Fossilized Marine Macroalgae used as a Hygienic
Soil Biology Enhancement Amendment and Contaminant Remediation Device
in Pakistani Rice Cropping Systems.
by Donald W. Trotter
Abstract
Environmental
Health Science Corporation of Provo, Utah (EHS) was tasked by Pacific
Alliance International Marketing Ltd. of British Columbia,
Canada to determine whether the fossilized marine macroalgae (Kelzyme)
material mined by EHS for use as an agricultural soil enhancement
amendment worldwide would have a remediation effect on soils contaminated
with elevated levels of Fluorine (F) from nitrogen applied to rice
crops of the "Green Revolution" generation rice cultivars
in Pakistan. EHS undertook the task at the Southern California Research
Station (SCRS) along with information gathered from documentation
of rice cropping systems where Kelzyme has been in use for several
years. The information gathered from these test shows significant
precipitation of Fluorine as well as an overall increase in soil
biological activity that indicates a potential for enhanced hygienic
microbial activity while decreasing pathogenic microbial populations.
From this testing it is herein documented that Kelzyme has the capacity
to remediate fluorine contaminants while improving the soil food
web's vigor.
Introduction
Fluorine (F) contamination of agricultural soils from various nitrogen
sources has been seen more often in this era of increased soil health
monitoring. Precipitation of Fluorine with Calcium Oxide (CaO) or
burned lime has in the past been the only effective method of scrubbing
Fluorine from environments where it has reached unacceptable levels.
CaO is often not a viable solution to this contamination in agricultural
soils due to lack of economically accessible material. Presently
little is known about biological remediation of Fluorine contaminated
soils, and due to the high solubility of F it can easily enter into
the food or drinking water supply. Fluorine is toxic to humans in
small amounts and can enter into plant tissues when complexed to
iron as FeF6, which is highly soluble in water. High concentrations
of Fluorine in soils where food crops are cultivated may result
in unacceptable levels of Fluorine or Fluorine complexes entering
into the food supply through a staple crop. Although it is not presently
known what the levels of Fluorine contamination exist in the soils
of the country of Pakistan, it is known that high levels of Fluorine
in the diet is a threat to the health of those consuming contaminated
foods (Fournier, et al, 1998). It is hypothesized that a reliable
source of CaO along with supplementation of trace minerals to contaminated
soil will precipitate the elevated levels of Fluorine while providing
essential mineral nutrients to renew hygienic biological (Bowen
and Rovira, 1966) activity in these soils as it provides renewed
mineral diversity to the soil in order to sustain renewed vigor
to crops.
Methods and Materials
The Kelzyme mineral was obtained from the deposit in Nevada, USA
and transported to the test site in Encinitas, California in San
Diego County, USA. Testing was done on twelve individual 3ft by
2ft beds of rice plants (Oriza sativa L). Muck soil was created
(Parr, Hornick, 1993) so that each test bed had the same basic chemistry
and physical structure. Twelve inches of the created muck soil was
placed into each bed then each bed was filled with ultra violet
light sterilized water to a depth of four inches above soil level.
To create movement in the water each bed was aerated with an airstone
to mimic typical levels of dissolved oxygen in a rice field (Rackocy,
Doelle, 1997). Each test bed was pH balanced using peat moss in
the muck soil mixture to achieve an aggregate pH of 5.8-6.0. Six
rice plant seedlings of equal size and weight were placed into the
muck soil.
The
control beds (A, B) were left alone. Beds (C, D) were inoculated
with Urea Formaldehyde nitrogen and 100-PPM elemental Fluorine.
Beds (E, F) were inoculated with Urea Formaldehyde nitrogen and
200-PPM elemental fluorine. Beds (G, H) were inoculated with anhydrous
ammonia and 100 PPM elemental fluorine and beds (I, J) were inoculated
with anhydrous ammonia and 200 PPM elemental fluorine. Beds (K,
L) were inoculated with emulsified fish solids and 100-PPM elemental
fluorine and beds (M, N) were inoculated with fish solids and 200-PPM
elemental fluorine. Beds (B, D, F, H, J, L, and N) were also inoculated
with Kelzyme at a rate equal to 340kg per acre.
Testing began on April 3, 2000 and were concluded on July 28,2000.
This allowed for one planting and the second generation of tests
is currently underway using the same methodology. Total microorganisms
were estimated by the plate count method. Bacteria and actinomycete
populations were counted on egg albumin agar (Tadao, 1984). Total
fungi were counted on rose bengal agar (Martin, 1950). Azotobacter
were isolated on nitrogen-free mannitol broth agar (Harrigan and
Margaret, 1966). Clostridia were isolated on media described by
Sheldon (1970). Lactobacillus spp. were counted on Rogosa agar (Harrigan
and Margaret, 1966). Starch digesting bacteria were counted using
the method of Sheldon (1970). Agrobacterium, Erwinia, Pseudomonas,
and Xanthomonas spp. were counted on D1, D3, D4, and D5 selective
media, respectively (Kado and Heskett, 1970). Fusarium was counted
on Komada's medium (Tadao, 1984); Verticillium on alcohol agar medium
(Mathew and Chester, 1959); and Thievalopsis on RBM2 medium (Tsao,
1964).
Soil bulk density and porosity were determined according to methods
described by Henry (1984), using 1cm diameter cores from each plot
taken to a depth of 4 cm. Soil porosity was calculated from the
ration of pore space and soil volume. Soil aggregation was determined
by the pipette method of Hormers and Parker(1961).
Testing for F was calculated by mass spectrometry of soil and plant
tissues. Testing was conducted offsite at independent testing laboratories,
Expert Chemical Analysis of Del Mar, California and San Diego State
University, San Diego, California.
Results
Changes in Fluorine Contamination
The inoculated beds (C, E, G, I, K, and M) remained high in F contamination
while beds (C and E) actually tested higher in F contamination than
the inoculated rate. In each case of inoculation with Kelzyme the
amount of elemental F in each sample taken was lower than the inoculation
rate by an average of 17.2%. Fluorine had been complexed to the
CaO in the Kelzyme into Calcium Fluoride CaF2, which exhibits a
very low solubility product of 3x10^-08.
Changes in Soil Microflora
In most cases, the numbers of bacteria, fungi, and actinomycetes
increased after the soil was treated with Kelzyme fossilized marine
algae, although the numbers of actinomycetes were lower in site
(G) than the unfertilized control. It was interesting that the lowest
number of actinomycetes occurred when the soil was treated with
urea formaldehyde fertilizer only (beds C and E).
Generic analysis of the bacterial flora in the soil due to Kelzyme
treatment is shown. In most cases the Kelzyme treatment markedly
increased the number of Enterobacter spp. and starch digesting bacteria
over that of the unfertilized control (A), but had little effect
on enhancing the numbers of Lactobacillus spp. The highest numbers
of Azotobacter and Clostridium species were attained with the fertilized
control (N), while the lowest number of each occurred with the unfertilized,
untreated control (A). The highest number of Xanthomonas and Erwinia
species were found in the fertilized control (G), the highest number
of Agrobacterium from the combination of cold process fish emulsion
and Kelzyme (N), and the highest number of Pseudomonas from anhydrous
ammonia (I).
Change in Soil Physical and Chemical Properties
Soil aggregation was significantly higher for all Kelzyme treatments
than either the control (A) or the fertilized control (B). Soil
aggregation actually decreased in the fertilized control (B). There
was little difference in the effect of Kelzyme treatment or the
unfertilized controls on soil pH. However humus content was markedly
increased which is assumed to be caused from the organic matter
in many of the treatments including Kelzyme. Nitrate levels were
slightly higher in treatments and ammonium levels were unremarkably
higher in the Kelzyme treatments. Potassium was also slightly increased
by an average of 7% by the Kelzyme treatments. The most dramatic
effects on the Kelzyme treatments were the elevated levels of calcium,
Ca and the increased levels of inorganic (plant available) phosphorus,
which was higher than the unfertilized control in all cases.
Discussion
The reduction of Fluorine contamination in each of the tests indicates
a positive aspect of using the Kelzyme mineral in order to reduce
the problems associated with this element in contaminated soils.
It is evident from the test beds that fertilization with urea based
nitrogen sources can exacerbate the problem of F contamination and
may in fact be the cause of the existing conditions in Pakistan.
The lowest number of actinomycetes occurred in soil treated with
anhydrous ammonium suggesting that these microorganisms may somehow
have been suppressed, either directly or indirectly, by the fertilizer
components. Beliaev (1958) found that continuous application of
ammonium fertilizer without calcium can suppress the actinomycetes
since the ammonium is oxidized to nitric acid by microbial action.
The resultant decrease in soil pH from can cause unfavorable growth
conditions where ammonia is used.
The
generic analysis of the bacterial flora showed that fermentative
bacteria such as Enterobacter, starch digesting bacteria, Azotobacter,
and Clostridia, are present in soil treated with Kelzyme and the
fertilized control (B), but to a lesser extent in the unfertilized
control. This may have been due to the effect of some specific nutrient
requirement for the growth of fermentative bacteria. Gyllenberg
(1956) reported seasonal variations in which the relative abundance
of Aa grouping bacteria increased with a decrease in the abundance
of Ba grouping bacteria. It remains unexplained whether the increase
in the relative abundance of the Aa grouping bacteria was accompanied
by the accumulation of specific nutrients such as amino acids.
At present there is no clear relationship between Kelzyme treatments
and the number of soil disease bacteria, e.g., Xanthomonas, Erwinia,
Agrobacterium, and Pseudomonas, as shown in Table 2. But in the
preliminary experiment it appeared that treatment of soils along
with certain organically based nitrogen source (beds K, L, M, N)
is associated with a rather low population of disease bacteria.
The effect of Kelzyme on fungal populations is soil indicated that
soil treated with only fertilizer had low numbers of Penicillium
and Trichoderma. These beneficial fungi can play an important role
in inhibiting or suppressing soil borne microbial plant pathogens
through their antagonistic activities. Large numbers of plant disease
pathogens were found in both of the control treatments.
The effect of Kelzyme on soil physical properties suggests that
Kelzyme can induce plant roots to penetrate soil more effectively.
Soil treated with Kelzyme becomes more friable and porous, less
compact, and promotes deeper cultivation. Microorganisms, particularly
fungi, can bind soil particles into more stable aggregates. Bacteria
can synthesize cementing agents in the form of gums and polysaccharides
that also help to promote good aggregation. Lynch (1981) found that
Azotobacter chroococcum, Lipomyces starkeyi, and Pseudomonas spp.
can promote the stabilization of soil aggregates.
Insoluble soil phosphorus compounds (both organic and inorganic)
are largely unavailable to plants, however many microorganisms can
solubilize these compounds and make them available for uptake. Martin
(1961) found that one-tenth to one-half of the bacterial isolates
he tested were capable of solubilizing calcium and phosphorus. Fungal
species of the genera Pseudomonas, Myobacter, Micrococcus, Flavobacterium,
Penicillium, Sclerotium, Aspergillus, and others are also known
to solubilize insoluble phosphates to plant-available forms.
References:
Beliaev, G.N., 1958,
Mikrobiologiya, 27: 472-477
Bowen, G.D. and Rovira,
A.D., 1966, Microbial Factor in Short Term Phosphate Uptake Studies
with Plant Roots, Nature (London), 211:665-666
Brown, M.E., 1974, Seed
and Root Bacterisation, Annual Review Phytopathology, 12:181-197
Elad, Y., 1985, Mechanisms
of Interactions Between Rhizosphere Microorganisms and Soil Borne
Plant Pathogens, p. 42-72,
In V. Jansen, A Kjoller, and L.H. Sorenson (ed.), Microbial Communities
in Soil, Elsevier Applied Science, New York.
Gyllenberg, H.G., 1956,
Seasonal Variation in the Composition of the Bacterial Soil Flora
in Relation to Plant Development, Canadian Journal of Microbiology,
3:131-134
Harrigan, W.F., 1984,
and E.M.C. Margaret, 1996, Laboratory Methods in Microbiology. Academic
Press, London.
Henry, D.F., 1984, Fundamentals
of Soil Science, 7th Edition, John Wiley and Sons, New York.
Higa, T., 1986, Studies
on the Application of Microorganisms in Farming, 6th IFOAM Conference,
August 18-21, 1986, University of California, Santa Cruz.
Kado, C.I. and M.G. Heskett,
1970, Selective Medium for Isolation of Cornyebacterium, Erwinia,
Pseudomonas, and Xanthomonas, Phytopathology, 60: 969-976.
Marois, J.J., D.J. Mitchell
and R.M. Sonoda, 1981, Biological Control of Fusarium Crown Root
of Tomato Under Field Conditions, Phytopathology, 71: 1257-1260.
Martin, J.P. and S. A.
Waksman, 1940, Influence of Microorganisms on Soil Aggregation and
Erosion II, Soil Science 42: 29-46 Martin J.P., 1950, Use of Acid
Rose Bengal and Streptomycin in the Plate Method for Estimating
Soil Fungi, Soil Science, 52: 29-40
Mathew, J.N. and E.H.
Chester, 1959, An Alcohol Agar Medium Selective for Determining
Verticillium microsclerotia in Soil, Phytopathology, 49: 527-528
Mishustin, E.N., 1970,
The Importance of Non-symbiotic Microorganisms in Agricultural Plants,
Plant and Soil 32: 545-554.
Rubenchick, L.I., 1963,
Azotobacter and its Use in Agriculture, Israeli Program for Scientific
Translations, Jerusalem, Israel.
Sheldon, A., 1970, Experimental
Microbial Ecology, Academic Press, New York.
Tadao, U.I., 1984, Handbook
of Soil Borne Disease, Japan Plant Protection Association, Tokyo.
Tsao, P.H., 1964, Effect
of Certain Fungal Isolation Agar Media on Thielaviopsis basicola
and on its Recovery in Soil Dilution Plates, Phytopathology, 54:
548-555
The number of fungal species after Kelzyme treatment of this soil
are shown. The highest number of Trichoderma species was found after
treatment with fish solids and Kelzyme (N) and the highest number
of Penicillium with fish solids and Kelzyme (N). However, the lowest
number of specimens in these genera resulted from the anhydrous
ammonia only treatment (G, I). The highest number of Verticillium
species was observed in the urea fertilized beds (C, E). But the
combination of cold process fish solids and Kelzyme appeared to
suppress the numbers of this soil borne plant pathogen. The highest
number of Fusarium species resulted from treatment with the urea
fertilized control (C, E), while the combination of cold process
fish solids and Kelzyme markedly suppressed the numbers of this
particularly destructive plant pathogen.
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