Composition of cell wall phenolics and polysaccharides of the potential bioenergy crop –Miscanthus

The composition and concentrations of cell wall polysaccharides and phenolic compounds were analyzed in mature stems of several Miscanthus genotypes, in comparison with switchgrass and reed (Arundo donax), and biomass characteristics were correlated with cell wall saccharification efficiency. The highest cellulose content was found in cell walls of M. sinensis‘Grosse Fontaine’ (55%) and in A. donax (47%) and lowest (about 32%) in M. sinensis‘Adagio’. There was little variation in lignin contents across M. sinensis samples (all about 22–24% of cell wall), however, Miscanthus×giganteus (M × g) cell walls contained about 28% lignin, reed – 23% and switchgrass – 26%. The highest ratios of cellulose/lignin and cellulose/xylan were in M. sinensis‘Grosse Fontaine’ across all samples tested. About the same total content of ester‐bound phenolics was found in different Miscanthus genotypes (23–27 μg/mg cell wall), while reed cell walls contained 17 μg/mg cell wall and switchgrass contained a lower amount of ester‐bound phenolics, about 15 μg/mg cell wall. Coumaric acid was a major phenolic compound ester‐bound to cell walls in plants analyzed and the ratio of coumaric acid/ferulic acid varied from 2.1 to 4.3, with the highest ratio being in M × g samples. Concentration of ether‐bound hydroxycinnamic acids varied greatly (about two‐three‐fold) within Miscanthus genotypes and was also the highest in M × g cell walls, but at a concentration lower than ester‐bound hydroxycinnamic acids. We identified four different forms of diferulic acid esters bound to Miscanthus cell walls and their concentration and proportion varied in genotypes analyzed with the 5‐5‐coupled dimer being the predominant type of diferulate in most samples tested. The contents of lignin and ether‐bound phenolics in the cell wall were the major determinants of the biomass degradation caused by enzymatic hydrolysis.     

Effect of mannan oligosaccharide elicitor and ferulic acid on enhancement of laccases production in liquid cultures of basidiomycetes

The effects of mannan oligosaccharides preparation (MO), as elicitor, and ferulic acid inducer for enhancement in laccases production in liquid cultures of three strains of basidiomycetes, Pycnoporus sanguineus, Coriolopsis polyzona and Pleurotus ostreatus was studied using a full factorial 3² experimental design. MO, either individually or combined with ferulic acid, enhanced laccases levels in the three different strains of the white-rot fungi. The enhancement of laccases production was species specific with the highest increase in liquid cultures of P. sanguineus (88-fold) followed by P. ostreatus (3-fold) and C. polyzona (2-fold). Separate additions of 75 mg/l of MO to the cultures of P. sanguineus and P. ostreatus caused the increase while a combined addition of 150 mg/l of MO and 1 mM ferulic acid resulted in the optimal production of laccases in the cultures of C. polyzona.     

Antibiotic potential of plant growth promoting rhizobacteria (PGPR) against Sclerotium rolfsii

High performance liquid chromatographic (HPLC) analysis of culture filtrates of plant growth promoting rhizobacteria (PGPR) and medium of inhibitory zone of interaction of Sclerotium rolfsii with PGPR, viz. Pseudomonas aeruginosa, Pseudomonas fluorescens 4, Pseudomonas fluorescens 4 (new) and Pseudomonas sp. varied from sample to sample. In all the culture filtrates of PGPRs, P. aeruginosa had nine phenolic acids in which ferulic acid (14.52 μg/ml) was maximum followed by other phenolic acids. However, the culture filtrates of P. fluorescens 4 had six phenolic acids with maximum ferulic acid (20.54 μg/ml) followed by indole acetic acid (IAA), caffeic, salicylic, o-coumeric acid and cinnamic acids. However, P. fluorescens 4 culture filtrate had seven phenolic acids in which salicylic acid was maximum (18.03 μg) followed by IAA, caffeic, vanillic, ferulic, o-coumeric and cinnamic acids. Pseudomonas sp. also showed eight phenolic acids where caffeic acid (2.75 μg) was maximum followed by trace amounts of ferulic, salicylic, IAA, vanillic, cinnamic, o-coumeric and tannic acids. The analysis of antibiosis zone of PGPRs showed fairly rich phenolic acids. A total of nine phenolic acids were detected in which caffeic acid was maximum (29.14 μg/g) followed by gallic (17.64 μg/g) and vanillic (3.52 μg/g) acids but others were in traces. In P. aeruginosa, antibiosis zone had seven phenolic acids where IAA was maximum (3.48 μg/g) followed by o-coumeric acid (2.08 μg/g), others were in traces. The medium of antibiosis zone of P. fluorescens 4 and P. fluorescens 4 new had eight phenolic acids in which IAA was maximum with other phenolic acids in traces.     

Novel laccase—producing ascomycetes

Screening of ascomycetes producing laccases during growth on agar medium or submerged cultivation in the presence of various natural sources of carbon and energy (grain crops and potato) was carried out. The conditions of submerged cultivation of the most active strains (Myrothecium roridum VKM F-3565, Stachybotrys cylindrospora VKM F-3049, and Ulocladium atrum VKM F-4302) were optimized for the purpose of increasing laccase activity. The pH-optima and substrate selectivity of laccases in the culture liquid of the strains in relation to ABTS and phenolic compounds (2,6-dimethoxyphenol, syringaldazine, ferulic acid, p-coumaryl alcohol, and coniferyl alcohol) were investigated. High laccase activity at neutral pH was shown for the culture liquids of M. roridum VKM F-3565 and S. cylindrospora VKM F-3049 strains that provides prospects for using laccases of these strains in various cell biotechnologies.     

Isolation of Enterobacteria able to degrade simple aromatic compounds from the wastewater from olive oil extraction

Summary Enterobacteria growing on wastewater from olive oil extraction were selected. Among this microflora, strains of Klebsiella oxytoca and Citrobacter diversus able to degrade simple monomeric aromatic compounds were isolated by enrichment culture of the effluent lacking simple sugars. In this preliminary investigation, the phenolic acids tested on solid and liquid media were gentisic, protocatechuic, p-hydroxybenzoic, benzoic, vanillic and ferulic. It was shown that the biodegradation of an aromatic acid is tightly dependent on both the type and the position of the radical substituted on the aromatic ring. Citrobacter was the most efficient strain in metabolizing ferulic acid in liquid medium at a concentration of 1.5 g/l. The substrate biodegradation yield achieved exceeded 86%.     

Enzymatic synthesis of structured phenolic lipids by incorporation of selected phenolic acids into triolein

The synthesis of structured phenolic lipids by lipase-catalyzed transesterification of selected phenolic acids, including p-hydroxyphenyl acetic, p-coumaric, sinapic, ferulic and 3,4-dihydroxybenzoic acids, with triolein was investigated. The highest enzymatic activity (248?nmol esterified phenolic acid/g solid enzyme/min) and bioconversion (62%) was obtained for the transesterification of p-hydroxyphenyl acetic acid with triolein. In addition, the transesterification of p-coumaric with triolein resulted in a higher enzymatic activity (87?nmol esterified phenolic acid/g solid enzyme/min) and bioconversion (46%) than those obtained for the transesterfication of ferulic and sinapic acids. The results also showed that using p-hydroxyphenyl acetic, p-coumaric and ferulic acids as substrate, the maximum bioconversion of phenolic monoacylglycerols was close to that of phenolic diacylglycerols. Although p-coumaric acid had very low radical scavenging activity (2%) compared to that of ferulic acid (62%), the p-coumaroylated lipids demonstrated a higher scavenging potency (16%) than that of the feruloylated one (10%).     

Development of printable bioactive paper containing laccase

Compatibility of Trametes versicolor and Trametes hirsuta laccases was studied with polymers used for flexographic inks. The aim was to produce bioactive paper with ability to change color. Optimum pH for the stability of Trametes versicolor and Trametes hirsuta laccases was determined during storage at room temperature for 60 days. The optimum pH for the stability of both laccases was 8–9. The stabilization effect of flexo printing inks on the enzymes was tested in liquid form and when coated on paper. Sulfo polyester resin HZ1100D stabilized the two laccases both in solution and on paper. For example, Trametes versicolor laccase remained stable for at least 8 weeks when coated with HZ1100D polymer. Furthermore, the adsorption of the flexo inks to cellulose was studied with quartz crystal microbalance with dissipation monitoring (QCM-D). It was observed that HZ1100D also adsorbs well on cellulose over a wide pH range. The results suggested that laccases are well suited to bioactive paper applications. Large scale manufacturing of bioactive paper products by flexo printing would be possible because of the compatibility of laccases with flexo inks.     

Laccases from Basidiomycetes: physicochemical characteristics and substrate specificity towards methoxyphenolic compounds

Laccases from the Basidiomycetes Coriolus hirsutus, Coriolus zonatus, Cerrena maxima, and Coriolisimus fulvocinerea have been isolated and purified to homogeneity and partially characterized. The kinetics of oxidation of different methoxyphenolic compounds by the fungal laccases has been studied. As laccase substrates, such methoxyphenolic compounds as 4-hydroxy-3,5-dimethoxycinnamic acid (sinapinic acid), 4-hydroxy-3-methoxycinnamic acid (ferulic acid), and 2-methoxyphenol (guaiacol) were used. The stoichiometries of the enzymatic reactions were determined: guaiacol and sinapinic acid are one-electron donors and their oxidation apparently results in the formation of dimers. It was established that k _cat/K _m, which indicates the effectiveness of catalysis, increases in the series guaiacol, ferulic acid, and sinapinic acid. This fact might be connected with the influence of substituents of the phenolic ring of the substrates. This phenomenon was established for fungal laccases with different physicochemical properties, amino acid composition, and carbohydrate content. This suggests that all fungal laccases possess the same mechanism of interaction between organic substrate electron donors and the copper-containing active site of the enzyme and that this interaction determines the observed values of the kinetic parameters.     

Efficient release of ferulic acid from sweet potato (<Emphasis Type="Italic">Ipomoea batatas</Emphasis>) stems by chemical hydrolysis

An efficient method for release of ferulic acid from sweet potato stems was developed. Ferulic acid along with phenolic compounds were released from stems by acid and alkaline treatments. The base hydrolysis with 0.1 N NaOH yielded the highest quantity of total extracts (471.1 mg/g). The stems released more phenolic compounds when 0.0125∼0.025 N NaOH was employed. Where as ferulic acid release was maximal with 0.05 N H₂SO₄ (0.32 mg/g). Ferulic acid was separated from phenolics by column chromatography. Among the elution solvents, ethyl acetate fractions (80%) contained ferulic acid. Ethyl acetate eluants were further fractionated with n-hexane/ethyl acetate/formic acid (100/50/0.5, v/v/v). All fractions showed ferulic acid and phenolic compounds. Fraction V among them was ascribed to ferulic acid with an yield of 5.41 mg/g of dry sweet potato tissue.     

Effect of lignin-derived phenolic monomers on the growth of the edible mushrooms Lentinus edodes,Pleurotus sajor-caju and Volvariella volvacea

Pleurotus sajor-caju was generally more tolerant to lignin-related phenolic monomers and tannin derivatives than Lentinus edodes and the straw mushroom, Volvariella volvacea. Several phenols, at up to 5 mM, enhanced mycelial growth of P. sajor-caju. No clear pattern was evident when the effects of phenols and tannins on the growth of V. volvacea and L. edodes were compared, but the lower concentrations of 4-hydroxybenzaldehyde and vanillin which were tested were markedly more toxic to the straw mushroom. The distribution of phenolic monomers and tannin derivatives in the agricultural wastes used for mushroom cultivation may be an important growth determinant. However, the differences in the growth inhibition profiles of L. edodes, P. sajor-caju and V. volvacea suggest that, alone, the effect of these compounds on fungal growth is unlikely to account for the varying abilities of the three mushroom species to grow and fruit on a particular lignocellulosic substrate.     

Optimized conditions for the synthesis of vanillic acid under hypersaline conditions by <Emphasis Type="Italic">Halomonas elongata</Emphasis> DSM 2581<Superscript>T</Superscript> resting cells

During growth on ferulic acid, Halomonas elongata DSM 2581^T was capable of promoting the formation of a significant amount of vanillic acid. The products were confirmed by high-performance liquid chromatography and gas chromatography mass-spectrometry analyses. To enhance the formation of vanillic acid and prevent its degradation, a resting-cell method using Halomonas elongata was developed. The growth state of the culture utilized for biomass production, the concentration of the biomass, the amount of ferulic acid that was treated and the reutilization of the biomass were optimized. The optimal yield of vanillic acid (82%) was obtained after a 10-h reaction using 10 mM ferulic acid and 5 g/l of cell pregrown on ferulic acid and harvested at the end of the exponential phase.     

Antioxidant activity of extracts from Thalictrum minus suspension culture

Low meadow-rue (Thalictrum minus L.) antioxidant complex was studied in cell extracts and culture medium. Its activity was expressed as total polyphenol content in ferulic acid equivalents. In these model systems (cell extracts and culture medium) the inhibition of lipid oxidation and diphenylpicrylhydrazine reduction (EC₅₀ = 12–15 μg/ml) were observed. At the phenolic compound concentration of 8–15 μg/ml, the reducing capacity of cell extracts was equivalent to 1.5 mM ascorbic acid. At the same time, berberine, a major alkaloid synthesized by the culture, manifested a low antioxidant activity. The analysis of phenolic acid composition in low meadow-rue showed that one of the main components of its antioxidant system were caffeic, gallic, chlorogenic, and ferulic acids.     

Production of lactic acid from paper sludge using acid-tolerant, thermophilic Bacillus coagulan strains

Production of lactic acid from paper sludge was studied using thermophilic Bacillus coagulan strains 36D1 and P4-102B. More than 80% of lactic acid yield and more than 87% of cellulose conversion were achieved using both strains without any pH control due to the buffering effect of CaCO₃ in paper sludge. The addition of CaCO₃ as the buffering reagent in rich medium increased lactic acid yield but had little effect on cellulose conversion; when lean medium was utilized, the addition of CaCO₃ had little effect on either cellulose conversion or lactic acid yield. Lowering the fermentation temperature lowered lactic acid yield but increased cellulose conversion. Semi-continuous simultaneous saccharification and co-fermentation (SSCF) using medium containing 100 g/L cellulose equivalent paper sludge without pH control was carried out in serum bottles for up to 1000 h. When rich medium was utilized, the average lactic acid concentrations in steady state for strains 36D1 and P4-102B were 92 g/L and 91.7 g/L, respectively, and lactic acid yields were 77% and 78%. The average lactic acid concentrations produced using semi-continuous SSCF with lean medium were 77.5 g/L and 77.0 g/L for strains 36D1 and P4-102B, respectively, and lactic acid yields were 72% and 75%. The productivities at steady state were 0.96 g/L/h and 0.82 g/L/h for both strains in rich medium and lean medium, respectively. Our data support that B. coagulan strains 36D1 and P4-102B are promising for converting paper sludge to lactic acid via SSCF.     

Inhibition of cellulose enzymatic hydrolysis by laccase‐derived compounds from phenols

The presence of inhibitors compounds after pretreatment of lignocellulosic materials affects the saccharification and fermentation steps in bioethanol production processes. Even though, external addition of laccases selectively removes the phenolic compounds from lignocellulosic prehydrolysates, when it is coupled to saccharification step, lower hydrolysis yields are attained. Vanillin, syringaldehyde and ferulic acid are phenolic compounds commonly found in wheat‐straw prehydrolysate after steam‐explosion pretreatment. These three phenolic compounds were used in this study to elucidate the inhibitory mechanisms of laccase‐derived compounds after laccase treatment. Reaction products derived from laccase oxidation of vanillin and syringaldehyde showed to be the strongest inhibitors. The presence of these products causes a decrement on enzymatic hydrolysis yield of a model cellulosic substrate (Sigmacell) of 46.6 and 32.6%, respectively at 24 h. Moreover, a decrease in more than 50% of cellulase and β‐glucosidase activities was observed in presence of laccase and vanillin. This effect was attributed to coupling reactions between phenoxyl radicals and enzymes. On the other hand, when the hydrolysis of Sigmacell was performed in presence of prehydrolysate from steam‐exploded wheat straw a significant inhibition on enzymatic hydrolysis was observed independently of laccase treatment. This result pointed out that the other components of wheat‐straw prehydrolysate are affecting the enzymatic hydrolysis to a higher extent than the possible laccase‐derived products. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:700–706, 2015     

Production of Substituted Styrene Bioproducts from Lignin and Lignocellulose Using Engineered Pseudomonas putida KT2440

Ferulic acid is a renewable chemical found in lignocellulose from grasses such as wheat straw and sugarcane. Pseudomonas putida is able to liberate and metabolize ferulic acid from plant biomass. Deletion of the hydroxycinnamoyl‐CoA hydratase‐lyase gene (ech) produced a strain of P. putida unable to utilize ferulic and p‐coumaric acid, which is able to accumulate ferulic acid and p‐coumaric acid from wheat straw or sugar cane bagasse. Further engineering of this strain saw the replacement of ech with the phenolic acid decarboxylase padC, which converts p‐coumaric and ferulic acid into 4‐vinylphenol and the flavor agent 4‐vinylguaiacol, respectively. The engineered strain containing padC is able to generate 4‐vinylguaiacol and 4‐vinylphenol from media containing lignocellulose or Green Value Protobind lignin as feedstock, and does not require the addition of an exogenous inducer molecule. Biopolymerization of 4‐vinylguaiacol and 4‐vinylcatechol styrene products is also carried out, using Trametes versicolor laccase, to generate “biopolystyrene” materials on small scale.     

New strains of basidiomycetes that produce bioethanol from lignocellulose biomass

Sixty six isolates were screened for ability of bioethanol production; dynamics of product accumulation and substrate utilization were investigated for two selected strains Trametes hirsuta MT-24.24 and Trametes versicolor IT-1. The strains’ efficiency was evaluated as bioethanol production by 1 g biomass. Strain T. versicolor IT-1 producing over 33 g/L of the ethanol for 9 d was selected. Direct conversion of Na-carboxymethyl cellulose, microcrystalline cellulose and straw was shown with ethanol yields of 2.1, 1.6 and 1.7 g/L, respectively, for 9 d fermentation time.     

Effect of Plant Growth-Promoting Rhizobacteria and Culture Filtrate of <Emphasis Type="Italic">Sclerotium rolfsii</Emphasis> on Phenolic and Salicylic Acid Contents in Chickpea (<Emphasis Type="Italic">Cicer arietinum</Emphasis>)

Two plant growth-promoting rhizobacteria (PGPR), viz., Pseudomonas fluorescens strain Pf4 and P. aeruginosa strain Pag, protected chickpea (Cicer arietinum) plants from Sclerotium rolfsii infection when applied singly or in combination as seed treatment. Pag gave the best protection to the seedlings, applied either singly (mortality 16%) or in combination with Pf4 (mortality 17%) compared with 44% and 24% mortality in control and Pf4 treatment, respectively. The two PGPR strains induced the synthesis of specific phenolic acids, salicylic acid (SA), as well as total phenolics at different growth stages of chickpea seedlings with varied amount. The maximum amount of total phenolics was recorded in all the aerial parts of 4-week-old plants. Gallic, ferulic, chlorogenic, and cinnamic acids were the major phenolic acids detected in high-performance liquid chromatography (HPLC) analysis. Induction of such phenolic acids in the seedlings was observed up to 6 weeks in comparison with control. Salicylic acid (SA) was induced frequently during the first 3 weeks of growth only. Between the two strains, Pag was more effective in inducing phenolic acid synthesis applied either singly or in combination with strain Pf4 during the entire 6 weeks of growth of chickpea. In the presence of a culture filtrate of S. rolfsii, the two Pseudomonas strains induced more phenolic acids in treated than in non-treated and control plants. The occurrence of salicylic acid was frequent in the first 24 h, but infrequent at 48 and 96 h. Foliar spray of Pseudomonas strains also enhanced the phenolic acid content as well as total phenolics within 24 h of application. Gallic, chlorogenic, and cinnamic acids were consistently discerned in the treated leaves, whereas SA was absent even up to 96 h of application. Resistance in chickpea plants by Pseudomonas strains through induction of phenolic compounds as well as induced systemic resistance via SA-dependent pathway was evident. Received: 1 April 2002 / Accepted: 4 May 2002     

Changes in Cell Wall-bound Phenolic Compounds and Lignin in Roots of Date Palm Cultivars Differing in Susceptibility to Fusarium oxysporum f. sp. albedinis

The roots of date palm contain four cell wall‐bound phenolic acids identified as p‐hydroxybenzoic, p‐coumaric, ferulic and sinapic acids. The ferulic acid represents the major phenolic compound since it constitutes 48.2–55.8% of cell wall‐bound phenolic acids. All these phenolic acids were present in the resistant cultivar (BSTN) and the susceptible cultivar (JHL). However, the pre‐infection contents of p‐coumaric, ferulic and sinapic acids were greater in the resistant cultivar than in the susceptible one. For the contents of p‐hydroxybenzoic acid, there was no significant difference between the resistant cultivar and the susceptible cultivar. Similarly, the pre‐infection contents of lignin were approximately equal for both cultivars. Inoculation of the date palm roots by Fusarium oxysporum f. sp. albedinis induced important modifications to the contents of the cell wall‐bound phenolic compounds and lignin, which made it possible to distinguish between resistant and susceptible cultivars. The post‐infection contents of cell wall‐bound phenolic compounds underwent a rapid and intense increase with a maximum accumulation on the tenth day for p‐hydroxybenzoic acid (1.54 μmol/g), p‐coumaric acid (2.77 μmol/g) and ferulic acid (2.64 μmol/g) and on the fifteenth day for sinapic acid (1.85 μmol/g). The maximum contents accumulated in the resistant cultivar were greater than those in the susceptible cultivar, namely, 11 times for p‐hydroxybenzoic acid, 2.6 times for p‐coumaric acid, 1.8 times for ferulic acid and 12.3 times for sinapic acid. In the susceptible cultivar, p‐coumaric acid and ferulic acid contents also increased after inoculation although they did not reach the pre‐infection contents of the resistant cultivar. The contents of p‐hydroxybenzoic acid in the susceptible cultivar roots did not present post‐infection modification and those of sinapic acid decreased instead. The lignin contents increased in both cultivars with a maximum accumulation on the fifteenth day. However, the maximum contents accumulated in the resistant cultivar roots were 1.5 times greater than those of the susceptible cultivar. These results showed clear differences between the resistant BSTN and the susceptible JHL cultivars. The implication of cell wall‐bound phenolic compounds and lignin in the resistance of date palm to F. oxysporum f. sp. albedinis appears to be dependent on the speed and intensity of their accumulation with greater contents in the first stage of infection.     

Ferulic acid may prevent infection of Cicer arietinum by Sclerotium rolfsii

High performance liquid chromatography analysis of different parts of Sclerotium rolfsii-infected and healthy seedlings of chickpea (Cicer arietinum) was carried out to examine the status of phenolic compounds. Three major peaks that appeared consistently were identified as gallic, vanillic and ferulic acids. Gallic acid concentrations were increased in the leaves and stems of infected plants compared to healthy ones. Vanillic acid detected in stems and leaves of healthy seedlings was not detected in infected seedlings. There was a significant increase of ferulic acid in those stem portions located above the infected collar region compared to minimal amounts in the roots of healthy seedlings. In vitro studies of ferulic acid showed significant antifungal activity against S. rolfsii. Complete inhibition of mycelial growth was observed with 1000 g of ferulic acid/ml. Lower concentrations (250, 500 and 750 g/ml) were also inhibitory and colony growth was compact in comparison with the fluffy growth of normal mycelium. Higher amounts of phenolics were found in the stems and leaves of S. rolfsii-infected seedlings in comparison to the healthy ones. A role for ferulic acid in preventing infections by S. rolfsii in the stems and leaves of chickpea plants above the infection zone is therefore feasible.     

Influence of Plant Phenolic Acids on Growth and Cellulolytic Activity of Rumen Bacteria

Isolated rumen bacteria were examined for growth and, where appropriate, for their ability to degrade cellulose in the presence of the hydroxycinnamic acids trans-p-coumaric acid and trans-ferulic acid and the hydroxybenzoic acids vanillic acid and 4-hydroxybenzoic acid. Ferulic and p-coumaric acids proved to be the most toxic of the acids examined and suppressed the growth of the cellulolytic strains Ruminococcus albus, Ruminococcus flavefaciens, and Bacteroides succinogenes when included in a simple sugars medium at concentrations of >5 mM. The extent of cellulose digestion by R. flavefaciens and B. succinogenes but not R. albus was also substantially reduced. Examination of rumen fluid from sheep maintained on dried grass containing 0.51% phenolic acids showed the presence of phloretic acid (0.1 mM) and 3-methoxyphloretic acid (trace) produced by hydrogenation of the 2-propenoic side chain of p-coumaric and ferulic acids, respectively. The parent acids were found in trace amounts only, although they represented the major phenolic acids ingested. Phloretic and 3-methoxyphloretic acids proved to be considerably less toxic than their parent acids. All of the cellulolytic strains (and Streptococcus bovis) showed at least a limited ability to hydrogenate hydroxycinnamic acids, with Ruminococcus spp. proving the most effective. No further modification of hydroxycinnamic acids was produced by the single strains of bacteria examined. However, a considerable shortfall in the recovery of added phenolic acids was noted in media inoculated with rumen fluid. It is suggested that hydrogenation may serve to protect cellulolytic strains from hydroxycinnamic acids.