Download Application of Free Living N-fixers in Agriculture

Application of Free Living
N-fixers in Agriculture
SOIL311 Literature Review
University of New England
School of Environmental and Rural Science
Jesse L. Fenn
March, 2015
Introduction
Nitrogen (N) is essential to maintaining complex lifecycles spanning all living organisms.
Biological N fixation is the process by which some 97% of new N inputs are incorporated
into unmanaged terrestrial ecosystems (Vitousek et al., 2002; Galloway et al., 2004, as cited
in Reed, Cleveland, & Townsend, 2011, p. 490). Plants require N for growth and
reproduction. In non-legume species, it is the most important mineral nutrient and in all
plants the most abundant, comprising of 2-4% of plant dry matter (Blair & Sale, 1996, p. 35).
While N is abundant in the atmosphere in various forms (Webb, 2015, para 2), in the soil
system N levels fluctuate and are often low enough to limit production due to the cyclic
nature of its various forms, mobility and numerous loss channels (McLaren & Cameron,
1996, p. 192-193).
Producers attempt to meet the plant’s requirements for N through the application of synthetic
fertilizers, recycled organic wastes (manures, etc) and the use and promotion of biological Nfixers (Orr, James, Leifert, Cooper, & Cummings, 2011, p. 911). As agriculture depends so
heavily on N, interest in these biological N-fixers is intensifying. Furthermore, with markets
demanding “organic” options, some producers are moving away from conventional synthetic
fertilizers altogether. This has initiated numerous studies on biological N-fixers seeking to
answer questions of quantity of N fixed, ecological interactions and management
considerations.
While these questions are being debated in the research realm, various products exist on the
market to inoculate soil systems with these diazotrophic microorganisms to boost populations
for the purpose of N-fixation. Rhizobium bacteria are known to form symbiotic relationships
with the root systems of leguminous species and their use is a commonly accepted method of
increasing N availability to plants under crop rotation systems. Less utilized are the freeliving bacteria capable of N-fixation, which have previously been viewed as largely noneffective for agriculture (Reid & Wong, 2005). The use of Free-living N-fixing bacteria
(FLNFB) in agriculture as well as management considerations found in the scientific
literature for the promotion of FLNFB will be reviewed.
Free Living N-Fixing Bacteria
The term “free-living” in relation to N-fixing bacteria refer to all bacteria that do not live in a
direct symbiotic association with vascular plants, both the true FLNFB and autotrophic and
heterotrophic organisms that fit this description (Cleveland, et al., 1999, p. 624). FLNFB are
present in nearly all terrestrial ecosystems and are often the primary pathway for the fixation
of atmospheric N into a plant available form (Reed et al., 2011, p. 489). A number of genera
have been identified as FLNFB (Stewart, 1969, p. 367), with strains capable of fixing N to
differing degrees. The most well known include Azotobacter, Biejerinckia and Clostridium,
all found in different climatic zones with preferences for specific soil conditions (acidic,
anaerobic etc.) (McLaren & Cameron, 1996, p. 195). Orr et al. (2011, p. 911) cite studies
where FLNFB fixation rates of up to 60 kg N ha-1 year-1 have been recorded.
Free Living N-Fixing Bacteria in Agriculture
The ecosystems where FLNFB are found are vast and varied, and Zechmeister-Boltenstern &
Kinzel (1990, p. 1079) state that the rates of fixation in forests and arable fields are relatively
low compared to saline and peat soils with studies documenting remarkable N2 fixation rates
under the latter conditions.
The contribution of the FLNFB is dependant on physiochemical properties where only a
proportion of the microbes capable of fixing N will be active under given conditions
(Wakelin, Gupta, & Forrester, 2010, p. 395, 397). In addition, overall microbial activity plays
a role, as shown in Patra et al. (2007, p. 155), where contribution of FLNFB was increased
when soil microbial activity decreased. Thus it can be deduced that FLNFB are less
competitive in the rhizosphere.
Still, studies have been undertaken to quantify the amount of N fixed by these bacteria in
agricultural systems. Herridge, Peoples, & Boddey (2008, p. 11) cited studies where it was
estimated that FLNFB fixed 25 kg N ha-1 year-1 in sugar cane crops and <5 kg N ha-1 year-1 in
crop lands other than used for legumes and rice; their conclusions on both studies was that
large variations were evident in the data or that suffient data was not available for decisive
values. While models exist to calculate potential N-fixation rates, measuring and predicitng
actual rates is extremely difficult.
These values are underwheling compared to the ~100 kg N ha-1 year-1 of N fertilizer applied
to many modern agriculture systems (Reed et al. 2011, p. 490-491). Using this rate as a
reference point, we are beginning to quantify FLNFB’s impact and contribution to an
agricultural production system.
Herridge et al. (2008, p. 11) then show in stark contrast the N-fixaton rates that occur in
leguminous species with the symbiotic relationship of the rhizobium bacteria. Not only are
the models used to predict fixation rates based on sound historical crop yield data, actual
fixaton rates are easier to measure in both cropping and pasture/fodder systems (cropping
more so) (p. 5). Herridge et al. offer the follow table summarizing the differences in fixation
rates (shown in Table 1):
Table 1 Summary of estimates of N fixed annually in agricultural systems comparing the agent by which it occurs (Herridge
et al., 2008, p. 11)
Agent
Agricultural system
Legume
rhizobia
-
Crop (pulse and oilseed legumes)
Rate of N2 fixation
(kg N ha-1 year-1)
115
Legume
rhizobia
-
Pasture and fodder legumes
110-227
12-25
Free-living
bacteria
Crop lands (other than legumes and
rice)
<5
<4
Free-living
bacteria
Extensive
grazing
<10
<14
savannas
used
for
Crop N fixed
(Tg year-1)
21
It is worthwhile noting however, that symbiotic associations cost the plant significant
photosynthates (De Mita, 2012, p. 885) and reduce the total yields of legumes.
Despite the lesser amount of N that FLNFB actually fix when comapred to common fertilizer
rates and symbiotic associations, products exist to help boost the numbers of these
micoorganisms to play their part (Webb, 2015, p. 4). While inoculating the soil with mass
numbers of these FLNFB may increase the amount of N they fix, it would seem unlikely that
it would ever match the amount of N required of other inputs for agricultural systems. Still,
increasing numbers and managing the soils to promote their activity can have beneficial
affects, as Wakelin et al., (2010, p. 391) show.
While acknowledging that the majority of N-fixation occurs in symbiosis with leguminous
plants, Wakelin et al. state that FLNFB play an important role between crop cycles,
contributing the N needed to decompose crop residues which generaly have wide C:N ratios.
So while their contribution to the N needed in agricultural production systems may not be
their primary role, they clearly contribute to overall soil fertility and nutrient cycling and thus
should not be ignored.
Management Considerations
Agricultural soils differ infinitely in the intricate physical and chemical properties where
microbial communities are found. These factors cause the spatial and temporal variation seen
in microbial populations (Gupta & Roper, 2010, p. 50) and introduce difficulties in sampling
for quantification studies (Orr et al. 2011, p. 918). Despite these differences, Wakelin et al.
(2010, p. 391) found that the effects of management practices often had a greater impact on
FLNFB than between-soil differences. This highlights the importance of management of soil
systems for continued productivity.
In the research conducted by Wakelin et al (2010, p.391-399), factors affecting diversity,
abundance and activity of FLNFB in Australian agricultural soils was studied. Based on the
previous conclusions regarding the role of FLNFB in agricultural systems, their findings are
helpful when considering practices to promote FLNFB.
Experiment 1 looked at the nifH gene (a component of the nitrogenase complex) levels in 20
agricultural sites of different management practices and soil types. The results showed of the
properties analyzed within these soils, particulate organic carbon (POC) levels were the most
correlated (ρ=0.502; P=0.001) with nifH structures. Conversely, Total C (or N) levels were
not strongly correlated, highlighting FLNFB's affinity for a type of C in the soil, not just the
total level. From a management perspective, POC is the component of soil organic matter
(SOM) that consists of recently added and semi-decomposed material, hence Wakelin et al.'s
previous conclusion of the role FLNFB play in nutrient cycling of crop residues.
Experiment 2 focused on heavily textured, high input agricultural soils with varying
management practices, details of which are presented in Table 2.
Table 2 Sites from Experiment 2 focusing on management practices and the affect on FLNFB (Wakelin et al. 2010, p. 392393)
Site
"Border Check"
Rotation
5 year; three cereals, canola and
faba bean
Management Practice
Stubble burnt, incorportated or left on
surface
"Contour"
Rice
Stubble burnt, incorportated or left on
surface
"Pivot"
3 year; two cereals and one
canola
Stubble incorporated
It was found that for the Border Check site, incorporating stubble increased N2-fixation
alomst four times over that of stubble burnt or left on surface. In contrast, there was no
difference in N2-fixation between management practice evident at the Contour site. This
difference could be attributed to the cultivation system (rice over cereal cropping) than
management practice. Finally, in the Pivot site, N2-fixation was more than 3.5 times higher (P
= 0.005) in the high input (irrigated) cropping area when compared with an adjacent nonirrigated fallow area when analysied by the acetylene reduction technique.
From this study, the management implications for broadacre cropping would be to
incorporate crop residue into the soil as the amount of C entering the system is directly
related to the activity and abundance of FLNFB (Wakelin et al., 2010, p. 397-398).
Orr et al., (2011, p. 911) and Wakelin et al. (2010, p. 397) cite studies showing that external
stress events, such as fire, salinity, liming and application of chemcial herbicides and
pesticides, can all have a negative affect soil microbe populations, including FLNFB. Gupta
and Roper (2010, p. 50) further investigate this understanding looking at factors that increase
the physical protection of FLNFB in the soil matrix from these common practices.
From a soil physics perspective, they found that with increased clay content, soil aggregation
was increased. Gupta and Roper (2010, p. 53) cite several studies which have found that
within these micro-habitats, FLNFB thrive and find protection from predation, environmental
extremes and exposure to toxic or biocidal compounds.
Roper and Smith (1991, as cited in Gupta & Roper, 2010, p. 53) found that clay soils
supported greater nitrogenase activity than sandy soils. The management implications are:


for clay soils, every effort should be taken to preserve the integrity of the soil;
avoiding compaction of susceptible clays, avoiding exposing sodic clays and
minimizing erosion wherever possible
for sandy soils, SOM can still act as a suitable micro-habitat for FLNFB, hence on
these soil types, the incorporation of stubble becomes paramount to the promotion of
microbial activity. This practice also increases the possibility of maintaining sufficient
moisture in the soil to support the soil microbes which itself presents as a significant
challenge where long-dry periods are encountered.
This conclusion support the findings of Lamb et al. (1987, as cited in ZechmeisterBoltenstern & Kinzel, 1990, p. 1081) where C2H2 reduction (a measure of microbial activity)
was greater in no-till systems than in conventional wheat-fallow systems due to higher soil
moisture and the presence of SOM in the no-till soils.
Finally, from a soil nutrients perspective, Reed, Cleveland, and Townsend (2013, p. 136) cite
several studies that show that P additions stimulate N-fixation, and thus there may be a P
limitation to N-fixation. This is particuraly important for Australian soils that are known to be
low in available P (Moody & Bollard, 1999, p. 187 - 188). Similarly, in the study conducted
by Orr et al. (2011, p. 911) it was found that FLNFB were more active in conventionally
fertilized plots over organically fertilized plots, however the effects are often subtle and short
lived (918). Finally, Reed et al. (2011, p. 501) cite Barron et al. (2008) in stating P fertilizers
coated and/or contaminated with Mo can confound interpretations showing Mo may also play
a necessary role and that responses vary significantly within and between ecosystems.
The management implications in regards to soil nutrients is to maintain the addition of
suitable fertilization or other appropriate soil amendments in order to maintain soil vitality.
This will differ from location to location and depend on crop/pasture composition, rotation,
past fertilizer regime and specific enterprise requirements. Soil tests are a valuable tool in
assessing possible nutrient limitations and when used skillfully, can save producers
significant money in amendment application.
Conclusion
It has been shown from the amount of N FLNFB actually fix that their role should not be over
emphasised, however their important contribution to soil fertility should not be ignored.
Assessing return from and contribution of applying FLNFB to a soil system would be
difficult in the field. Until more accurate and accessible methods for determining contribution
from FLNFB are developed, investing in this practice would be inadvisable. Nevertheless,
acknowledging the existence of, and managing for the promotion of FLNFB should
contribute to overall soil fertility leading to an increase in productivity.
References
Blair, G., & Sale, P. (1996). Plant Nutrition. Armidale, Australia: University of New
England.
Cleveland, C., Townsend, A., Schimel, D. … Wasson, M. (1999). Global patterns of
terrestrial biological nitrogen (N2) fixation in natural ecosystems. Global Biological Cycles,
13, 623-645.
De Mita, S. (2012). For better or for worse: cooperation and competition in the legumerhizobium symbiosis. New Phytologist, 194, 885-887.
Gupta, V., & Roper, M. (2010). Protection of free-living nitrogen-fixing bacteria within the
soil matrix. Soil Tillage and Research, 109, 50-54.
Herridge, D., Peoples, M., & Boddey, R. (2008). Global inputs of biological nitrogen fixation
in agricultural systems. Plant Soil, 311, 1-18.
McLaren, R., & Cameron, K. (1996). Soil Science - Sustainable production and
environmental protection. South Melbourne, Australia: Oxford University Press.
Moody, P.W., & Bollard, M.D.A. (1999). Phosphorus. In D.J. Reuter (Ed), Soil Analysis: an
interpretation manual (pp. 187-220). Collingwood, Australia: CSIRO Publishing.
Orr, C., James, A., Leifert, C., Cooper, J., & Cummings, S. (2011). Diversity and activity of
free-living nitrogen-fixing bacteria and total bacteria in organic and conventionally managed
soils. Applied and Environmental Microbiology, 77, 911-919.
Patra, A., Le Roux, X., Abbadie, L., Clays-Josserand, A., Poly, F., Loiseau, P., & Louault, F.
(2007). Effect of microbial activity and nitrogen mineralization on free-living nitrogen
fixation in permanent grassland soils. J. Agronomy & Crop Science, 193, 153-156.
Reed, S., Cleveland, C., & Townsend, A. (2011). Functional ecology of free-living nitrogen
fixation: a contemporary perspective. Annu. Rev. Ecol. Evol. Syst., 42, 489-512.
Reed, S., Cleveland, C., & Townsend, A. (2013). Relationship among phosphorus,
molybdenum and free-living nitrogen fixation in tropical rainforests: results from
observational and experimental analyses. Biogeochemistry, 114, 135-147.
Reid, G., & Wong, P. (2005). Soil Biology Basics. Retrieved from www.dpi.nsw.gov.au/
_data/assets/pdf_file/0017/41642/Soil_bacteria.pdf
Stewart, W. (1969). Biological and ecological aspects of nitrogen fixation by free-living
micro-organisms. Proc. Roy. Soc., 172, 367-388.
Wakelin, S., Gupta, V., & Forrester, S. (2010). Regional and local factors affecting diversity,
abundance and activity of free-living, N2 fixing bacteria in Australian agricultural soils.
Pedobiologia, 53, 391-399.
Webb, G. (2015). Nitrogen fixing bacteria in Agriculture. Retrieved from: www.activesoil.com/files/9513/7080/1105/TwinN_Nitrogen_Fixing_Bacteria_in_Agriculture.pdf
Zechmeister-Boltenstern, S., & Kinzel, H. (1990). Non-symbiotic nitrogen fixation associated
with temperate soils in relation to soil properties and vegetation. Soil Biol. Biochem, 22,
1075-1084.