Amino Acid: Its Dual Role as Nutrient and Scavenger of Free Radicals in Soil

: Ascorbic acid is a bacteriostatic agent; one of the many ways by which ascorbic acid hampers bacterial growth is by the production of hydrogen peroxide, which further converts into hydroxyl free radicals. Certain amino acids can counteract the inhibitory effect of hydroxyl free radicals by checking their oxidizing effect. Though ascorbic acid is bacteriostatic in nature, it facilitates prokaryotic respiration by decarboxylation. This study was carried out to understand how microbes from different horizons of the forest soil respond to the addition of a bacteriostatic agent (ascorbic acid) and growth promoting agent (amino acids), with respect to the soil respiration. We observed that the addition of either ascorbic acid or a combination of it with amino acid consistently results in increased soil respiration, and this increase is different for different soil types depending on soil composition and origin. Furthermore, we also found that beta alanine-induced maximum respiration in basic soils and L-glutamic in acidic soils. This study is signiﬁcant because it can be used to explain how a strong reducing sugar, i.e., ascorbic acid, affects the soil respiration mediated via soil microbes. To the best of our knowledge, it is the ﬁrst report that demonstrates the effect of bacteriostatic and the growth promoting agent together on microbe-mediated soil respiration.

Ascorbic acid is a reducing sugar with anti-oxidant properties [13] , and it shows bacteriostatic properties in soil microenvironments [14,15].It autoxidizes in the presence of air or oxygen to form hydrogen peroxide, and hydrogen peroxide reacts with a ferrous ion to produce the highly reactive hydroxyl ion via the Fenton reaction [16] (Figure 1).The hydroxyl free radical produced during auto oxidation has a bacteriostatic or bactericidal action which kills the microbes in the soil.This is one of the many ways by which ascorbic acid hampers bacterial growth [17].Certain amino acids can counteract the inhibitory effect of hydroxyl free radicals by checking their oxidizing action.Amino acids are abundantly present in the soil which are mainly derived from root exudates [18] and serve as an important source of nitrogen for microbes in the soil [19].
Ascorbic acid is a reducing sugar with anti-oxidant properties [13] , and it shows bacteriostatic properties in soil microenvironments [14,15].It autoxidizes in the presence of air or oxygen to form hydrogen peroxide, and hydrogen peroxide reacts with a ferrous ion to produce the highly reactive hydroxyl ion via the Fenton reaction [16] (Figure 1).The hydroxyl free radical produced during auto oxidation has a bacteriostatic or bactericidal action which kills the microbes in the soil.This is one of the many ways by which ascorbic acid hampers bacterial growth [17].Certain amino acids can counteract the inhibitory effect of hydroxyl free radicals by checking their oxidizing action.Amino acids are abundantly present in the soil which are mainly derived from root exudates [18] and serve as an important source of nitrogen for microbes in the soil [19].The aim of this study was to find out how and up to what extent amino acid can counteract the oxidative action of free radicals generated by the autoxidation of ascorbic acid.Efflux of CO2 from soil is an outcome of decomposition activities by both heterotrophic and autotrophic soil organisms [20].Out of these, heterotrophic soil respiration forms a major component of total soil respiration [21][22][23].It is mainly controlled by microbial decomposition of soil organic matter, which depends on a plethora of biotic and abiotic factors [24,25].In this study, we measured soil respiration, which is one of the many biological indicators of soil health [26,27].We examined the antioxidant property of different classes of amino acids against hydroxyl free radicals formed in the process of auto oxidation of ascorbic acid.
The physical and chemical properties of all soil sampling sites are listed in Table 1.The soil carbon and nitrogen ratio (C/N) was highest in Beech stand (old age, Dystric Luvisol, Oe horizon) and lowest in the Deciduous forest (old age, Rendzic Humic Leptosol, Bwk horizon).Soil pH measurement in water and 0.01 M CaCl2 are two common methods.However, the CaCl2 method is more accurate as soil electrolyte concentration is least affected.The pH of all soil samples was measured by both the methods.The pH of the Beech stands soils of Ae and Oe horizon was lowest.The aim of this study was to find out how and up to what extent amino acid can counteract the oxidative action of free radicals generated by the autoxidation of ascorbic acid.Efflux of CO 2 from soil is an outcome of decomposition activities by both heterotrophic and autotrophic soil organisms [20].Out of these, heterotrophic soil respiration forms a major component of total soil respiration [21][22][23].It is mainly controlled by microbial decomposition of soil organic matter, which depends on a plethora of biotic and abiotic factors [24,25].In this study, we measured soil respiration, which is one of the many biological indicators of soil health [26,27].We examined the antioxidant property of different classes of amino acids against hydroxyl free radicals formed in the process of auto oxidation of ascorbic acid.

Sampling and Processing
Seven soil samples were collected from four different forest sites (Table 1), namely: (1) Middle-aged mixed stand of deciduous trees (33 years, oak 60%, hornbeam 30%, beech 10%; 415 m a.s.l., N 49 After sampling, soil was sieved through a 5 mm sieve and stored in a refrigerator at 4 degrees centigrade.The dry matter content of each soil sample was calculated before the start of the experiments, and the moisture content of soil was maintained at 25%. The physical and chemical properties of all soil sampling sites are listed in Table 1.The soil carbon and nitrogen ratio (C/N) was highest in Beech stand (old age, Dystric Luvisol, Oe horizon) and lowest in the Deciduous forest (old age, Rendzic Humic Leptosol, Bwk horizon).Soil pH measurement in water and 0.01 M CaCl 2 are two common methods.However, the CaCl 2 method is more accurate as soil electrolyte concentration is least affected.The pH of all soil samples was measured by both the methods.The pH of the Beech stands soils of Ae and Oe horizon was lowest.

Treatments and Soil Respiration
Different portions of 40 g mineral soil (B,E,G) or 20 g organic soil (A,C,D,F) were added to a 140 mL air tight incubation box followed by the addition of 0 (control), 2.0 mg of ascorbic acid per g of dry soil and its combination with six different amino acids, beta Alanine, D-Glutamic acid, L-Glutamic acid, Citrulline, and L-Ornithine and D-Ornithine, in triplicates.A glass petri dish containing 2 mL 1 M NaOH was placed over the soil, tightly sealed with a lid, and incubated for 6 h at 22 • C.After the incubation, the lids were removed, and the NaOH was transferred to a beaker.Washings with distilled water were given to the petri dish to make sure that all of NaOH was completely removed.A saturated solution of BaCl 2 was added to the beaker, and the solution was titrated with phenolphthalein.After the titration, the intensity of respiration was calculated by the formula below [28].
Intensity of respiration was measured as R. where × 6005 (weight of soil) × (dry matter content) × (time of incubation) A = amount of HCl for titration of control (mL); B = amount of HCl for titration of sample (mL); c(HCl) = exact concentration of HCl (mol/L); 6005 = coefficient for recalculation (from V (HCl) to a µg C-CO 2 ).

Statistical Analysis
One-way analysis of variance (ANOVA) was carried out to determine significant differences between the different treatments on each site.Tukey's test was applied at 5% significance.

Results
There was always an increase in respiration after the addition of amino acids to the ascorbic acid pretreated soil.This increasing respiration trend was observed in all seven soil types (Figure 2).The highest increase in respiration was observed in the case of the beta alanine and ascorbic acid combination added to Deciduous forest, old-aged Oe horizon soil (Figure 2B).The soil with the smallest increase in respiration for all combinations of an amino acid with ascorbic acid was Old-aged beech stand Aa horizon (Figure 2F).
There was always a significant increase (p < 0.05) in respiration in all soil types after the addition of ascorbic acid, as compared to the basal respiration in untreated soil.The difference was also significant between basal respiration and respiration after the addition of a combination of an ascorbic acid with all six amino acids.(Figure 2).Beta alanine induced the maximum rate of increase in soil respiration in deciduous forest old age (Oe, Ahk, and Bw horizon) and in old beech stand (Aa horizon).L glutamic acid induced the maximum rate of increase in soil respiration in the deciduous forest middle age (Ahk horizon) and beech stand old age (Oe horizon), whereas L citrulline induced maximum respiration in spruce stand old age (Oe horizon) only.The minimum increment in soil respiration upon the addition of amino acids was found in the case of D ornithine for five soil samples out of seven in old-aged beech stand (Oe, Ah horizon), old-aged spruce stand (Oe horizon), old-aged spruce stand (Oe horizon), and middle-aged mixed stand of deciduous forest (Ah horizon), whereas D glutamic acid induced the minimum rate of increase in respiration for two soil samples of old age deciduous (Amk, Bw horizon).
All soil samples with basic pH value, e.g., B, C, D, show a maximum increase in respiration with Beta alanine treatment, whereas all the acidic soil samples, e.g., E, F, G, show a maximum increase in respiration on treatment with L-Glutamic acid.In particular, the ascorbic acid and L-glutamic acid combination caused the highest increase in soil respiration in three (old-aged beech stand Oe horizon, old-aged spruce stand Oe horizon, a middle-aged mixed stand of deciduous forest Ah horizon) of the seven soils.The detail showing percentage increase for each treatment is shown in Table 2.

Discussion
In the present study, we observed the respiration rates in seven types of soils before and after treatment with ascorbic acid and amino acids.The different basal respiration rates before any treatment in the soils studied here could be reasonably due to the different microbial activity.After the treatments, we observed an increase in the respiration rate, induced by the addition of ascorbic acid and amino acid in all the soil samples.The lowest increase in respiration was for agriculturally managed soil (beech stand, old age, Dystric Luvisol, Aa horizon) [27].
It has also been established in previous studies that the addition of ascorbic acid leads to an increased soil respiration that could vary depending on soil properties and the organic matter content of soil [29][30][31][32][33].The responses of microbial communities in soils on the addition of ascorbic acid were of the same order as those reported by other groups [34][35][36].
Increased soil respiration and CO 2 efflux were observed, along with increased microbial biomass in arid ecosystems upon the addition of nitrogen sources [37] (the increase was more prominent in bulk soil (0-10) cm depth).Amino acids in both and L-and D-forms can be considered as organic nitrogen and carbon sources for soil microbes [38,39] that can lead to an increase in respiration in soil [40].However, studies reveal that L-amino acids are more readily taken up by gram-positive bacteria in soil than their D form [38]. So, we conclude that the microbial composition of soil is an important consideration that decides the uptake of amino acids and their enantiomers.The addition of amino acids in soil leads to changes in microbial activity as a measure of microbial respiration, and the rate of this change in activity varies with soil horizon [41].Here again, microbial communities in different soil types could have affected the differential response for amino acids in our study.
The increased soil respiration, with the addition of ascorbic acid only, could be associated with the fact that ascorbic acid has a structural similarity with hexose sugars because of the presence of the dienol group, which might serve as a carbon source for bacterial respiration [42].A further increase in respiration upon the addition of amino acid is probably due to the reduction of amino acid itself by hydroxyl free radicals, which protects the microbial flora from getting oxidized (additionally, it acts as a nutrient [43]).
Interestingly, after the addition of the amino acid and ascorbic acid combination, the increase in respiration was different in all seven types of soil because amino acid behaves differently at different pHs.Soil pH plays a distinctive role in the action of an amino acid as an oxidizing or reducing agent.Amino acids with low oxidation potential are most easily oxidized and are an excellent scavenger of free radicals [44].Basic amino acid is easier to oxidise than acidic amino acid [45].
In our study, L-Glutamic acid induced the highest soil respiration rate in four out of seven soil samples.Glutamic acid is negative charge acidic amino acid.The titration graph of glutamic acid shows that at sufficiently low pH (between 4 and 5), it acts as a basic amino acid [46] and it can easily gain the proton from the hydroxyl free radicals, thereby turning into cation with a single positive charge.Soil samples A, E, F, and G have pH values between 5.1 and 4.1, so in these samples glutamic acid acts as a strong reducing agent and shows maximum power to poison the hydroxyl free radical.Hence, these soil samples show the highest respiration rate with glutamic acid treatment.
Beta alanine shows surprising results in soils B, C, and D. These soil samples have higher pH values (7.1-7.7),i.e., basic samples.The titration graph of beta alanine shows that at a higher pH, beta alanine shows an acidic nature and acts as an oxidizing agent [47].Notwithstanding, beta alanine treatment shows maximum respiration in soil samples B, C, and D. We explain that such an increase in the respiration activity is due to the high sensitivity of r-strategist microorganisms; their activity, latent before the treatment, became evident after the addition of carbon and nitrogen sources [48].

Conclusions
From this study, we conclude that ascorbic acid autoxidizes to form hydroxyl free radicals, which inhibits microbial growth in the soil.However, an increase in soil respiration due to the addition of amino acid proves that inhibition was not due to ascorbic acid, but it was due to free radical formation.
Another reducing agent can also be used, but the use of amino acid can be best explained in two ways: it has greater oxidation potential than peroxide to get quickly oxidized, and it acts as a nutrient for the soil microbe and plants.From our study, we can conclude that beta alanine induced maximum respiration in basic soils and L-glutamic in acidic soils, as compared to other treatments.

Figure 1 .
Figure 1.Auto oxidation of ascorbic acid and generation of hydroxyl free radicals.

Figure 1 .
Figure 1.Auto oxidation of ascorbic acid and generation of hydroxyl free radicals.

Figure 2 .
Figure 2. Amino acid-induced respiration rate in the seven different forest soil samples pretreated with 2.0 mg ascorbic acid g-1 soil: (1) no treatment; (2) treated with ascorbic acid only; (3) treated with 2.0 mg beta Alanine acid g-1 soil and Ascorbic Acid; (4) treated with 2.0 mg D-Glutamic acid g-1 soil and Ascorbic Acid; (5) treated with 2.0 mg L-Glutamic acid g-1 soil and Ascorbic Acid; (6) treated with 2.0 mg L-Citrulline g-1 soil and Ascorbic Acid; (7) treated with 2.0 mg L-Ornithine g-1 soil and Ascorbic Acid; and (8) treated with 2.0 mg D-Ornithine g-1 soil and Ascorbic Acid.Different letters indicate significant difference (confidence level of 95%) during the incubation time (one-way analysis of variance (ANOVA), followed by the least significant difference test).Error bars represent the standard deviation of replicate analyses (n = 3).(A) Deciduous forest (middle age, Ah horizon); (B) Deciduous forest (old age Oe horizon); (C) Deciduous forest (old age, Ahk

Table 1 .
Physical and chemical properties of tested soil.Those parameters which are not available are represented by NA.

Table 2 .
Highest percentage increase in respiration in different soil types in response to different amino acid applications.