J . Chern. Tech. Riotechnol. 1980,30, 345-350

Production of Aromatic Acids During Anaerobic Digestion of Citrus Peel

Alan G. Lane

CSIRO Division of Food Research, North Ryde, Australia

(Paper received 22 June 1979 and accepted 13 February 1980)

Commercially prepared citrus oils, distilled citrus oils, limonene and the non-volatile fraction of lemon oils were all found to be toxic to the anaerobic digestion process for conversion of citrus waste to methane. Toxicity was characterised by appearance of benzoic, phenylacetic and phenylpropionic acids in the digestion liquors, though these acids were not in themselves toxic. The bulk of the phenylpropionic acid was derived from flavonoids.

1. Introduction

Anaerobic digestion can be used to convert solid wastes from fruit and vegetable processing into methane fuel.' However, citrus wastes are known to be toxic to the anaerobic process and cannot be utilised in this way.2.3 During investigations into the mechanism of this toxicity, we detected benzoic, phenylacetic and phenylpropionic acids in liquors from anaerobic digestions fed citrus peels. These acids have not been previously reported in such liquors. This paper describes experi- ments aimed at identifying the precursors of these acids and at establishing the relationship between their formation and the toxicity of citrus peel in the anaerobic process.

2. Materials and methods 2.1. Digesters Small-scale digestions were carried out in 150-cm3 conical flasks containing digesting sludge (100 crn3), incubated at 37°C and fed daily with 2 cm3 of slurry containing 0.2 g (dry weight) of solids, (i.e. 2 kg dry solids fed m-3 of sludge day-1). Gas production was monitored by trapping the gas by displacement of water in a graduated cylinder.

Larger-scale digestion experiments were carried out using 1 0-dm3 microbiological fermenters (L. H. Engineering, Stoke Poges, Bucks, UK), containing digesting sludge (8 dm3) stirred at 200 rev min-l and maintained at 36°C. Gas was collected in a football bladder and the volume produced daily was measured by discharging it through a gas meter.

Pilot plant digestion experiments were carried out in a 4540-dm3 digester which has been described previously.1

In all experiments, digestion was said to have 'failed' when the pH of the sludge fell to 6.4.

2.2. Estimation of oil content

A sample of peel, flavedo or albedo (100 g fresh weight) was homogenised with water (200 cm3) in a Waring blender and the mixture was extracted with petroleum ether (3 x 30 cm3). The pooled ether extracts were evaporated to dryness at room temperature and the residue was weighed. Although this procedure measured total petroleum ether extractables in the sample, it afforded an approxi- mate estimate of the oil content.

Ol42-03S6/80/0600-0345 S02.00 0 1980 Society of Chemical Industry 345

346 A. G. Lane

2.3. Gas chromatography 2.3.1. Aromatic acids

Benzoic, phenylacetic and phenylpropionic acids were initially identified in samples of digestion supernatant fluid by gas chromatography-mass spectroscopy (g.c.-m.s.). The acids were subse- quently methylated, separated by g.c. and estimated by comparison of their peak areas, using authentic methyl esters as standards.

A sample ( 5 cm3) of supernatant fluid from centrifugation of digestion fluid was made alkaline with 1~ NaOH (0.1 cm3) and dried with a jet of air on a waterbath at 70°C. The acidic components were methylated by heating at 55°C for 0.5 h with methanol (2 cm3) and sulphuric acid (0.2 cm3). Water (2cm3) was then added and esters were extracted with chloroform (0.5 cm". Aliquots (5 cm3) of standard solutions of each of the aromatic acids (50 mg dm-3) were mixed before being similarly methylated.

Samples (20 nm3) of the chloroform extracts were analysed by g.c. using a Perkin-Elmer Gas Chromatograph and a flame ionisation detector (setting 10 x 102) and using nitrogen (60 cm3 min-1) as the carrier gas. The column (2000 x 3 mm. stainless steel) used routinely was packed with 5 % FFAP on Chromosorb W HP (80-100 mesh). The identity of peaks was verified in selected samples using a column packed with 3 % SE 30 on Chromosorb W HDCS (80-100 mesh). Tempera- tures of column, detector and block were 150,250 and 240°C, respectively, with both columns. The limit of detection for each acid was a concentration of about 0.1 mg dm-3.

2.3.2. Limonene Limonene was estimated by comparison of peak areas from g.c. runs at 70°C on a stainless-steel column ( 2 0 0 0 ~ 3 mm) packed with 5 % SE 30 Chromosorb W (80-100 mesh). Other conditions were as described in section 2.3.1 for aromatic acids. standard peaks were obtained by chromato- graphing samples (0-1 nm3) of a solution of limonene (b.p. range 175-180°C, 10 nm3) in ethanol (2.5 cm3). The limit of detection was 2 x nm3. Limonene in distillation residues was estimated by diluting the residue to the original volume of the oil with ethanol and injecting lO-nm3 samples of the solution.

2.3.3. Feehtocks Waste whole peel was obtained from a commercial orange juice plant, comminuted (8-mm screen), enriched with nitrogen and phosphorus by addition of (NH&HP04 (25 g kg-1 dry wt of peel) and stored at - 20°C.

Orange albedo was obtained from another plant operating an oil recovery process in which the oil-containing flavedo was removed from the fruit before juice extraction, using Brown peel-shaving equipment (Brown International Corp, USA); it was treated as described for whole peel.

Samples of hand-peeled orange albedo were also prepared and treated as described for whole peel. Pelletised orange peel was a commercial product, (Mildura Citrus Products, Mildura, Australia),

prepared as a cattle feed by a process involving addition of lime to fresh peel, pressing, drying and pelletising. The required weight of pellets were reconstituted in water to a volume equal to 5 "/, of the digester capacity before being fed to the digesters. Nitrogen and phosphorus were added as (NH.&HP04 at the rate of 25 g kg-l dry pellets.

Inorganic nutrients were made up in 0 . 1 ~ HCI at the following concentrations (g dm--3): MgC12.6Hz0, 250; CaC12.2Ha0, 250; FeC13, 5 ; ZnO dissolved in 2~ HCI, 2; MnClz.4H20, 0.5; (NH4)6Mo7024-4H20, 0.5; CuSO4, anhydrous, 0.5. The solution was added to the daily feed at the rate of 0.2 ml dm-3.

The whey-peptone-cellulose medium (WPC) used as a citrus-free feedstock contained (g dm-3) : whey powder (Kraft Foods, Melbourne, Australia), 50; cellulose powder (Whatman, England), 50; NaH2P04, 2.25 and peptone ('Lablemco', Oxoid, UK), 6.5.

Production of aromatic acids during anaerobic digestion 341

2.3.4. AaUitives Cold-pressed orange oil (CPOO) was donated by Cenco, Gosford, Australia. Cold-pressed lemon oil (CPLO) and distilled orange oil (DOO) were obtained from Lanfranconi Sud, Reggio Calabria, Italy. Limonene (b.p. range 175-180°C) was prepared from recovered orange oil by laboratory distillation. Distilled lemon oil (DLO) was prepared by vacuum-distilling cold-pressed oil (8 cm3) to yield 75 % volatile fraction and 25 % tarry residue. The residue was dissolved in ethanol (8 cm3) to provide an additive (DR) representing non-volatile oil components on a v/v basis. All the above additives were used at the rate of 0.1 cm3 100 6111-3 of digesting sludge day-'.

Quercetin (Fluka AG, Buchs SG, Switzerland) and hesperidin (Sigma Chemical Co, St Louis, USA; Grade I) were used as suspensions (2.5% w/v) in 0 . 0 5 ~ NaOH at the rate of 0.4 cm3 of suspension 100 cm-3 of sludge day-1.

Phenylpropionic acid (a laboratory preparation of CSIRO Division of Applied Organic Chemistry, Fishermans Bend, Victoria), and benzoic and phenylacetic acids (analytical grade reagents, E. Merk, Darmstadt) were used as aqueous solutions containing 5 mg cm-3 of each acid.

3. Results and discussion

Digestions fed with orange peel at 2 kg (dry weight) m-3 of sludge day-I invariably failed after 2-3 weeks. Failure was characterised by a sudden fall in pH from the normal level of 6.8-7.2 to levels below 6.4, with a simultaneous drop in gas production from the normal level of 1.0-1.3 volumes of gas volume-' of sludge day-1 to less than 0.5 v/v day-* and by appearance in the digestion liquors of benzoic acid (BA), phenylacetic acid (PAA) and phenylpropionic acid (PPA), typically at concentrations of 7.4, 17.7 and 77.8 mg d m 3 , respectively (Table 1). PAA (0.4 mg dm-3) and PPA (1.7 mg dm-3) were also detected in 4540-dm3 digestions fed pelletised peel at 2 kg m-3 of sludge day-' but none of the three acids was detected in WPC digestions. However the level of PPA in pelletised peel digestions increased as the loading approached the maximum, reaching 11.2 mg dm-3

Table 1. Aromatic acids in digestions" fed orange peel or pelletised orange peel with and without aromatic acids

Aromatic acid content (mg d r 3 ) . .~ ~ .___ Sampling timeb

Feed (days) Benzoic Phenylacetic Phenylpropionic . - .~ . . .. .- .- .- ~ - ___

Orange peel 4 N D 3.0 N D 8 N D 4 . 4 7 . 2

16c 7 . 4 17.7 77.8 Pelletised peel 4 N D 1 . 5 N D

7 0 . 9 I . 7 2 . 3 8 ND 2 . 2 N D

17 N D 2 . 9 N D Pelletised peel + benzoic acid" 4 4 . 6 2 . 5 5 . 5

7' 2 . 2 84.6 2 3 . 0 7 + 3 . 5 h 0 . 9 67.3 11.6

8 ND 31.6 ND Pelletised peel + mixed acids" 4 ND 39.4 5 5 . 0

7e I . 2 I35 142 7 + 3 . 5 h 1 .2 100 99.8

8 1 . 1 61 . 5 7.6

R100-cm3 volumes of digesting sludge fed daily with 0 .2 g of solids. "Days from commencement; sample taken 24 h after preceding feed, unless otherwise indicated. "Digestion failed. dAcids added daily to concentrations of: benzoic 50; phenylacetic, 50; phenylpropionic, 100 nig dm-3. 'Sampled 10 rnin after addition of acid(s). N D = N o t detected.

348 A. C. Lane

at a feed rate of 4 kg m-3 day-1 and 103.5 mg dm-3 at a feed rate of 5 kg m-3 day-1. The digestion showed symptoms of overloading at the higher feed rate and after 1 week feeding was discontinued for 4 days. During this period the PPA concentration fell to 68.4 mg dm-3, indicating microbial degradation of PPA in agreement with Balba and Evans.*

These results suggested that high levels of aromatic acids were not the primary cause of citrus peel digestion failure. Instead, the acids appeared to be a symptom of toxicity due to other components of citrus peel and this was confirmed when digestions were fed pelletised orange peel and added benzoic acid (50 mg d w 3 day-l) or benzoic, phenylacetic and phenylpropionic acids (200 mg dm-3 day-1 of a 1 : 1 : 2 mixture). These added acids appeared to be degraded quite rapidly and the digestions showed no symptoms of toxicity after 17 days, though the levels of aromatic acids were comparable with or higher than those observed in orange peel digestions (Table 1).

In a subsequent experiment, PPA, previously shown by g.c. to be free from BA and PAA, was added daily to a WPC digestion at concentrations which increased from 20 to 100 mg dm-3 over the period of 1 week. The concentrations of acids 6.5 h after the final addition of PPA were: BA, 1.6; PAA, 1.4; PPA, 62.7 mg dm-3. It was concluded that the small amounts of BA and PAA detected

Table 2. Aromatic acids in digestions" fed orange peel or WPCb and additives

Feed

WPC Orange peel WPC+ hesperidin WPC + limonene WPC + CPLOd WPC + CPLO +hesperidin WPC+CPLO+quercetin WPC+ DLOC WPC+ DLO+hesperidin W PC + DRf WPC+DR + hesperidin WPC+ CPOOS WPC+CPOO+ hesperidin

Time to failure' (days)

- 19

17 28 12 13 21 I5 1 1 II 24 22

-

Aromatic acid concentration (mg dni-3)

Benzoic Phenylacetic Phenylpropionic ~ -. .. ~ _.___

. . . .. . . . . . . . - . .~

N D N D ND 5 . 4 58.3 89.9

N D N D N D I . 4 7 . 2 N D

26.3 72.9 3 . I 4 . 5 60 .8 70.8 3 . 3 65.8 59.0 4 . 5 93 .2 I .7 2.7 90 .8 84.3 2 . 4 59.2 I . 3 I . 9 57.5 43.4 1 .8 30.5 3 . 5 9 . 5 41.4 I44

0lOO-cm3 volumes of digesting sludge, fed daily with 0 . 2 g of solids; samples taken 24 h after feeding. bWPC=whey powder, 50 g; peptone, 6 g; cellulose powder, 50 g; NaZHPOr, 2 .25 g d n r J in water. 'pH 6 . 4 or less. "CPLO =cold-pressed lemon oil. cDLO=distilled lemon oil. JDR= residue from distillation of lemon oil, dissolved to original oil volume in ethanol. C P O O = cold-pressed orange oil. N D = Not detected.

in digestions fed PPA were due to microbial conversion of the PPA, but that this conversion was unlikely to give rise to the levels of BA and PAA seen in peel digestions.

The three aromatic acids have also been reported in the rumens of livestock; dietary phenylalanine was indicated as the major precursor of phenylacetic acid but dietary tyrosine as only a minor precursor of phenylpropionic acid, which was presumed to be derived from some other p recu r s~r .~ The levels of the three aromatic acids observed in the present study cannot be explained in terms of breakdown of aromatic amino acids present in the citrus peel or peel oils.

It seemed possible that the high levels of aromatic acids seen in peel digestions might have been derived from Aavonoids in the feed materials. However the addition of the flavonoids hesperidin and quercetin to digestions fed WPC and CPLO, CPOO or DR caused an increase in the level of PPA, but not of the other two acids (Table 2). It is probable, therefore, that the major part of the

Production of aromatic acids during anaerobic digestion 349

phenylpropionic acid found in peel digestions was derived from the flavonoids. Cleavage of the hesperidin nucleus would be expected to yield 3-methoxy-4-hydroxy-Bphenylpropionic acide and of the quercetin nucleus 3,4-dihydroxyphenylacetic acid.’ Subsequent reactions involving addition and removal of hydroxyl, methyl and methoxy groups can give rise to a variety of substituted aromatic acids.e-ll These were not detected in the present study, suggesting that dehydroxylati~n~ and demethoxylationeJo proceeded more rapidly than ring cleavage,ll allowing the unsubstituted acids to accumulate. The observation that addition of quercetin to digestions increased the levels of phenylpropionic rather than phenylacetic acid (Table 2) suggests that the pathways outlined previously by some authors5-10 do not fully explain the reactions taking place in the anaerobic digestion system.

The BA and PAA in failed digestions fed WPC and limonene or DLO could not have resulted from breakdown of flavonoids, since flavonoids are not volatile under conditions used to distill limonene and DLO and would not have been present in these feed additives. However some of the coumarins, also normally present in citrus oils, may be volatile under the conditions of distillation used and, if so, could have given rise to tht? BA and PAA seen in digestion liquors.

The pathway for the anaerobic decomposition of benzoate to methane and carbon dioxide has been elucidated4Jl and possible pathways for the degradation of phenylpropionate and phenylacetate to methane and carbon dioxide have been proposed (Evans, W. C., private communication).

Anaerobic digestion is known to be inhibited by citrus oils in citrus processing waste waters and the oils must be removed before the effluents can be treated in this w a ~ . ~ , ~ This inhibition was attributed to the toxicity of D-limonene, which caused digestion failure with symptoms of toxicity similar to those observed in digestions fed orange juice or processing waste water. However, in the present study a 25% ethanolic solution of non-volatile residue from distillation of CPLO (DR) proved to be at least as toxic (v/v) as limonene and resulted in a similar drop in pH and in the appearance of the aromatic acids (Table 2). This toxicity was not due to the presence of residual limonene in the non-volatile fraction, since the proportion of limonene in the alcoholic solution of the residue was shown by g.c. to be less than 2 x 10-7 (v/v). Apparently the lemon oil contained both volatile and non-volatile components which exerted similar toxicity to the anerobic digestion process.

It seemed possible that commercial oil recovery processes might reduce the oil in the peel to non- toxic levels. However, a digestion fed such commercially prepared orange albedo failed after 9 days and the concentrations of the aromatic acids (mg dm-3) were then benzoic, 28.2; phenylacetic, 48.1; and phenylpropionic, 644. The albedo was found to contain 13.2 mg of oil 100 g-l (fresh weight), corresponding to 37.4% of the oil in the whole peel. In contrast, a digestion fed hand- prepared orange albedo containing 2 mg of oil 100 8-1 showed no symptoms of toxicity after 21 days and the aromatic acids were not detected in the supernatant liquor.

These results indicate that toxic inhibition of citrus peel digestions and the production and degradation of aromatic acids are complex, dynamic processes which change over the course of an experiment. Variations between experiments would also be expected to result from differences in the initial microbial flora of the anaerobic sludges and to seasonal and varietal variations in composition of the citrus oils, flavonoids and other components of citrus peel. Elucidation of the microbial interactions and biochemical pathways involved will require carefully designed experiments which take these sources of variability into account.

4. Conclusions

The toxicity of citrus processing wastes and effluents for the anaerobic digestion process has been previously attributed to the presence of D-limonene in citrus oils. I n the present study, however, both cold-pressed and distilled orange and lemon oils and both volatile and non-volatile fractions of lemon oil were found to be toxic, causing digestion failure with production of benzoic, phenylacetic and phenylpropionic acids. The toxic component of these oils apparently interferes with the break- down of the flavonoids, and possibly coumarins, and most of the phenylpropionic acid observed in ‘failed’ peel digesters is probably derived from flavonoids.

350 A. G . Lane

Acknowledgements

The author wishes to express appreciation to Dr F. B. Whitfield for his identification of the aromatic acids by g.c.-m.s., for gifts of authentic methyl esters and for many helpful and stimulating dis- cussions, and also B. Crowley for his capable technical assistance throughout this work.

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