Hydroxyl free radicals are formed due to auto oxidation of ascorbic. These free radicals are lethal to the soil microbial community. Ascorbic acid helps in the regulation of the developmental processes in plants [1
]. It serves as a cofactor of many enzymes [3
], such as prolyl and lysyl hydroxylases [5
]; 1-aminocyclopropane-1-carboxylate oxidase [7
]; and 2-oxoacid-dependent dioxygenases [3
]. High levels of ascorbic acid inside plant cell help in its defense against pathogens [10
]. Ascorbic acid helps in the biosynthesis and signaling of many plant hormones like ethylene, gibberellic acid (GA), and abscisic acid (ABA).
Ascorbic acid is a reducing sugar with anti-oxidant properties [13
] , and it shows bacteriostatic properties in soil microenvironments [14
]. 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
]. It is mainly controlled by microbial decomposition of soil organic matter, which depends on a plethora of biotic and abiotic factors [24
]. In this study, we measured soil respiration, which is one of the many biological indicators of soil health [26
]. 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.
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 2
B). 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 2
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
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
]. 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
Increased soil respiration and CO2
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
] 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
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.