PRODUCTION AND DEPLOYMENT OF

PHOTOSYNTHETIC NITROGEN-FIXING
BIOFERTILIZERS

Timothy Flynn, M.S., Ph.D.

Primordial Solutions, Inc.
463 Gunnison Ave., Suite 2
Grand Junction, CO 81501

69
 BIOGRAPHICAL SKETCH

Timothy Flynn, M.S., Ph.D.

Timothy Flynn is a graduate of Western State College in Gunnison, Colorado, where

he studied biology, botany, and microbial ecology. Flynn then earned an M.S. and Ph.D. in
fungal microbiology (mycology) at Virginia Tech. Postdoctoral experiences include enzymatic
studies of the human pathogenic yeast, Cryptococcus neoformans, at the department of
Biochemistry and Anaerobic Microbiology (Virginia Tech) under the direction of Walter
Niehaus. Another effort entailed manipulating the photoproduction of hydrogen by the alga,
Chlamydomonas reinhardtii, directed by Maria Ghirardi. Following these experiences, Dr.
Flynn taught courses in biology and chemistry at Mesa State College in Grand Junction,
Colorado. Underlying this background and history is the desire to repair damaged soils. Dr.
Flynn is now the Vice President for Research at Primordial Solutions, Inc. The company
produces living microbial biofertilizers that accelerate the restoration of disturbed arid soils.

70
 PRODUCTION AND DEPLOYMENT OF
PHOTOSYNTHETIC NITROGEN-FIXING
BIOFERTILIZERS

Timothy Flynn, M.S., Ph.D.

Primordial Solutions, Inc.

463 Gunnison Ave., Suite 2
Grand Junction, CO 81501

ABSTRACT

The task of revegetating the delicate arid landscapes is a challenging proposition.

The major limiting factors are water, nitrogen fertility and easily eroded sandy soils. The
current art often includes the application of seeds, chemical fertilizers, tackifiers, and mulch.
If inadequate precipitation or poor nutrient status prevents the establishment of vascular
plants, these soil amendments are often reapplied, creating additional expense.

In contrast, Primordial Solutions, Inc., has developed a technology that is designed

to restore soil fertility, water retention, and reduce erosion without relying on the presence of
vascular plants. Instead, we exploit photosynthetic microorganisms, called cyanobacteria or
“blue-green algae,” which convert solar energy to stored chemical energy, and fix
atmospheric nitrogen (N 2) into usable ammonia (NH3). The fixed nitrogen is ultimately
incorporated into proteins and nucleic acids, biopolymers essential to life.

Throughout the world, cyanobacteria, lichens, bacteria, and fungi, establish self-
sufficient microbial communities called “Biological Soil Crusts” (BSC). In the American
southwest, BSC are responsible for 99% of the nitrogen input, represent up to 70% of the
living ground cover, improve the nutritional value of forage plants, improve water retention,
and control erosion. However, following a disturbance, the BSC are slow to recover.
Recovery estimates range from 4 to 4000 years depending on the climate, inoculum proximity
and the definition of recovery.

Primordial Solutions’ approach for restoring soil fertility involves isolating

cyanobacteria that are native to the disturbed area, mass-producing the microorganisms and
applying the mixed culture to the disturbed soil. This process establishes a self-perpetuating,
living, green-manure that becomes active when hydrated. Once established, the restored
BSC creates a substrate suitable for the survival of vascular plants.

A preliminary experiment was done to compare inoculated soil with an uninoculated

control. Following 18 months of incubation under ambient climate, the inoculated soil had a
twelve-fold increase in nitrogen content compared to the control treatment. Additionally, the
cyanobacterial biomass increased almost ten-fold from the initial 500 mg C m-² to 4788 mg
C m-² (dry weight biomass). These results show that the cyanobacteria are alive and retain

71
 the ability to fix and secrete nitrogen into the soil. We plan to describe the 2005 field
experiments that are being conducted in the desert west of Grand Junction, Colorado. This
work was supported by the Small Business Innovation Research program of the U.S.
Department of Agriculture, grant #2003-33610-13086.

Key Words: biological soil crust; cyanobacteria; biofertilizer; nitrogen fixation

INTRODUCTION to fixing and secreting nitrogen into the soil, many of

The term, Biological Soil Crusts (BSC), is one of
the many terms used to describe terrestrial microbial
communities that are composed of cyanobacteria,
cyanolichens, phycolichens, fungi, eukaryotic algae,
bacteria, bryophytes, and protozoa. A comprehensive
review of the literature is found in Belnap and Lange
(2001) and an excellent primer on BSC biology is
found at www.soilcrust.org. Most of the cyanobacteria
and all of the cyanolichens are azototrophic, meaning
that they fix their own nitrogen. Nitrogen secretion
rates on the cool desert of the Colorado Plateau are on
the order of 10 kg N ha-1 yr-1 (Belnap, 2001). In addition
these organisms are essentially slime-covered
filaments that cause the soil particles to bind together.
These biological characteristics make BSC
communities the major contributor to the fertility and
physical stability of desert soils. Figure 1 shows a well
developed native crust community in southeastern
Utah. Figure 2 shows steep slopes being stabilized by
native BSC at the Gunnison Gorge Wilderness Area
(Delta Co., Colorado).
However, given these desirable attributes, the
major handicap of BSC communities, just like all
organisms in arid environments, is slow recovery rates

Figure 1. Biological soil crust community in southeastern Utah (photo courtesy of

www.soilcrust.org).

72
 Figure 2. Steep slopes being stabilized by native biological soil crust in western
Colorado.
following disturbances such as fire, overgrazing,
mineral or energy extraction, and unmanaged
recreation. Depending on the definition of recovery,
proximity to inoculum, and precipitation, estimates of
recovery range from 4 to 4000 years. The reason for
the slow recovery is that sufficient moisture and
adequate temperatures rarely occur at the same time
in arid ecosystems. Therefore, naturally occurring
inocula are more likely to blow away before they can
become established. In order to defeat this problem,
we are mass-producing the native BSC cyanobacteria
in a photobioreactor. This inoculum, produced at the
pilot-plant scale, reflects the native BSC community
structure and the local ecotype. When the inoculum is
installed to disturbed soils, the rate of BSC recovery is
accelerated. We expect that once the microbial soil
community is restored, the recovery of the native
vascular flora will follow.
Environmentally, we established 11 plots in five
study areas. The study plots address the questions of
soil chemistry and the floristic in response to various
cyanobacterial inoculation rates. The results of this
study are still being generated. However, a preliminary
study done in the same area revealed supportive
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results. Compared to the control, the inoculated plots
increased the total nitrogen content twelve-fold. The
algal biomass was 4800 times greater than the control,
and the inoculated biomass increased almost ten-fold
compared to the initial density over an 18-month
period.
The technology we have developed is designed to
perform as a living biofertilizer and erosion control
agent. It can either replace or enhance current soil
remediation techniques such as the use of mulches,
tackifiers, geotextiles, and synthetic fertilizers. Potential
applications or markets include repairing the results of
energy and mineral extraction, public lands that were
damaged by fire, overgrazing, recreation, or be used
as a cover crop to reduce wind erosion in dry-land
agricultural regions (dust-bowl states).
RESULTS AND METHODS
Inoculum
The culture (TF1015) was isolated from a well
developed BSC sample taken fro the desert of Bang’s
Canyon near Grand Junction, Colorado. The
organisms are supplied with light, inorganic nutrients
and 2% CO2. The species composition consisting of
Microcoleus, Scytonema, Nosotc, Trichormus ( = Ana-
baena), and others. Scytonema is illustrated in Figure
3. All of the listed cyanobacterial genera, except for
Microcoleus, are nitrogen fixers.
Tubular Photobioreactor
The tubular photobioreactor (Figure 4) a major
advance of our efforts. It is designed to mass-produce
cyanobacteria in a semicontinuous manner. The
photostage (Figure 4) was constructed from Clear PVC
tubing (7.5 cm inner diam) giving a total length of 100
m and a 415 L working volume. A pump, placed
between the photostage and the resting-tank,
circulates the culture to give a photostage residence
time of 15 minutes. After the culture has circulated
through the photostage, it is discharged into the resting
tank (265 L working volume) where the excess oxygen
is degassed to the atmosphere. Two sets of probes are
used to measure dissolved oxygen, pH, and
temperature at the beginning and end of the
photostage. The difference in dissolved oxygen values
at the beginning and end of the photostage measures
the rate of photosynthesis. Likewise the difference in
pH values measures the rate of CO2 uptake. All of the
probes are connected to a controller and the data are
logged into a computer. In addition to the data
acquisition, the controller holds the pH and dissolved
Figure 3. Scytonema from TF1015.
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CO2 constant through control of a solenoid valve.
When the solenoid valve is open, pure CO2 is
introduced into the beginning stage using a ceramic
diffuser (13 mm (diam) x 30 cm) at a rate of 25 mL C
min-1.
In the current system, the pH is held at 7.5, and
the total alkalinity, or the ability to absorb protons,
expressed as CaCO3, was found to be 218 mg C L-1 by
titration. Multiplying 218 mg CaCO3 C L-1 time the
conversion factor 0.061 (Cole, 1979) yields the 13.3
mg C L-1 of free CO2. On bright sunny days the rate of
photosynthetic oxygen evolution is about 520 :moles
O2 C mg Chl-1 C h-1. This is estimated from the
difference of dissolved oxygen from the beginning and
end of the photostage, the residence time, and
chlorophyll concentration.
Figure 5 shows the cumulative mass that was
harvested from the photobioreactor over a 41-day
period. The current harvesting method entails stopping
the pump and isolating the resting tank from the
photostage. After 15 minutes, the cells have settled
from the 195 L working volume to 20 L at the bottom of
the tank. Another valve is opened and the thick algal
slurry is collected into a bucket. The slurry is then
dewatered by gravity filtration requiring roughly two
hours. The dewatered algal mass has a color and
consistency of steamed, pureed spinach. The algal
mass is 7.66% dry weight +/- 0.57% (7.66 g dry = 100
Figure 4. Photobioreactor with growing culture.

Figure 5. Photobioreactor productivity.

75
g wet). Day zero (Figure 4) is defined as the first
harvest following two weeks of initial growth. The wet-
weight productivity was about 1 kg per day for the first
5 harvest days.
FIELD EXPERIMENTS
Study Area and Floristics
Eleven plots distributed among five study areas
were installed on BLM public land known as the
“Rabbit Valley Recreation Management Area.” It is
located off exit #2 of I-70 30 miles west of Grand
Junction, Colorado. Plot #2 at area #1 (code: plot 1.2)
is exactly located at 39E 11.812' N, 108 E 01.874' W.
The five areas range in elevation from 1430 to 1504 m.
The soils are alkaline, pH around 8.2, and range from
sandy to sandy-clay. The main variable that
distinguishes the areas is the level of disturbance, and
the dominance of undesirable invasive exotic species.
Historically, Rabbit Valley was a “sheep driveway”
where thousand of sheep were moved twice a year
from summer to winter pasture. In addition, the
introduction of Bromus tectorum (Cheat Grass) has led
to hot destructive fires and area #3 had a tremendous
flood in 1996 that removed the topsoil. Table 1 shows
the altered species composition and the total absence
of BSC. Compared with these data, non-disturbed
areas are dominated by Biological Soil Crusts (BSC)
representing up to 70% of the living cover, the
perennial grasses including Oryzopsis, Stipa, and
Hillaria, and the shrubs Sarcobatus andAtriplex. These
native species have high nutritional value and are
important to livestock and wildlife. In contrast, Bromus
tectorum is a fire hazard and has no nutritional value,
Table 1. Community Structure at the Disturbed Areas of Rabbit Valley.
Cover Type Area 1
BSC
Bare Soil 26.8
Bromus tectorum 68.0
Eremopyrum triticeum
Hillaria jamesii 4.1
Stipa comata
Oryzopsis hymenoides
Salsola australis
Halogeton glomeratus
Gutierrezia sarothrae 1.1
Atriplex confertifolia
* Estimated by the line-intercept method
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Salsola (Russian Thistle) is unpalatable, and
Halogeton (Wienerleaf) is poisonous.
Experimental Design
The field experiments were designed to answer
two primary questions. Firstly, to determine the effects
that cyanobacterial populations have on soil fertility.
These parameters were assayed by soil chlorophyll
and secreted nitrogen. The second goal was to
observe whether cyanobacterial populations can
promote changes in the floristic composition. The
second hypothesis comes from the observation that
invasive exotic weeds do not coexist with Biological
Soil Crusts.
The plot design is similar for the two experiments,
but the questions are different. The “soil plots” and the
“vegetation plots” are 1.5 x 20 m. A 30 cm buffer zone
of inoculated soil surrounds each plot’s perimeter and
1-meter walkways were placed between. There are
four inoculation levels for both experiments; 0, 30, 100,
and 300 mg C m-² expressed as cyanobacterial dry
mass. The placement of the treatment levels was
randomly assigned throughout. The “soil plots” were
further divided into a Cartesian-like system so that 100
cm² sampling units were randomly chosen by having
the grid locations printed on small pieces of paper and
choosing four replicates from the “hat.” Permanent 30
cm pieces of 0.5" PVC pipes were imbedded flush with
the soil surface to mark the corners of each treatment
1.5 x 20 m plot, and the ends were sealed with cork
stoppers. The randomly selected Cartesian
coordinates were then located on the ground by
installing two parallel tape measures to the corner PVC
markers using a “surveyor’s arrow.” The parallel tapes
% Cover*
Area 3 Area 5
41.7 37.3
56.0
6.3
0.4
0.6
21.5
30.5
0.3
0.1
give the linear centimeter mark and a labeled wooden
rod identified the “a, b, c, d” the x-axis locations. The
main response variables are the levels of NH4+, NO3-,
NO2-, which measure the contribution to soil fertility,
and soil chlorophyll, which is related to cyanobacterial
biomass. All of these assays are measured using a
spectrophotometer, and we request a newer machine
with multi sample capability to increase the rate of data
acquisition.
The vegetation plots were installed in similar
fashion, except the locator pipes were centered along
the long axis. A tape measure was attached to the
permanent pipes and the plants that touch the tape
were counted. The permanent locator pipes allow us to
detect any significant changes in floristic composition.
Photographs are also taken to give a visual record of
potential change.
Soil Chemistry
The soil chemistry data from the field experiment
described above currently is being collected. However,
a preliminary inoculation experiment was initiated in
July 2003 adjacent to plot #1.3. The experiment had
two treatment levels of cyanobacterial inoculum, 0 and
500 mg C m-² on a dry mass basis. The values in Table
2 are based on four replicated measurements and are
significantly different at the 99% level. The data in
Table 2 show that soil inoculated with terrestrial
cyanobacteria increased the total nitrogen content
twelve-fold compared to the control. The dominant
species of nitrogen in the inoculated soils are NO3- - N
and NH4+ - N, with nitrate nitrogen being almost four
times greater than ammonium nitrogen, and the nitrite
nitrogen is only 1.6% to the total nitrogen in the
inoculated soil. The distribution of nitrogen species
followed a different pattern in the uninoculated control
soil. In this case the nitrate and nitrite nitrogen levels
were about equal and represented 81% of the total
nitrogen with only 19% being represented by
ammonium nitrogen. The ammonium results are
Table 2. Soil Response to Cyanobacterial Inoculum.
Parameter (mg C m ²) -
NO3- - N
NH4+ - N
NO2- - N
Total Nitrogen
Soil Chlorophylla
Algal Biomass
* less than the detection limits.
consistent with the fact that nitrogen secreted by
cyanobacteria is in the form of ammonia.
Soil chlorophyll (Table 2) was used to estimate the
presence of cyanobacteria. The detection limit of our
equipment is 0.5 mg Chl @ m² or 50 ng C cm², and the
conversion from chlorophyll to dry biomass for cultured
cyanobacteria is 2.59 :g Chl C mg-1 cyanobacteria (dry
weight). The chlorophyll assays show that
cyanobacteria are absent in the uninoculated soil, and
microscopic examination has failed to reveal a single
cyanobacterial cell. The chlorophyll content of the
inoculated soil, however, achieved about 20% of the
chlorophyll content of well-established native
cyanobacterial soil crusts (32 to 64 mg Chl C m-²) after
only 18 months in the field. Further, our estimate of
cyanobacterial biomass had increased almost ten-fold
over the initial concentration. We find these results to
be extremely encouraging given the current drought.
Physical Environment
These results, in our view, are impressive when
the physical environment is taken into consideration.
Colorado and the western United States, over the last
6 years, had experienced the most severe drought in
written history. Historic fires and the draining of Lake
Powell attest to the drought’s extent. We have installed
two data-logging weather stations at study areas #1
and #5. These areas are 8 linear miles apart (12.9 km).
These weather stations measure air temperature 30
cm above the ground; and soil temperature at two
depths, 15 cm and at 0.5 cm that is essentially the soil
surface. The tipping- bucket rain gauges are calibrated
at 0.2 mm liquid water per event. The data from area
#5 was lost because some rodent or Lagomorph
(rabbit) ate the wires. We solved the problem by
running the wires through conduit.
The data interval was from 30 July to 19 October
2004 or 81 days. Only 7 mm of precipitation was
measured the first 45 days. An additional 24 mm was
Inoculation Rate (mg C m-² dry weight)
0 500
19.2 452
9.01 121
19.0 9.74
47.3 583
< 0.5* 12.4
< 0.193* 4788
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recorded from day 46 to day 133 with the wettest
period being in late October. The normal average
precipitation for the Grand Junction airport is about 275
mm @ yr-1. So if one makes the false assumption of
even precipitation throughout the year, the observed
precipitation rate is only 31% of normal.
In addition to little precipitation, the terrestrial
cyanobacteria experience extreme temperatures.
Figure 6 plots the daily thermal maxima and minima at
the soil surface (5 mm depth). We did not expect that
the diurnal temperature difference was so large. For
instance, the temperature difference on 3 and 31
August was 40E C, with the surface maxima being 60E
C and 54E C, respectively. This trend continued
through the middle of September and then declined to
a high and low of 31 E C and 0E C on 15 October.
These trends are reflected in the air and deep soil
probe, where the summertime differential of air was
31E C and the differential of the deep soil probe 10E C.
Figure 6. Daily thermal maxima and minima at the soil surface (7/30–9/8, 2004).
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CONCLUSION
We find these results to be encouraging. We have
shown that the important cyanobacterial species,
isolated from native biological soil crust, can be mass
produced. When these cultures are installed on
disturbed arid soils, the cyanobacteria survive the
treatments, grow, and secrete nitrogen. Given these
results, we believe that this novel approach will provide
a valuable addition to the control of soil erosion.
REFERENCES
Belnap J., and O. L. Lange. 2001. Biological Soil
Crusts: Structure, Function, and Management.
Ecological Studies 150, Springer-Verlag, New
York, New York.
Cole, G. A. 1979. Textbook of Limnology. C.V. Mosby
Company, St. Louis, Missouri.