Phenolic utilizing microbes

What is needed at this point are quantitative data on phenolic acid utilizing microbes (e.g., bacteria, fungi, actinomycetes) in field soils + phenolic acid enrichment during the spring, summer, and fall for various crop and forest systems. In addition, we need quantitative data describing the relationships between bulk-soil and rhizosphere phenolic acid utilizing microbes and the observed phytotoxicity of phenolic acids for sensitive species, i.e., a form of dimension analysis utilizing equations to predict useful and/or consistent relationships. 

Utilization of phenolic acids by microbes can be substantial as long as conditions are appropriate, e.g., adequate nutrition, moisture, pH, and temperature. However, a variety of other factors can also influence microbial utilization of phenolic acids. For example, phenolic acid utilization by microbes was reduced when more readily available carbon sources were present in the soil such as glucose and phenylalanine (Fig. 2.24 Blum et al. 1993 Pue et al. 1995) or other sources of available carbon, i.e., roots (Blum et al. 1999a). The presence of methionine, however, did not influence the rate of phenolic acid utilization by soil microbes (Pue et al. 1995). Differential utilization of carbon sources by soil microbes is fairly common (Martin and Haider 1979 Harder and Dijkhuizen 1982 Papanastasiou 1982 Sugi and Schimel 1993). Thus the relationship between phenolic acid-utilizing microbes and their carbon environment is complex. There is yet another aspect that adds to this complexity. Initial microbial breakdown products of phenolic acids are frequently other phenolic acids. For example, ferulic acid is converted to caffeic acid or vanillic acid and these are converted to protocatechuic acid. Next the ring structure of the... 

Fig. 3.19 Effects of total phenoUc acid composed of a 4-equal-molar mixture of p-coumaric acid, ferulic acid,p-hydroxybenzoic add, and vanillic acid on absolute rates of leaf expansion (cm /day r = 0.44) of 12 day-old cucumber seedlings and microbial populations (CFU/g sod F = 0.49) that can utilize phenolic acids as a sole carbon source in Cedi A soil (a). Relationships between phenolic add-utilizing microbes (CPU, colony forming units) and percent inhibition of absolute rates of leaf expansion for cucumber seedlings are presented in (b). Values for (b) were calculated from (a). Figures based on regressions from Blum et al. (2000). Plenum Publishing Corporation, regressions used with permission of Springer Science and Business Media...

However, if phenolic acid from tissues are primarily utilized ( used up ) to stimulate phenolic-acid utilizing microbes within the bulk soil and/or the rhi-zosphere/rhizoplane, then the inhibition of cucumber seedlings could be due to a promoter/modifier/inhibitor complex dominated by other inhibitors and any remaining phenolic acids and phenolic acid breakdown products, and... 

To Determine Relationships Between Phenolic Acids-Utilizing Microbes and Phenolic Acid Inhibition (Section 2.4.6)... 

Con 2 Selection and induction of phenolic acids-utilizing microbes in soil occur readily when phenolic acids are present. 

Pro 2 Selection and induction of phenolic acid-utilizing microbes are not evident unless high concentrations of phenolic acids are supplied to soil systems over time and environmental conditions are appropriate. However, what is really more important here is the activity of these microbes, i.e., utilization rates for phenolic acid (see 3). 

Reversible sorption of phenolic acids by soils may provide some protection to phenolic acids from microbial degradation. In the absence of microbes, reversible sorption 35 days after addition of 0.5-3 mu mol/g of ferulic acid or p-coumaric acid was 8-14% in Cecil A(p) horizon and 31-38% in Cecil B-t horizon soil materials. The reversibly sorbed/solution ratios (r/s) for ferulic acid or p-coumaric acid ranged from 0.12 to 0.25 in A(p) and 0.65 to 0.85 in B-t horizon soil materials. When microbes were introduced, the r/s ratio for both the A(p) and B-t horizon soil materials increased over time up to 5 and 2, respectively, thereby indicating a more rapid utilization of solution phenolic acids over reversibly sorbed phenolic acids. The increase in r/s ratio and the overall microbial utilization of ferulic acid and/or p-coumaric acid were much more rapid in A(p) than in B-t horizon soil materials. Reversible sorption, however, provided protection of phenolic acids from microbial utilization for only very short periods of time. Differential soil fixation, microbial production of benzoic acids (e.g., vanillic acid and p-hydroxybenzoic acid) from cinnamic acids (e.g., ferulic acid and p-coumaric acid, respectively), and the subsequent differential utilization of cinnamic and benzoic acids by soil microbes indicated that these processes can substantially influence the magnitude and duration of the phytoxicity of individual phenolic acids (Blum, 1998). 

There is some confusion in the literature as to when it is appropriate to apply the term allelochemical to phenolic acids. Since phenolic acids and their derivatives are found essentially in all terrestrial soils, it should be understood that the presence of phenolic acids in soil does not automatically imply that these phenolic acids are functionally allelochemicals. In theory, phenolic acids in soils, depending on their chemical state, concentrations, and the organisms involved, can have no effect, a stimulatory effect, or an inhibitory effect on any given plant or microbial process. For phenolic acids in the soil to be classified as allelochemicals requires that a) the phenolic acids are in an active form (e.g., free and protonated), b) they are involved in chemically mediated plant, microbe, or plant/microbial interactions and c) the concentrations of the active forms in the soil solution are sufficient to modify plant or microbial behavior, either in a positive or negative manner.8,49 However, changes in microbial behaviour associated with the utilization of phenolic acids as a carbon or energy source would not qualify as an allelopathic response. 

As with soil microbes, the data available for microbes in the rhizosphere that can utilize phenolic acids as a carbon source are also very limited. Phenolic acid utilizing microbial populations observed within the rhizosphere of cucumber seedlings and mature wheat plants range from 107 to 1013 CFU/g root (Table 3.2). 

For both the soil and the rhizosphere data (Tables 3.1 and 3.2) the bacteria that can co-metabolize phenolic acids but not utilize phenolic acids as a sole carbon source are not included in these numbers. Other soil and rhizosphere microbes (e g., fungi and actinomycetes) that can utilize phenolic acids as a carbon source have also not been determined. The present data base for phenolic acid utilizing microorganisms in the bulk-soil and the rhizosphere is clearly inadequate. 

Implications of Mobility on the Availability and Degradation of Pesticides in Soil. Repeated application of 2,4-dichlorophenol, p-nitrophenol, and salicylic acid (as observed in current studies) and carbofuran phenol (20) has induced enhanced microbial degradation of their parent compounds. Rf values of these hydrolysis products indicate intermediate to high mobility in soils. The p-nitrophenol, 2,4-dichlorophenol, and salicylic acid were utilized as energy sources by microbes, and their availability in soil may contribute to the induction of rapid microbial metabolism. Carbofuran phenol did not serve as a microbial substrate but also enhanced the degradation of its parent compound, carbofuran (20). Carbofuran phenol is freely available in anaerobic soils, but the significance of its availability is yet to be understood. 

Type III polyketide synthases are particularly relevant to this chapter because they catalyze the formation of phenolic compounds. This group of polyketide synthases consists of CHSs, stilbene synthase (STS), and curcuminoid synthase (CUS), which perform decarboxylative condensations between a starter unit, either p-coumaroyl-CoA 19 or cinnamoyl-CoA 18, and an extender unit, malonyl-CoA 10. CHS, STS, and CUS convert the substrate molecules into flavo-noids (Cg-Cs-Cg), stilbenoids 8 (Cg-C2-Cg), and curcuminoids 9 (Cg-C7-C6), respectively [59]. Stilbenoids 8 and curcuminoids 9 are out of the scope of this chapter but possess medicinal properties as well resveratrol is a well-known stilbenoid 8 associated with longevity, and curcumin is a common curcuminoid 9 that is responsible for the yellow color in turmeric and can be utilized as a natural pigment possessing antioxidant and anti-inflammatory properties [60-63]. For an in-depth treatment of plant polyketide production in microbes, the reader is directed to a recent comprehensive review by Boghigian et al. [64]. 

For our soils water, EDTA, citrate, NaOH, etc. extractions were carried out on individual samples or subsamples of soils with or without amendment phenolic acid(s). Thus the recovery for each extractant comprised the sum of the free , reversibly sorbed, and/or fixed (not immediately available to roots or microbes) phenolic acids that were recoverable by a given extractant. Differences between water extractions (primarily free phenolic acids) and EDTA or citrate ( free and reversibly sorbed phenolic acids) extractions were utilized to estimate reversibly sorbed phenolic acids. Differences between EDTA or citrate extractions and NaOH extractions were utilized to estimate the fraction of fixed phenolic acids that could be recovered by NaOH. For additional details see Section 2.4.3. This approach has been criticized because free phenolic acids were not removed from soil samples before reversibly sorbed phenolic acids were extracted (Ohno and First 1998). However, in a preliminary study extracting our soils by the traditional method (removing free ... 

Cucumber seeds and seedlings have associated with them substantial microbial populations that are difficult to eliminate because microbes are not only found on and in the cutinized surface of the seed coat but can also be found internally within the seed (Leben 1961 Mundt and Hinkle 1976). Depletion of phenolic acids from nutrient solutions thus represent uptake by roots and microbial utilization. By replacing the nutrient solution (control) and nutrient-phenolic acid solutions (treatments) every other day, microbial populations were kept in check and phenolic acid concentrations were brought back to the original treatment concentrations. However, since phenolic acid treatments changed microbial populations on the rhizoplane (Fig. 2.9)... 

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