Plant and Soil 167:305-311, 1994.

© 1994 KluwerAcademic Publishers. Printed in the Netherlands.

Enrichment of some B-vitamins in plants with application of organic fertilizers

A. M o z a f a r
Division of Agronomy, Institute of Plant Sciences, Swiss Federal Institute of Technology (ETH), ETH Zentrum,
CH-8092 Ziirich, Switzerland

Received 18 March 1994. Accepted in revised form 18 August 1994

Key words: barley, cyanocobalamin, dung, manure, organic fertilizer, spinach, soybean

Abstract

A review of the literature showed that plants grown with organic fertilizers often contain higher concentrations of
vitamins B1 (thiamin) and B12 (cyanocobalamin) as compared with plants grown with inorganic fertilizers. Since
plant roots were recently shown to be able to absorb B1 and B12, it was thus suspected that organic fertilizers
(such as manure of diverse sources or sewage sludges which often contain relatively high concentrations of several
vitamins) introduce additional vitamins into the soil which in turn leads to increased vitamins in the plants. This
possibility was studied by measuring the B 12 content in the seeds of soybean and barley and in the leaves of spinach
plants grown in soils amended with pure B12 or cow dung (which is naturally rich in B12). The addition of pure BI2
or cow dung did not alter the B12 content in the soybean seeds but significantly increased that in the barley kernels
and in the spinach leaves. For example, the addition of cow dung at the rate of 10 g kg-1 increased the B 12 content
in barley kernels by more than threefold (from 2.6 to 9.1 ng g-1 DW) and in spinach leaves by close to twofold
(from 6.9 to 17.8 ng g-1 DW). Long-term addition of organic fertilizers to the soil also significantly increased the
soil content of this vitamin. Since plants cannot synthesize B12 and thus plant foods are normally fully devoid of
(or have very low concentrations of) this vitamin, the finding that plants grown with organic fertilizers may contain
relatively higher concentrations of this vitamin may have nutritional consequences in that the consumption of these
plants by humans would inadvertently increase their intake of this vitamin. This may be of special benefit to people
living by choice or by necessity on strict vegetarian diets who are known to be in danger of B12 deficiency.

Introduction and millet seeds obtained from plots fertilized with

manure grew much better than those fed seeds from
Organically grown fruits and vegetables are gaining plots fertilized with inorganic fertilizers. This observa-
increasing popularity, partly because of the public per- tion led to the conclusion that seeds from organically
ception that they may be of higher quality than those grown plots must have contained higher concentra-
grown by "conventional" methods. Organically grown tions of B vitamins. Later, Rowlands and Wilkinson
plants, besides low or no exposure to protection chem- in 1930 reported that vitamin-deficient rats who were
icals, are usually fertilized solely with organic fertiliz- fed grass seeds grown with pig manure gained weight
ers (various animal wastes, composts, etc.) rather than at twice the rate as rats fed seeds grown with inorgan-
with inorganic fertilizers. It is thus of interest to know ic fertilizers, an observation that suggested that seeds
whether use of these organic substances as such would from manure-fertilized plots must have contained more
affect some measurable quality factor in plants such as B vitamins. In 1950, Antoniani and Monzini noted
their vitamin content (Mozafar, 1994). that irrigation of fodder plants with uncleared sewage
A review of the literature showed that the effect water, as compared with cleared water, increased the
of organic fertilizers on plant vitamins was noted a vitamin B1 content of plant tops by approximately six-
long time ago. McCarrison and Viswanath noted in fold (1368 vs. 235/zg 100 g-l respectively).
1926 that vitamin-deficient pigeons raised on wheat
306

Wilberg (1972) reported that organically grown Agricultural School in the vicinity of the laboratories.
spinach contained more than twice the vitamin B1 of Dung was first air-dried (25-35°C for 3--4 d), broken
spinach grown by conventional methods (400 vs. 180 into small pieces and finally sieved through a 5-mm
#g 100 g-l DW). Leclerc and coworkers (1991) noted sieve. Control soils were mixed with equal amounts of
that carrots purchased from organic farms in France quartz sand. The soil used was a parabrown loam (pH
tended to have higher concentrations of vitamin B1 = 6.5) from the fields of Eschikon Experiment Station,
and niacin; the differences were, however, not statis- Switzerland. Plants were seeded in pots containing ca.
tically significant. On the other hand, Singh and Dhar 2.5 kg soil.
(1986) reported that green gram and wheat grown with Soybean and barley were planted at a density of one
cow dung or cane-bagasse contained significantly more plant/pot, and spinach at a density of four plants/pot.
vitamin B1 than the control plants. Plants were grown form seeds planted directly into
Despite the different assay methods used in the the soil in the greenhouse (16/8 h day/night photoperi-
above cited works, the relatively consistent observa- od with photon flux density of 550 #E.m-2.s -l from
tion of higher B vitamins in the organically fertilized Phillips high-pressure sodium lamps SON/T and tem-
plants is noteworthy. The question is whether the high- peratures of 22-24/18-19°C, respectively) and were
er content of vitamins in the plants grown with organ- watered twice a week with a l/5-strength Hoagland
ic fertilizers is due to a higher synthesis of vitamins solution throughout the growth period. No additional
by plants or to the increased absorption of "soil vita- fertilization of the soil was made. Experiments were
mins" by the roots? Early investigators, without any replicated five times.
direct experimental evidence, suspected that some of Effect of long-term addition of organic wastes on
the vitamins found in the plants may be of soil origin, the B12 content of soil was investigated by measuring
i.e., they are absorbed by the plant roots (Grosbeck and the concentration of this vitamin in the soil samples
Salonen, 1976; Lilly and Leonian, 1939; Robbins et taken from long-term split plot experiments at T~inikon
al., 1950). Recently, reports indicate that under water Experiment Station in Switzerland, where for the pre-
culture conditions, plant roots are able to absorb var- vious 16 years the effects of rotation, inorganic versus
ious vitamins and transport them to other plant parts mixture of inorganic and organic fertilizers and herbi-
(Mozafar and Oertli, 1992a, b, 1993). The purpose of cides on crop yield have been investigated (Srzednicki,
this study was thus to investigate the effect of adding 1977). In the plots assigned as mixed organic and inor-
organic fertilizer (cow dung) to the soil on the content ganic fertilizers, plant residues were finely chopped
of vitamin B 12 (hereafter B 12) in plants since any detec- and worked into the soil and additionally cow dung
tion of this vitamin within the plants (which apparently (mixture of feces and cereal straw) was added every
cannot synthesize this vitamin) would mean that it was five years (before the potatoes in the rotation). Every
taken up by the roots. year enough inorganic mineral fertilizers was added to
each plot bringing the total calculated amounts of N, P,
K (in all the fertilizers added) to the approximate lev-
Material and methods el which was added to the plots which received only
inorganic fertilizers. We took soil samples (from 0-20
Uptake of B 12from soil amended with pure B 12(Roche, cm) from the subplots which had received the lowest
Basel, Switzerland) or with cow dung was studied level of triazine herbicide. The soil samples were taken
using soybean (Glycine max Merr. cv. Maple Arrow), during October 1989 and were stored at 4°C till ana-
barley (Hordeum vulgare cv. Atos) and spinach (Spina- lyzed, which was done during the three months after
cia oleracea L. cv. Monnopa) as test plants. The treat- the sampling.
ments were 0 and 10 mg of B12 or 10 g of dry cow For measuring the vitamin content, plant parts were
dung per kg room-dried soil. Mixing of soil with vita- first thoroughly washed with freshly distilled water
min or cow dung was achieved by placing the amount to remove the presence of any surface soil. Samples
of soil required for each treatment plus the appropriate of plant parts, weighing ca. 0.2 to 0.4 g fresh, were
amount of vitamin (mixed with ca. 30 g of 1-mm quartz weighed and frozen with liquid nitrogen and stored at
sand per kg of soil) or cow dung into a small (180-L) -80°C until analyzed. Plant extracts were obtained by
cement mixer and mixing them for 12 hours. Cow dung crushing the frozen plant materials and homogenizing
(manure not mixed with any extra straw) was collect- them with 20 to 40 mL of a 0.4 mol L - l acetate buffer
ed fresh from the dairy farm of Strickhof Vocational (pH = 4.6, 3 mmol L -~ KCN) as described by Oes-
 307

terdahl and coworkers (1986), using a Waring blender

Table 1. Concentration of B12 (ng g - t DW) in soy-
fitted with a micro-container model MC-1 or MC-2.
bean and barley seeds and in leaves of spinach grown in
Soil extracts were obtained by using one gram of soil soils amended with cow dung (10 g kg- 1 dry soil) or pure
(air-dried and sieved through a 1-mm sieve) to 50 mL Bl2 (10 mg kg - | dry soil). Control plants were grown
of acetate buffer and mixing for 10 minutes at room under similar conditions but without any amendment
temperature and then autoclaving for 30 minutes (to
Treatment Soybean Barley Spinach
release the bound B12). After cooling the extracts the
total volume was brought to 50 mL with buffer solu- Control 1.6aI 2.6a 6.9a
tion. Cow dung 2.9a 9.1b 17.8b
Autoclaving of samples was found to give the high- Pure BI2 3.1a 10.8b 235.0c
est degree of extraction B12 from soil. For the plant l Values in each column followed with different letters
samples, autoclaving did not increase the Bi2 extrac- are significant at the 0.05 level.
tion, thus indicating that most, if not all, of the BI2 in
the plant may be in the free form. After adjusting the
pH of plant and soil extracts to 6.8 with NaOH, they
were slowly filtered (Whatman, No. 1), and then were Results and discussion
either analyzed for B12 immediately or stored at -80°C
till analyzed. Aliquots of these extract, after appropri- The addition of pure B 12did not alter the B 12 content in
ate dilution, were used for vitamin determination by the soybean seeds but significantly increased that in the
the RID. barley kernels by more than threefold (2.6 vs. 10.8 ng
(Radioisotope Dilution, also called protein binding) g - 1 DW) and in the spinach leaves by 34 times (235 vs.
method using the test kit CT.302 supplied by Amer- 6.9 ng g- 1 DW). Addition of cow dung at the rate of l0
sham of England as described elsewhere (Mozafar g kg-1 increased the B12 content in barley kernels by
and Oertli, 1992a). Vitamin B12 measured by the RID more than threefold (from 2.6 to 9.1 ng g-l DW) and
method using purified hog intrinsic factor shows close in spinach leaves by close to twofold (from 6.9 to 17.8
correlation with the results obtained by the Euglena ng g-1 DW) (Table 1). Plant growth was not affected
test (Bennink and Ono, 1982; Casey et al., 1982; Oes- by the addition of cow dung or pure BI2 presumably
terdahl et al., 1986; Richardson et al., 1978; Stenman because the plants' nutritional needs were covered by
and Puutula-R~is~en, 1980). In short the B12 is first their regular irrigation with nutrient solution.
released from its binding proteins by mixing the sample Long-term amendment of soil with organic fertiliz-
with a given amount of tracer (57Co-cyanocobalamin) ers was noted to approximately double its B12 content
and incubating with a denaturing agent in the pres- as compared with soil treated with inorganic fertilizers.
ence of potassium cyanide. Following denaturation, A crop rotation in which maize occupies a large share
the sample is incubated with a solution containing a was noted to decrease the B12 content in soil when
new binding protein (purified hog intrinsic factor) on compared with other rotations (Table 2).
the surface of which the BI2 from the sample and the
radioactively labelled B12 molecules compete for the Soil vitamins
limited number of binding sites available. The protein
bound fraction is now separated from the free vitamin Soils usually contain several vitamins originating from
fraction by means of coated charcoal. By measurement different sources: a) soil microbial activities (Kozlo-
of 57C0 activity in the protein fraction and appropriate va, 1970; Krasil'nikova, 1962; Lochhead and Burton,
standard curves, the amount of BI2 in the sample can 1957; Lochhead and Thexton, 1951; Strzelczyk and
be calculated. According to the technical information R6z_z_z_~cki, 1985), b) organic wastes of plant or ani-
accompanying the kit, the intrinsic factor used in this mal origin (Coates, 1968; Schmidt and Starkey, 1951;
kit is essentially free of 'R' protein binding activity Starkey, 1944) and c) root exudates (Rovira and Har-
and thus the assay has a high selectivity for B12 and ris, 1961; Sch6nwitz and Ziegler, 1982; Sulochana,
not for its analogues. 1962). Soil is also inhabited by microorganisms which
need some vitamins for their survival (Lochhead, 1958;
Lochhead and Burton, 1954, 1957; Lochhead and
Thexton, 1951, 1952). Soil concentration of B12 is
very close to that of beef and thus soils can be regard-
308

Table 2. Content of B12 in soils of experimental plots in Switzerland after

16 years of receiving inorganic fertilizer or a mixture of inorganic and organic
fertilizers under different rotation treatments (see Srzednicki, 1977 for details)

Fertilizer Rotation 1 BI2 (/zg kg - l dry soil)

Inorganic Conventional 9b 2
Inorganic Maize (intensive) 5a

Organic + inorganic Conventional 14c

Organic + inorganic Maize (intensive) 10b

1Conventional: wheat, potato, barley, forage, forage.

Maize (intensive): wheat, faba beans, maize, maize, maize.
2Values with different letters are significant at the 0.05 level.

ed as one of the richest natural sources of B12 (Table Microbial decomposition of vitamins can take place
3). Even 1:1 or 1:2 aqueous soil extracts may have during composting of plant material (Starkey, 1944)
as much as 2 to 8 #g L-1 of this vitamin which is as and under soil conditions (Schmidt and Starkey, 1951).
high as that in cow's milk (Robbins et al., 1950; Smith, In soil, for example, more than 50% of the riboflavin
1960). added disappeared during a three-day period (Schmidt
Microorganisms are also the primary source of B12 and Starkey, 1951). Although information as to the
for higher animals. In fact, bacterial synthesis of this fate of other vitamins in soil is very rare, B12 appears
vitamin in the gastrointestinal tract of animals, and to be extremely stable under natural conditions (Pratt,
its subsequent uptake by the intestinal membranes, 1972). Thus, long-term addition of animal manure to
accounts for a large portion of this vitamin found in the soil could conceivably raise the concentration of
animal tissues (Coates, 1968). A relatively large por- B12 in the soil either by direct addition of vitamin to
tion of the B I2 produced in the gastrointestinal tract is, the soil or indirectly through increased soil organic
however, excreted through feces or urine. In sheep, for matter and thus increased activity of soil microorgan-
example, 95% of the B12 produced in the gastric tract isms. It is also conceivable that increased microbial
is not absorbed and is excreted by the feces (Friedrich, activity may lead to co-metabolism (a process whereby
1975), which in most cases lands on soil. Therefore, microorganisms transform a substance while growing
organic substances such as manure and sludge contain on another substrate) of B12 in the soil. The finding that
considerable amounts of vitamins which may be sever- soil from fields fertilized for 16 years with a mixture
al orders of magnitude higher than that in most soils. In of organic (farmyard manure) and inorganic fertilizer
cow dung, for example, the concentration of B12 is 10 contained more B12 than soil from fields fertilized only
to 100 times and that of vitamin B 1 and B2 (riboflavine) with inorganic fertilizers (Table 2), however, supports
several times higher than that in soil (Table 3). Acti- the view that Bl2 is relatively stable under soil con-
vated sludge is so high in Bt2 that it comes very close ditions and addition of cow dung leads to a direct or
to one of the richest known sources of this vitamin, indirect increase in the concentration of this compound
i.e. liver (Table 3). With such a high vitamin content, in soil.
it is conceivable that fertilization of soils with organic As to the state of B12 (or its analogues) in soil, there
wastes or spreading of sewage sludge on agricultural is no information available. In other ecosystems, B12
land could have temporary or long-term effects on the is almost bound to proteins (Chanarin, 1969). To what
content of some vitamins and especially that of B12 in extent the soil B12 is bound to the soil's organic or
soil (Table 3). It is furthermore conceivable that this mineral fraction is a matter of speculation which needs
change in soil vitamins could have short- or long-term to be investigated. Since the free cobalamins are more
influences on the activity of those soil microorganisms readily degraded than the bound form in other systems
which require this or other vitamins for their survival, (Gr~sbeck and Salonen, 1976), one may speculate that
a situation which in turn could affect the soil fertility. soil-bound B I2 may be less subject to microbial degra-
 309

Table 3. Concentration (/zg kg -1) of vitamins B12 (or biologically active corrinoids), B1 (thiamin) and B2
(riboflavine) in soil, organic wastes and some other materials 1

Source B 12 Thiamin Riboflavin Reference

Soil2 0.2 6 Vukhrer and Mirdzhalilova, 1971

Soil3 2 Avakyan and Afrlkyan, 1981
Soil4 10 Smith, 1960
Soil2 14 This study
Soil3 15 19 9800 Kononova, 1966
Soil2 53 Friedrich, 1975
Soil3 98 Kirakosyan et al., 1970
Soil 3 45 980 Krasil'nikov, 1962
Soil 3 50 Hagedorn, 1967
Soil 2 400 Epanchinov, 1975
Soil extract5 2 Lochhead and Thexton, 1951
Soil extract5 5 Smith, 1960
Soil extract5 8 Robbins et al., 1950
Sewage sludge:
Banglore 3,6 165 Sathyanarayana et al., 1959
Banglore 2,7 754 8000 9800 Srinath and Pillai, 1966
Ziirich2'7 1590 20500 21600 Kocher and Corti, 1952
Unknown2 7000 Smith, 1960
Philadelphia2,7 9300 Hoover et al., 1951
Bavaria 2,7 29100 41000 Lemmer and Nitschke, 1994
Peoria3,7 47000 Kraus, 1942
Sewage Waters 0.2 Antoniani and Monzini, 1950
Sewage waters ,s 386 116 Lemmer and Nitschke, 1994
Cow dung2 130 Bonner and Greene, 1938
Cow dung3 140 Sathyanarayana et al., 1959
Cow dung2 1000 Friedrich, 1975
Sheep feces2 1860 Dawbarn et al., 1957
Milk4 6 Smith, 1960
Milk (dry) 2 40 Gr'~beck and Salonen, 1976
Beef* 27 Gr~beck and Salonen, 1976
Liver (cow) 2 2140 Friedrich, 1975

IData from different authors are obtained by different methods of analysis and may not be valid for comparison
purposes. In cases where a range of concentration was cited by the authors, the maximum value was used for this
table. Some of the values are rounded off. 2Dry weight basis. 3It is not clear whether the data are on fresh or
dry weight basis. 4Fresh weight basis. 5,ug liter -1. 6Raw sludge. 7Activated sludge. 8Water phase of a digested
sludge.

dation in soil and thus the increase observed by the to soil was also noted to significantly increase the con-
cow dung addition. centration of this vitamin in these plant parts (Table 1).
These data are in contrast to the views that plants do
Vitamin uptake by plants not contain B12 at all and that the B12 activity found
in plants (Robbins et al., 1950) seems to be due to
In soybean, addition of pure B I2 tended to increase the contamination of plants with soil bacteria (Chanarin,
Bt2 content of the seeds but it was not statistically sig- 1969; Gr/isbeck and Salonen, 1976). Considering the
nificant. Addition of B 12to the soil, however, increased reports that plant roots and leaves can absorb the rel-
the concentration of B12 in the seeds of barley and in atively large molecules of Bl2 from nutrient solutions
spinach leaves by severalfold. Addition of cow dung and transport them to other plant parts (Mozafar and
310

Oertli, 1992a) and the belief that plants cannot synthe- of soil types and of organic and conventional methods
size this vitamin (Friedrich, 1987; Lehninger, 1977; of farming on the vitamin concentration in plants is
Smith, 1960), it seems that the observed increase in presented elsewhere (Mozafar, 1994).
the concentration of B12 in barley seeds and spinach
leaves fertilized with cow dung is mostly (if not fully)
due to the uptake of this vitamin by the roots from Acknowledgements
the soil and not due to superficial contamination or an
increased synthesis of this vitamin within the plant. This work was financed by Hoffmann-La Roche
If we further assume that vitamin B12 is present in of Basel and a Swiss government grant for the
a bound form in the soils, these findings appear to advancement of industry (KWF). The author grateful-
indicate that plant roots are in a position to separate ly acknowledges the technical assistance of Mr Roland
this vitamin from its absorbing complex and/or trans- Waldvogel. The cordial supply by W G Sturny of soils
port the whole B12- complexing protein by their roots. from the long-term fertilizer trials for the vitamin mea-
Whether plant roots differ from microorganisms or the surements is greatly appreciated.
cells of intestinal tracts which process special recep-
tors to absorb the cobalamin-intrinsic factor complex
(Gr~beck and Salonen, 1976) remains to be seen. References
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