J . Sci. Fd Agric. 1978, 29, 822-830

Biogas Production by Anaerobic Digestion of Fruit and Vegetable Waste. A Preliminary Study

Wieger Knol, Michael M. van der Most and Jacobus de Waart

Central Institute Jbr Nutriiion and Food Reseurch TNO, Zeisr, The Ne~heria~ds

(Manuscript received 23 January 1978)

Waste of apples, asparagus, carrots, green peas, French beans, spinach and straw- berries from a canning factory have been screened on mesophilic anaerobic digestion in 90-day experiments at loading rates varying between 0.80 and 1.60 kg volatile solids (VS) m-3 day-l at a retention time of 32 days. Average biogas yields varied from 0.30 to 0.58 m3 kg-l VS day-1. High percentages of reduction in VS, carbohydrate and crude fibre were obtained in most experiments. Some waste materials showed un- balanced digestion, as might be expected from carbohydrate-rich substrates. In those cases alkali addition, feed interruption and mixing with a nitrogen-rich substrate were used to overcome unbalanced digestion. Residual solids in the digested sludges were removed by flocculation with a polyelectrolyte and centrifugation; liquids with lower CODs remained after flocculation.

1. Introduction

Biogas production by anaerobic digestion of waste materials has been in use for more than a century. In some Asian countries biogas obtained from animal and domestic waste provides the fuel needed by small farmers.l In developed countries interest in anaerobic digestion revived when circumstances made energy supply uncertain; for example in Germany shortly after World War 11.2

Although biogas production is an important aspect of anaerobic digestion, the process has other advantages in waste treatment, such as reduction in dry matter, smell and pathogenic organisms and the better dewatering properties of the digested sludge. Therefore, it has been used to stabilise sewage sludge in domestic sewage plants for at least 60 years.

It is not surprising that in the last decennium anaerobic digestion has come into focus again. Rising fuel prices have led to serious consideration of the profit to be expected from practical appli- cation of the process. On the other hand, adequate treatment of growing quantities of agricultural and domestic waste materials is becoming more and more urgent in present day society. In this connection, much attention has been given in recently published papers to the disposal of the enormous quantities of animal waste from intensive pig

In the fruit and vegetable canning industry, about 10% of the raw material is, generally speaking, wasted in the course of production, the actual amount depending on the product involved. Part of the relevant waste can be used as feed. The rest is transported to a municipal dump site. In view of the increasing charges for this kind of disposal, production of biogas by anaerobic digestion would be an interesting alternative. The biogas produced might cover an important part of a factory's energy demand. While in domestic sewage sludge digestion approximately 50 % of the biogas yield is needed to heat the digester contents in order to obtain optimal digestion, in the fruit and vegetable industry cooling water might serve this purpose so that the total gas production could be utilised, e.g. for the generation of steam to be applied directly in the factory. However, detailed data on the anaerobic digestion of fruit and vegetable waste are not available. The aim of the present study was to get preliminary information on this matter.

Many papers have been published on the microbiology and biochemistry of anaerobic digestion

0022-5142/78/0900-0822 $02.00 0 1978 Society of Chemical Industry 822

Biogas from fruit and vegetable waste 823

Waxes Plastics Hydrocarbon 011s

Nan-protein N Corbohydrates T SO4 NO3

Long chain sugars SH NH3

V o l a t i l e f a t t y

7

Bacteria, sa l ts

residues

Figure 1. Reactions involved in anaerobic digestion according to Hobson et aL6

in digesters as well as in the rumen of herbivores. In the survey by Hobson et a1.6 these topics are discussed in detail. Briefly, anaerobic digestion can be defined as a two-stage process, carried out by a mixed bacterial population in a continuous culture, the optimal temperature for mesophilic digestion being approximately 32°C. The first stage, defined as ‘liquefaction’, comprises the hydrolysis of high molecular weight compounds into metabolites, from which volatile acids, hydrogen and carbon dioxide are formed. In the second stage, defined as ‘methanogenesis’, the intermediates are further transformed to methane and residual carbon dioxide (Figure 1). The two stages are balanced so that little of the intermediate acids accumulate.

Addition of a new substrate to a stable digestion could for a time result in unbalanced growth until a new bacterial population is established. Therefore, new substrates may need a certain adaptation period. Recovery of a balanced flora may be prevented when rapid acid formation lowers the pH from its optimum of about 7.2, caused by much easily digestible carbohydrate in the substrate, as in fruit and vegetable waste. Methane fermentation is then disturbed and even complete digestion failure may occur. Digestion of nitrogenous compounds leads to ammonia which is partly converted to bacterial protein. Accumulation of ammonia, due to much nitrogen in the substrate, will have a neutralising effect upon excess acids. Inhibition by accumulated ammonia is not likely to O C C U ~ . ~ A better balanced digestion may be obtained by mixing wastes high in carbohydrate with wastes high in nitrogen. A temporary increase in acidity may be counteracted by cessation of loading or addition of alkali.638 These measures were applied in this study.

In addition, this investigation comprises preliminary experiments concerning removal of residual solids from the digested wastes and the pollutional value of the remaining sludges.

2. Experimental 2.1. Materials Samples of fruit and vegetable waste were collected at Veluco Conservenfabrieken BV, Gelder- malsen, The Netherlands. From each sample an aliquot was analysed for ash, crude protein, total carbohydrates and crude fibre (Table 1). Directly after arrival the samples were homogenised, frozen and stored at - 20°C. The samples were thawed and diluted to feed the digesters at a loading rate of approximately 1 kg volatile solids per m3 digester volume per day and to maintain a retention time of 32 days.

Digested sludge from the anaerobic digesters of the municipal sewage works at Zeist, The Nether- lands, was used to start the experiments.

2.2. Anaerobic digestion The experiments were carried out in 1 litre digesters. Stirring speed was set at 200 rev min-l. Each digester was heated by a 33°C water bath. Biogas production was measured with Mariotte bottles

824

Table 1. Origin and chemical composition of the fruit and vegetable wastes

W. Knol et al.

Waste samplea Origin, definition

Total carbohydrates

( %) Crude protein Crude fibre

( %) ( %)

Group I Spinach-waste Raw and blanched spinach,

Asparagus-peels Peels removed by hand Group 2 French bean-waste Mixture of samples collected

from waste containers along production line

Strawberry-slurry Strawberry waste with small amounts of asparagus and peas

Apple-pulp Remains after blanching and pressing in apple-sauce production

Apple-slurry Mainly apple particles with small amounts of asparagus

Carrot-waste Mixture of carrot-skin-sludge and rejected carrots

Group 3 Green peas-slurry Mainly whole and damaged

peas

dropped along production line 12.0 1.74

8.9 0.72

13.4 1.49

11.5 1.75

23.2 0.46

6.7 0.29

5.3 0.48

12.8 0.45

0.66 (0.05)b

0.50 (0.06)

4.01 (0.29)

2.42 (0.21)

5.57 (0.24)

1.41 (0.21)

1.58 (0.29)

5.70 (0.44)

4.22 (0.35)b 2.11

1.98 (0.22) 2.14

2.37 (0.18) 2.10

2.25 (0.19) 2.38

1.28 (0.05) 3.74

0.60 (0.08) 1.29

0.38 (0.07) 0.67

3.49 (0.27) 1.68

fC Waste materials are classed under groups 1, 2 or 3 on account of their carbohydrate content in the dry weight. 1’ Figures in brackets are the percentages on dry weight basis.

connected with the gas outlet of the digester and filled with acidified water to prevent solubilisation of carbon dioxide (Figure 2). The digesters were filled initially with 1 litre digested sewage sludge. Each working day, except Friday, the digesters were fed 40 ml influent sludge, i.e. 160 ml diluted waste over 4 days. Then half the amount for 3 days, i.e. 60 ml was added. In this way, an average liquid retention time of 32 days was obtained. Simultaneously with loading the same volume of digested waste was discharged without allowing air to enter the digester. Biogas production was recorded every day. Examinations of the effluents were carried out periodically and comprised determination of pH, total solids, ash, volatile fatty acids; the amount of CH4 in the biogas was also estimated. The duration of the experiments was about 90 days, i.e. three times the retention time. The loading rate of each experiment varied as is indicated in Table 2. At the end of the experi- ments the digested waste was dried for analysis.

In addition, preliminary experiments were carried out with regard to the removal of residual solids and the pollutional value of the digested waste. Residual solids were removed by (a) floccula- tion of 100 ml final sludge with 15 ml of a 0.1 % solution of a polyelectrolyte (Preastol 444K) and (b) centrifugation of 100 ml sludge at 1500 xg. Chemical oxygen demand of the filtrate and super- natant was determined.

2.3. Analysis Definitions of the terms to be used are given below to make comparison with other investigations possible. Total solids content (TS) is defined as the percent dry weight of residue obtained after drying the sludges for 16 h at 105°C. Ash is the weight of material remaining after ashing of dry matter and heating of the residue for 3 h at 550°C. VolatiZe solids (VS), i.e. organic matter, is calculated as total solids minus ash. Total nitrogen was estimated according to the Kjeldahl method. Crude protein was calculated by multiplying total nitrogen content by 6.25.

Biogas from fruit and vegetable waste 825

Total carbohydrate estimation was carried out according to Van de K a ~ n e r . ~ Tn this procedure the material is boiled with water and subsequently exposed to pancreatin amylase. After acid hydrolysis the reducing sugars obtained are determined. Total carbohydrate is expressed as glucose. Crude fibre analyses were performed according to the methods of the Rijkslandbouwproefstation, Maastricht, The Netherlands,lo a modification of the Weender method based on successive boiling in dilute sulphuric acid and dilute sodium hydroxide, washing and drying. Loss of weight after incineration of the dry material at 550°C is reported as crude fibre. Volatilefatty acids (VFA) were estimated according to a method developed at the institute (Wijsman, J. A., personal communication), and carried out as follows. After removing the solids by centrifu- gation, alkali is added to the supernatant. The salts of the acids are dried and suspended in organic solvents. By addition of concentrated phosphoric acid the fatty acids are reformed and the different acids (CZ-CS) are quantitatively and qualitatively estimated by gas chromatographic analysis.

7

Figure 2. One-litre digester. (1) Funnel and tube-clamp for charge; (2) tube and clamp for discharge; (3) gas outlet; (4) Mariotte bottle; (5 ) heat exchanger connected to water bath. The digester was immersed in a water bath at 32°C.

Extent of decomposition. The percentage decomposition of volatile solids, total carbohydrates and crude fibre is derived from the analysis of the waste materials before and after digestion. The pH was measured with an Electrofact pH meter. Methane. The methane content of the produced biogas was determined on a Carlo Erba gas chroma- tograph, type Fraktovap M, with a 80-100 mesh silica gel column and a hot wire detector.

Chemical oxygen demand (COD) was determined by the bichromate sulphuric acid procedure, according to the methods of the Dutch Normalisation 1nstitute.ll

3. Results and discussion 3.1. General In Table 1 the chemical compositions of the different fruit and vegetable waste samples are given. The characteristics of the various experiments are listed in Table 2.

Tab

le 2

. C

hara

cter

istic

s of

the

expe

rim

ents

and

ext

ent

of d

econ

lpos

ition

by

anae

robi

c di

gest

ion

Ext

ent

of d

ecom

posi

tion

(%)

Load

ingb

B

ioga

s V

FA

(mg

litre

-')"

kg V

S m

-3

m3 k

g-lV

S bi

ogas

P

H

-

Aft

er

Tot

al

rate

pr

oduc

tion

CH

I in

TS (

%)

Exp

erim

enta

da

y-1

day-

' (%

) In

fl.

Effl.

In

A.

Effl.

T

otal

50 d

ays

VS

carb

ohyd

rate

s C

rude

fibr

e

Gro

up I

Sp

inac

h-w

aste

A

spar

agus

pee

ls

Gro

up 2

Fr

ench

bea

n-w

aste

St

raw

berr

y-sl

urry

A

pple

-pul

p A

pple

-slu

rry

Car

rot-

was

te

Gro

up 3

G

reen

pea

-slu

rry

Mixe

d su

bstr

ates

Pi

g m

anur

e + a

pple

pul

p

0.83-1.18

0.74-1.06

0.40

0.30

77-80

64-82

3.1-4.5

2.5-3.6 1.3-2.0

I .7-2.4

4.9

4.3

7.2-7.5

7.0-7.2

15-1580

430-3740

15-220

430-2670

70

40

80

90

70

20

0.47

0.34

0.49

0.38

0.58

95

95

95

95

100

60

55

20

55

70

0.96-1.15

1.02-1.15

1.02-1.60

0.80-0.90

0.83-1.15

71-75

72-82

5&75

72-75

71-73

3.444.0

3.9-4.3

3.5-5.1

2.7-3.8

2.8-3.2

1 .5

-1.

9 2.0-2.7

2.6-2.8

1.5-2.2

1.0-1.5

4.5

4.7

4.1-7.0

3.8-7.0

4.2

7 .O-7.2

6.4-7.0

5.3-7.1

6.5-7.1

6.6-7.0

7-165

92-4090

1560400

0-16

10

43-3300

7-150

92-2370

2570-3350

36-490

0-340

70

50

40

60

75

0.87-1.25

0.42

66-8 1

2.8-4.0

1 .2-1.6

4.5

5.6-6.9 1750-5460

3930-5350

75

95

80

0.90-1.12

0.45

72-75

3.2-4.3

2.1-

2.9

6.9

7.1-7.3

22-1430

22-1 55

50

95

60

a W

aste

mat

eria

ls a

re c

lass

ed u

nder

gro

ups 1,

2 o

r 3

on a

ccou

nt o

f th

eir

carb

ohyd

rate

cont

ent.

See

text

. * M

axim

um a

nd m

inim

um v

alue

s ar

e gi

ven,

exc

ept f

or th

ose

of b

ioga

s pr

oduc

tion,

bei

ng a

vera

ge v

alue

s.

c V

FA a

re g

iven

in

two

colu

mns

; in

the

first

col

umn

VF

A d

urin

g th

e w

hole

exp

erim

ent,

in th

e se

cond

vol

ume

VFA

aft

er a

n as

sum

ed a

dapt

atio

n pe

riod

of

50 d

ays.

Biogas from fruit and vegetable waste a21

To facilitate the discussion of the results the waste samples have been divided into three groups. The division is based on the carbohydrate content in the dry weight of the materials, because the carbohydrate content may affect digester functioning, as stated in the introduction.

Spinach-waste and asparagus-peels can be classed under group 1, the carbohydrate content in the dry weight being 0.05 and 0.06 %. The larger group 2 comprises apple-pulp, apple-slurry, carrot- waste, French bean-waste and strawberry-slurry with a carbohydrate content varying between 0.21 and 0.29%. Group 3 consists of green pea slurry only, having a carbohydrate content in the dry weight of 0.44 %.

3.2. Waste samples with 0.05 to 0.06% carbohydrate in dry weight (group 1) Table 2 shows that spinach-waste was well digested. Good biogas production with a high CH4 content in the biogas was obtained, while considerable reductions in VS, carbohydrate and crude fibre were produced. After approximately 30 days VFA decrease to low concentrations. In Table 3

Table 3. Volatile fatty acids (VFA) found during anaerobic digestion of spinach-waste and asparagus-peels

Acetic Propionic i-Butyric n-Butyric i-Valeric n-Valeric Days acid acid acid acid acid acid Total VFA

Spinach waste 17 89 24 57 31 82 38 120 46 85 52 20 59 15 66 I1 73 11 94 45 Asparagus-peels 15 1800 36 198 43 77 50 70 57 41 64 40 71 68 18 91 85 110 92 67 99 86

1000 1270 905 120

18 2

210 105

A

-

1200 1800 21 80 1360 1510 2090 1980 2040 1560 830 265

155 170

71 96 94

195 265 175 120

31 -

360 22 3

4 3 1 2 7 1

-

280 32 6 2 1

225 265 295

3 3 60 290 305 110 86 35 27

29

95 110 77

-

79 12 15 30 28 23

6 5 7 3

50

1580 1530 1090 350 180 22 15

220 120 56

3740 2450 2660 1660 2270 2620 2480 2250 1770 935 430

-=Not detected. All values are in mg litre-'.

the course of the different VFA during the experiment is given. With asparagus-peels less efficient digestion was obtained. Biogas production was the lowest of all experiments and small decomposi- tion of VS and crude fibre was found, which could be due to the fact that the peels have a woody structure. The crude fibre will consist of strongly linked polysaccharides with much lignin, and be difficult to degrade. High concentrations of VFA-mainly propionic acid-were present in the digester contents (Table 3), indicative of an unbalanced digestion. For this reason feeding was stopped for 1 or 2 days after 50, 70 and 75 days. After 85 days the average production increased from 0.25 to 0.41 m3 kg-1 VS day-1.

Obviously, a proper flora had developed at the end of the experiment. Whether this was caused by feed interruption or by the fact that asparagus-peels actually needed a longer adaptation period cannot be deduced from the results.

828 W. Knol et al.

3.3. Waste samples with 0.21 to 0.29% carbohydrate in dry weight (group 2) In some of the experiments with this group a pH decreasing effect is observed, which can be attributed to excess acid production from carbohydrates.

French bean waste and carrot-waste were very well digested, as expressed in the reduced figures on VS, carbohydrate and crude fibre, as well as in biogas production and VFA-amounts. Figure 3 shows the biogas production from French bean waste, as example of regular gas production. The somewhat lower pH (average 6.8) during digestion of carrot-waste did not affect stable digestion in this case.

During the first 2 months of the experiment with strawberry slurry pH was rather low (6.3 to 6.5) and VFA were high, varying from 2370 to 4090 mg litre-1. Then, pH increased spontaneously to 7.0 and VFA decreased to 92 mg litre-l in the end, while a slight improvement in gas production was noticed. Obviously, strawberry-waste needed a longer adaptation time.

Anaerobic digestion of apple-pulp showed the expected pattern of carbohydrate-rich substrates. During the first weeks of this experiment pH decreased from 7.0 to 5.3. Alkali was added several times, without interruption of feeding. Then a rapid increase in biogas production was observeu and high biogas yields continued for several weeks. When pH decreased once more, i.e. to 6.3, an attempt was made to improve the situation by alkali addition and feed interruption, but this timc without success and biogas production almost stopped. Finally, after adding new digested sewago sludge and also neutralising the influent, a slight improvement was noticed. Figure 4 shows the

$ 1 I I I I m

20 40 60 80

Time (days)

Figure 3. Biogas production from French bean-waste.

Time (days)

Figure 4. Biogas production from apple-pulp. (- - -) Irregular feeding.

Biogas from fruit and vegetable waste 829

irregular gas production pattern of this experiment. However, the average biogas production rate of 0.49 litre g-1 VS day-1 (Table 2) is one of the highest. This indicates that the average gas produc- tion rate alone can be misleading and is not a measure of stable digestion.

It might have been expected that digestion of apple-slurry would give similar results. However, the course of this experiment was different. Although the pH-range was rather low (6.7 to 7.1), the amount of VFA (varying from 36 to 490 mg litre-1) was not high and biogas production was fairly stable. Only after 10 weeks did the pH decrease to 6.5. In this case feed interruption and neutralisa- tion of the influent was applied with success. That the irregular course of the experiment with apple- pulp compared with apple-slurry was probably caused by the high loading rate of the apple-pulp (1.02 to 1.60 kg VS m-3 day-1) is suggested by the results of the experiment with carrot waste. In this experiment the loading rate was lowest, amounting to 0.80 to 0.90 kg VS m-3 day-l, while the highest biogas yield was obtained during a stable digestion. The crude protein contents in the dry weight of apple-pulp, apple-slurry and carrot-waste (Table 1) are remarkably low. Therefore, a neutralising capacity by accumulated ammonia cannot be expected. This might explain the trouble with apple-pulp and apple-slurry but is in contrast with the results of the carrot-waste experiment. Possibly, stable anaerobic digestion of waste materials with unfavourable proportions of carbo- hydrates and nitrogenous substrates can be obtained after all by using low loadings, though probably only until adaptation is reached.

3.4. Waste sample with 0.44% carbohydrate in dry weight (group 3) In the first weeks of the experiment with green pea slurry-the only waste of this group-biogas production was low, i.e. average 0.17 m3 kg-1 VS day-1, pH decreasing to 5.6. After pH had been set at 6.9 by alkali addition, biogas production suddenly increased to average 0.42 m3 kg-1 VS day-1. From that time the pH remained constant. Obviously, bacterial activity was suppressed by the low pH. Pohland et a1.8 described a similar effect in digester experiments with sewage sludge. In spite of high VFA-levels during the whole experiment, biogas yield, CH4 content, or extent of decomposition were not lower than those of other satisfactory experiments. One may assume that the addition of alkali as well as the higher content of crude protein in the dry weight (Table 1) effected the rather stable digestion.

3.5. Application of mixed substrates An additional experiment was carried out to check the possibility of using mixed substrates as a method of preventing suboptimal anaerobic digestion of carbohydrate-rich substrates. A mixture of apple-pulp and pig manure in a total solid ratio of 1 : 1 was tested. In previous investigations on anaerobic digestion of pig manure4, l2 the authors found no easily digestible carbohydrates, but much nitrogen. The present results (Table 2) show that satisfactory digestion of the mixed substrates can be effected. A more stable digestion was obtained than in the case of apple-pulp alone. The pH did not fall below 7.1 and the gas production was found to be regular. Laura et al.13 ascertained that digestion of a mixture of sugar cane and cow dung resulted in a better biogas yield with a higher CH4 amount if an alkaline agent like urea or CaC03 was added. Significantly higher CH4 amounts in the biogas were not found in the author’s present experiment. Obviously, however, the better results with regard to mixed substrates obtained by Laura and by the authors must be attributed to the fact that addition of urea, CaC03 and manure respectively resulted in maintaining a neutral pH, thus creating favourable conditions for the activity and growth of methanogenic bacteria.

3.6. Preliminary experiments on removal of residual solids after anaerobic digestion The COD-values of the digested sludges before further treatment were not determined but could be calculated to amount to 10 000 to 20 000 mg litre-1, based on the VS-content of the final sludges. The results presented in Table 4 show that removal of residual solids from the digested sludges by flocculation with a polyelectrolyte leads to lower COD-values in the effluents than those obtained by centrifugation. One might assume that additional purification by conventional aerobic treatment of the effluents obtained will cause no problems on account of the low COD values.

830 W. Knol et ui.

Table 4. Chemical oxygen demand (COD) of effluent after removal of solids

COD in mg litre-' after

Waste sample Centrifugation Flocculation

Spinach-waste French bean-waste Apple-pulp Carrot-waste Asparagus-peels Apple-slurry Strawberry-slurry Green pea-slurry Pig manure+ apple-pulp

3880 21 50

12500 2300 5470 2120 850

1800 2400 1

530 320

625 -

- - 220 810

1030

- =Not estimated. For methods, see text.

4. Conclusions

The results of this study show that anaerobic digestion would be a suitable process for the treatment of waste materials from the fruit and vegetable industry. Although the composition of certain carbohydrate-rich substrates tend to suboptimal digestion, alkali addition, adjustment of loading rate and use of mixed substrates proved to be suitable correction measures. The residual solids in the sludges after digestion are almost completely removed by flocculation with polyelectrolytes, leaving a liquid that is not likely to cause problems in subsequent conventional aerobic purification.

However, several aspects need further investigation. Longer experimental times would ensure long-term stability of digestion of the substrates as a result of complete adaptation of the digester flora. Further tests will probably show that high loading rates and shorter retention times can be used when stabilised digestion has actually been obtained. In addition, the composition of the waste from a particular factory varies with the season, depending on the harvest of crops and fruits. Assuming that an anaerobic digester would be in operation all through the production season, the eventual effects of successive addition of varying amounts and kinds of waste on digester functioning have to be investigated. Furthermore, studies on digestibility, acceptance and nutritional value will be needed to prove whether or not the separated residual solids can be used as a feed additive.

Acknowledgement The authors are grateful to Veluco Conservenfabrieken BV for their contribution to this study.

I . 2, 3. 4. 5 .

6. 7. 8. 9.

10. 11.

12. 13.

References

Economic and Social Commission for Asia and the Pacific (ESCAP) Newsletter, Bangkok, Thailand, 1976. Tietjen, C.; Dt . Landwirt. Presse 1954, 77 (22), 31 1. Hobson, P. N.; Shaw, B. G. Wat. Res. 1973, 7, 437. Waart, J. de; Most, M. M. van der; Knol, W. Voedingsrniddelentechnol. 1976, 9, No. 35. Catania, P. J. Food Fuel Fertilizer, Canadian Plain Proceedings. 2. Symp. Uses of Agricultural Wastes, Saskat- chewan, 1974. Hobson, P. N. ; Bousfield, S . ; Summers, R. Crit. Rev. Envir. Control 1974, 4 (2), 131. Velsen, A. F. M. van. Ned. J. agric. Sci. 1977, 25, 151. Pohland, F. G.; Ghosh, S. Biotechnol. Bioengng. Symp. no. 2, 1971, 85. Kamer, J. H. van de. Chem. Weekbl. 1941,38, 286. Methods of the Landbouwproefstation, Maastricht, The Netherlands, No. RC-2. Determination of Chemical Oxygen Demand, Netherlands Institute for Nornialisation, Test methods for Waste Water, 1972, NEN 3235, 5.3. Waart. J. de; Most, M. M. van der; Knol, W. Fifth International Fernientation Symposium Berlin, 1976. Laura, R. D.; Idnani, M. A. J. Sci. Fd Agric. 1971, 22, 164.