted in an unrealistically low energy requirement for biomass
production (γ). Therefore, it is highly likely that the sessile biomass had a different
composition from the suspended biomass. This would entail glucose expenditure on
N-lean biomass as a carbon sink. This carbon sink could consist of intracellular polymeric
substances (IPS), such as ATP-independent poly-3-hydroxybutyrate [27], and/or EPS. This
competing process was deemed a challenge for future experiments.
This study investigated the consortium in non-sterile conditions to facilitate more
realistic soil microbial interactions as compared to pure culture studies. Thus, exact
metabolic pathways and survival mechanisms were not determined due to the complexity
of the consortium. The carbon feed consisted of glucose as it is a simple model compound
and would be suitable to determine the potential for nitrogen fixation.
Processes 2021, 9, 2039 12 of 15

3.2. C:N Ratios

The TOC analysis and total nitrogen (TN) tests for all runs were performed at the
glucose depletion point. Averages of the results at each condition were calculated to find
an overall C:N ratio. Both TOC and TN showed low values of <200 mg/L and <20 mg/L,
respectively. This was deemed excellent from a water treatment point of view and indicates
a high selectivity towards biomass production. Figure 7 shows the resulting C:N ratios.
These results confirm the hypothesis that much of the carbon was converted to sessile
biomass, IPS, or EPS.

Figure 7. The C:N ratios at each condition were between 1–2. Differences between conditions could
be attributed to varying malic acid concentrations and ETC down-regulation, which would increase
carbon dioxide formation. The error bars indicated the standard deviations of each set of results.

3.3. ngDNA Sequencing

For each culture sample 50 to 100 different species were identified. There were, how-
ever, few species which dominated the microbial consortium. These species made up
60–70% of the cultures. The dominant species matched across experimental conditions,
however, depending on oxygen-availability they were present in slightly different per-
centages. Table 3 shows a summary of the species that made up the largest part of the
consortia. See the Supplementary Materials for the complete reports received from In-
qaba Biotec, where samples were labelled A, B, and C, for condition O2 _21, O2 _35, and
O2 _7, respectively.
From the read count in the metagenomic information, it was clear that Chryseobac-
terium ssp. and Flavobacterium ssp. made up approximately 50% of the bacterial com-
munities. This showed that the experimental conditions targeted nitrogen-fixing species
successfully. In addition, the composition of the cultures showed that most of the dominat-
ing species were most commonly found in soil [28–31] with intrinsic sensitivities to oxygen
therefore negating the likelihood of air-borne contamination. This strongly indicated that
the cultured bacteria were indeed retrieved from the soil samples. The Bary-Curtis dissimi-
larity index (Equation (5)) was calculated over at least 84% and 87% of the total species
count of the two aerobic culture data sets (O2 _21 and O2 _35), where counts of unknown
species were omitted. The index was computed as 0.284 which implies there was a sig-
nificant similarity (71.6%) between the data sets. The high similarity indicates that the
consortium was relatively stable at these conditions. Due to the low read count measured
for the O2 _7 run sample (Supplementary Materials) the Bary-Curtis dissimilarity index
could not be computed for this condition, however, the ngDNA report indicated very
Processes 2021, 9, 2039 13 of 15

similar species were identified as the aerobic runs providing qualitative support for the
stability of the consortium under low aeration conditions.

Table 3. Summary of most prominent bacteria species in the experimental cultures.

Species Description Environment N2 -Fixing References

Chryseobacterium Gram-negative, yellow bacterium commonly Aerobic Yes [28,32]
found in soil. The bacterium also produces plant-
promoting growth hormones.
Flavobacterium Gram-negative, rod-shaped bacterium found in Aerobic Yes [29]
soil and fresh water.
Pseudoxanthomonas Gram-negative, rod-shaped bacterium Aerobic No [33]
Azorhizophilus Rhizobial bacterium capable of fixing nitrogen Aerobic Yes [31,34]
both symbiotically and in free-living conditions.
Microbacterium Gram-positive, heterotrophic bacterium Aerobic Yes [35–37]
Pseudomonas Plant-growth promoting bacterium. Posseses Aerobic Yes [34,38]
adaptation strategy for anaerobic environments.
Brevundimonas Root colonizing bacterium which also facilitates Aerobic Yes [30,39]
effective phosphate solubization for enhanced
plant growth.
Agrobacterium Gram-negative, rod-shaped bacterium Aerobic Yes [40–42]
Acidovorax Gram-negative with nitroaromatic degradation ability Aerobic Yes [43,44]
Klebsiella Gram-negative, heterotrophic soil bacterium Aerobic Yes [45,46]

4. Conclusions
In the current study a locally obtained soil diazotrophic microbial culture was suc-
cesfully cultured in a non-sterile bench-scale bioreactor. This study provides one of the
only known investigations evaluating the effect of operational conditions on BNF in this
type of bioreactor. A repeatable culture was obtained and the effect of oxygen availability
on the growth profile of the culture was investigated. From the experimental data, it was
concluded that aeration feed composition had a significant effect on the growth profile
of the diazotrophic culture studied. A mass-transfer limited regime was reached for all
conditions, where oxygen availability directly controlled the growth rate. Increased oxygen
availability, however, did not proportionally increase the growth rate as the energy require-
ments were affected. This was likely due to nitrogenase protection mechanisms against
oxygen-stress and ETC down-regulation at high oxygen availability. The most efficient
growth occured at condition O2 _21. The supernatent was found to be virtually clean,
with little to no carbon and nitrogen-compounds present. This confirmed that part of the
glucose was channelled to a carbon sink in the form of IPS or EPS. The metagenomic data
showed that the largest portion of the microbial population were aerobic nitrogen-fixers.
The dominating species were Chryseobacterium ssp. and Flavobacterium ssp.
Since this study utilized glucose as its carbon-source which is sub-optimal from an
economic perspective, it is recommended to explore alternative carbon-sources. In soil,
diazotrophs form symbiotic relationships with plants where plant exudates are utilized as
carbon sources. Alternative carbon-sources with a high C:N ratio, such as lignocellulosic
waste, could be explored. Co-inoculation with lignocellulosic digesting organisms could,
therefore, be a potential research direction. This approach would improve the sustainability
of the process. Lastly, the loss of glucose towards carbon sinks is a challenge. The ability of
the consortium to utilize the carbon sinks at a later stage for growth should be investigated.

Supplementary Materials: The following are available online at https://www.mdpi.com/article/10

.3390/pr9112039/s1.
Author Contributions: Conceptualization, A.Y.S.d.Z., I.L.v.R., W.N. and H.G.B.; methodology,
A.Y.S.d.Z.; software, A.Y.S.d.Z.; validation, A.Y.S.d.Z., W.N. and H.G.B.; formal analysis, A.Y.S.d.Z.;
Processes 2021, 9, 2039 14 of 15

investigation, A.Y.S.d.Z. and J.C.B.; resources, W.N.; data curation, A.Y.S.d.Z. and J.C.B.; writing—
original draft preparation, A.Y.S.d.Z.; writing—review and editing, A.Y.S.d.Z., I.L.v.R., W.N. and
H.G.B.; visualization, A.Y.S.d.Z., W.N. and H.G.B.; supervision, W.N. and H.G.B.; project administra-
tion, W.N.; funding acquisition, W.N. All authors have read and agreed to the published version of
the manuscript.
Funding: This research received no external funding.
Data Availability Statement: The data presented in this study are openly available in the University
of Pretoria Research Data Repository at 10.25403/UPresearchdata.17005651.
Acknowledgments: Devesh Devroop for technical support in the design of the bench-scale reactor
regarding electronic equipment and the control system.
Conflicts of Interest: The authors declare no conflict of interest.

References
1. Population Action International. Why Population Matters to Food Security; Population Action International: Washington, DC, USA,
2011; pp. 1–4.
2. Hester, R.; Harrison, R. Soils and Food Security, 1st ed.; Royal Society of Chemistry: Cambridge, UK, 2012; pp. i–vi.
3. Kozai, T.; Niu, G.; Takagaki, M. Plant Factory: An Indoor Vertical Farming System for Efficient Quality Food Production, 1st ed.;
Elsevier Science and Technology: Amsterdam, The Netherland, 2015; pp. 1–10.
4. OECD. Human Acceleration of the Nitrogen Cycle: Managing Risks and Uncertainty; IWA Publishing: London, UK, 2018.
5. Barnard, J. Biological Nutrient Removal—Quo Vadis. In Proceedings of the 93rd Annual Conference—Michigan Water Environment
Association, Boyne Falls, MI, USA, 24–27 June 2018.
6. Chen, S.; Perathoner, S.; Ampelli, C.; Centi, G. Chapter 2—Electrochemical Dinitrogen Activation: To Find a Sustainable Way to
Produce Ammonia. Stud. Surf. Sci. Catal. 2019, 178, 31–46.
7. Mitsushima, S.; Hacker, V. Fuel Cells and Hydrogen, 1st ed.; Elsevier: Amsterdam, The Netherland, 2018; pp. 243–255.
8. Kuypers, M.; Marchant, H.; Kartal, B. The microbioal nitrogen-cycling network. Nat. Rev. 2018, 16, 263–276.
9. Mahanty, T.; Bhattacharjee, S.; Goswami, M.; Bhattacharyya, P.; Das, B.; Ghosh, A.; Tribedi, P. Biofertilizer: A potential approach
for sustainable agricultural development. Environ. Sci. Pollut. Resolut. 2018, 24, 3315–3335. [CrossRef]
10. Oelze, J. Respiratory protection of nitrogenase in Azotobacter species: Is a widely held hypothesis unequivocally supported by
experimental evidence? FEMS Microbiol. Rev. 2000, 24, 321–333. [CrossRef]
11. Pankievicz, V.; Irving, T.; Maia, L.; Ané, J. Are we there yet? The long walk towards the development of efficient symbiotic
associations between nitrogen-fixing bacteria and non-leguminous crops. BMC Biol. 2019, 17, 99. [CrossRef]
12. Singh, R.; Ryu, J.; Kim, S. Microbial consortia including methanotrophs: Some benefits of living together. J. Microbiol. 2019,
57, 939–952. [CrossRef]
13. Brewin, B.; Woodley, P.; Drummond, M. The Basis of Ammonium Release in nifL Mutants of Azotobacter vinelandii. J. Bacteriol.
1999, 181, 7356–7362. [CrossRef]
14. Barney, B.; Plunkett, M.; Natarajan, V.; Mus, F.; Knutson, C.; Peters, J. Transcriptional Analysis of an AmmoniumExcreting Strain
of Azotobacter vinelandii Deregulated for Nitrogen Fixation. Appl. Environ. Microbiol. 2017, 83, e01534-17. [CrossRef] [PubMed]
15. Bali, A.; Blanco, G.; Hill, S.; Kennedy, C. Excretion of Ammonium by a nifL Mutant of Azotobacter vinelandii Fixing Nitrogen.
Appl. Environ. Microbiol. 1992, 58, 1711–1718. [CrossRef] [PubMed]
16. Romero-Perdomo, F.; Camelo-Rusinque, M.; Criollo-Campos, P.; Bonilla-Buitrago, R. Effect of temperature and pH on the biomass
production of Azospirillum brasilense C16 isolated from Guinea grass. Pastos Y Forraje 2015, 38, 231–233.
17. Merck KGaA. Nitrogen (Total) Cell Test. 2021. Available online: https://www.sigmaaldrich.com/ZA/en/product/mm/100613
(accessed on 6 October 2021).
18. PacBio. Procedure & Checklist—Full-Length 16S Amplification, SMRTbellR Library Preparation and Sequencing. 2018. Avail-
able online: https://dnatech.genomecenter.ucdavis.edu/wp-content/uploads/2018/07/PacBio-Full-Length-16S-Sequencing-
Protocol-June2018.pdf (accessed on 6 October 2021).
19. Somerfield, P. Identification of the Bray-Curtis similarity index: Comment on Yoshioka. Mar. Ecol. Prog. Ser. 2008, 372, 303–306.
[CrossRef]
20. Smercina, D.; Evans, S.; Friesen, M.; Tiemanna, L. To Fix or Not To Fix: Controls on Free-Living Nitrogen Fixation in the
Rhizosphere. Appl. Environ. Microbiol. 2019, 86, e02546-18. [CrossRef] [PubMed]
21. Kövilein, A.; Kubisch, C.; Cai, L.; Ochsenreither, K. Malic acid production from renewables: A review. J. Chem. Technol. Biotechnol.
2020, 95, 513–526. [CrossRef]
22. Wang, D.; Xu, A.; Elmerich, C.; Ma, L. Biofilm formation enables free-living nitrogen-fixing rhizobacteria to fix nitrogen under
aerobic conditions. ISME J. 2017, 11, 1602–1613. [CrossRef]
23. Lexow, W.G.; Sekgetho, C.M.; Brink, H.G.; Nicol, W. Identifying Energy Extraction Optimisation Strategies for A. succinogenes.
Catalysts 2021, 11, 1016. [CrossRef]
24. Bailey, W. Biofilms: Formation, Development and Properties; Nova Science Publishers: New York, NY, USA, 2011.
Processes 2021, 9, 2039 15 of 15

25. Nielsen, J.; Villadsen, J. Bioreaction Engineering Principles; Springer: Boston, MA, USA, 1994.
26. Iwata, K.; Azlan, A.; Yamakawa, H.; Omori, T. Ammonia accumulation in culture broth by the novel nitrogen-fixing bacterium,
Lysobacter sp. E4. J. Biosci. Bioeng. 2010, 110, 415–418. [CrossRef]
27. de Souza Pinto Lemgruber, R.; Valgepea, K.; Tappel, R.; Behrendorffb, J.; Palfreyman, R.; Plan, M.; Hodson, M.; Simpson,
S.; Nielsen, L.; Köpke, N.; et al. Systems-level engineering and characterisation of Clostridium autoethanogenum through
heterologous production of poly-3-hydroxybutyrate (PHB). Metab. Eng. 2019, 53, 14–23. [CrossRef]
28. Dhole, A.; Shelat, H.; Deepak, P. Chryseobacterium indologenes A Novel Root Nodule Endophyte in Vigna radiata. Int. J. Curr.
Microbiol. Appl. Sci. 2017, 6, 836–844.
29. Giri, S.; Pati, B. A comparative study on phyllosphere nitrogen fixation by newly isolated Corynebacterium sp. & Flavobacterium sp.
and their potentialities as biofertilizer. Acta Microbiol. Immunol. Hung. 2004, 51, 47–56.
30. Naqqash, T.; Imran, A.; Hameed, S.; Shahid, M.; Majeed, A.; Iqbal, J.; Hanif, M.; Ejaz, S.; Malik, K. First report of diazotrophic
Brevundimonas spp. as growth enhancer and root colonizer of potato. Sci. Rep. 2020, 10, 12893. [CrossRef]
31. Kaminski, P.; Mandon, K.; Arigoni, F.; Desnoues, N.; Elmerich, C. Regulation of nitrogen fixation in Azorhizobium caulinodans:
Identification of a fixK-like gene, a positive regulator of nifA. Mol. Microbiol. 1991, 5, 1983–1991. [CrossRef] [PubMed]
32. Calderón, G.; García, E.; Rojas, P.; García, E.; Rosso, M.; Losada, A. Chryseobacterium indologenes infection in a newborn: A case
report. J. Med. Case Rep. 2011, 5, 10. [CrossRef] [PubMed]
33. De Donno Novelli, L.; Moreno, S.; Rene, E. Polyhydroxyalkanoate (PHA) production via resource recovery from industrial waste
streams: A review of techniques and perspectives. Bioresour. Technol. 2021, 331, 124985. [CrossRef]
34. Jurat-Fuentes, J.; Jackson, T. Chapter 8—Bacterial Entomopathogens, 2nd ed.; Academic Press: Cambridge, MA, USA, 2012;
pp. 265–349.
35. Gtari, M.; Ghodhbane-Gtari, F.; Nouioui, I.; Beauchemin, N.; Tisa, L. Phylogenetic perspectives of nitrogen-fixing actinobacteria.
Arch. Microbiol. 2012, 194, 3–11. [CrossRef] [PubMed]
36. Osman, Y.; Elrazak, A.; Khater, W. Bioprocess Optimization of Microbial Biopolymer Production. J. Biobased Mater. Bioenergy
2016, 10, 119–128. [CrossRef]
37. Orozco-Medina, C.; López-Cortés, A.; Maeda-Martínez, A. Aerobic Gram-positive heterotrophic bacteria Exiguobacterium
mexicanum and Microbacterium sp. in the gut lumen of Artemia franciscana larvae under gnotobiotic conditions. Curr. Sci. 2009,
96, 120–129.
38. Ali, I.; Jamil, N. Biosynthesis and genetics of polyhydroxyalkanoates by newly isolated Pseudomonas aeruginosa IFS and 30N
using inexpensive carbon sources. Int. J. Environ. Sci. Technol. 2017, 14, 1879–1888. [CrossRef]
39. Bhuwal, A.; Singh, G.; Aggarwal, K.; Goyal, V.; Yadav, A. Isolation and Screening of Polyhydroxyalkanoates Producing Bacteria
from Pulp, Paper, and Cardboard Industry Wastes. Int. J. Biomater. 2013, 2013, 752821. [CrossRef]
40. Nojima, S.; Mineki, S.; Iida, M. Purification and characterization of extracellular poly(3-hydroxybutyrate) depolymerases
produced by Agrobacterium sp. K-03. J. Ferment. Bioeng. 1996, 81, 72–75. [CrossRef]
41. Van Montagu, M.; Zambryski, P. Agrobacterium and Ti Plasmids; Elsevier: Amsterdam, The Netherlands, 2017.
42. Earth Observing System. Nitrogen Fixation: N-Fixing Plants And Bacteria. 2021. Available online: https://eos.com/blog/
nitrogen-fixation/ (accessed on 20 September 2021).
43. Rai, S.; Solanki, A.; Anal, A. Modern Biotechnological Tools: An Opportunity to Discover Complex Phytobiomes of Horticulture Crops;
Academic Press: Cambridge, MA, USA, 2021; pp. 85–124.
44. Copeland, A.; Lucas, S.; Lapidus, A.; Barry, K.; Detter J.C.; Glavina del Rio, T.; Dalin, E.; Tice, H.; Pitluck, S.; Chertkov, O.; et al.
Proteomes—Acidovorax sp. (Strain JS42). UniProt Database. 2006. Available online: https://www.uniprot.org/proteomes/UP000
000645 (accessed on 20 September 2021).
45. Feng, Y.; Feng, J.; Shu, Q. Isolation and characterization of heterotrophic nitrifying and aerobic denitrifying Klebsiella pneumoniae
and Klebsiella variicola strains from various environments. J. Appl. Microbiol. 2018, 124, 1195–1211. [CrossRef]
46. Apparao, A.; Krishnaswamy, V. Production of Polyhydroxyalkanoate (PHA) by a Moderately Halotolerant Bacterium Klebsiella
pneumoniae U1 Isolated from Rubber Plantation Area. Int. J. Environ. Bioremediation Biodegrad. 2015, 3, 54–61.