Document Outline

10.1016/j.biombioe.2012.02.018

0961-9534/$

doi:

industrial scale can lead to a limited residence time of the
material in the digester (after which energy efficiency starts to
decline); this can produce a digestate that is not completely
exhausted in terms of easily-degradable organic compounds.
This may generate problems during storage (odour emission,
production of toxic compounds, pathogen re-growth and
phytotoxicity) and cause unfavourable impacts on the soil-
plant system, thus limiting the potential fertilising value of
digestate when unstable materials are added to soil

[7,8]

These can cause an extensive range of deleterious effects on
crops, such as prevention or delay of seed germination, plant
death or marked reductions in growth, which must be
addressed. Bioassays incorporating plant material are simple,
reproducible, rapid and economic tests that identify phyto-
toxic materials.

On the other hand, the agricultural use of digestates should

be considered as a recovery process instead of a simple
disposal method. But, the market demand for digestates as
soil conditioners or fertilisers depends on the compliance with
quality standards, as regulated by European and national
guidelines

[9,10]

as well as by quality protocols for digestate

application in Germany

b i o m a s s a n d b i o e n e r g y 4 0 ( 2 0 1 2 ) 1 8 1

and the United Kingdom

[12]

. In

Spain, no specific, applicable standards for digestates, which
would favour their use in agriculture, have been developed -
which represents a major barrier for the development of
anaerobic digestion.

In this study, the main agrochemical characteristics of

a number of digestates, including phytotoxicity, have been
determined, by analysing twelve samples from several,
representative co-digestion processes carried out in Spain,
and then compared to the quality criteria established in order
to assess their potential use as fertilisers in agricultural
systems.

Materials and methods

2.1.

Digestate origin and sampling

After the anaerobic co-digestion of twelve mixtures incorpo-
rating pig (PS) or cattle (CS) slurries as major components, the
corresponding,

representative

digestate

samples

were

collected for analysis. According to the origin of the digestate,
the samples were classified into the following four groups
(on a fresh mass basis):

- From PS plus energy crop residues (PS-EC):

þ9.6% rape

residue (PS-EC1),

þ4.5% sunflower residue (PS-EC2) and

þ5.4% corn residue (PS-EC3).

- From PS plus animal by-products (PS-AB):

þ0.6% pasteurised

slaughterhouse

residues

(PS-AB1),

þ3.8% pasteurised

slaughterhouse residues (PS-AB2) and

þ1.0% sludge from

a slaughterhouse wastewater treatment plant

þ6.5% bio-

diesel wastewaters (PS-AB3).

- From CS plus glycerine (CS-G):

þ4% glycerine (CS-G1) and

þ6% glycerine (CS-G2 and CS-G3).

- From CS plus agroindustrial residues (CS-AW):

þ5% orange

peel residue (CS-AW1),

þ10% orange peel residue (CS-AW2)

and

þ4.3% cattle manure þ11.6% maize-oat silage

(CS-AW3).

As shown in

Table 1

, these samples included two diges-

tates derived from industrial processes (PS-AB3 and CS-AW3),
while the rest came from laboratory-scale experiments run in
order to optimise biogas production by using co-substrates
such as glycerine or orange peel residues (for CS) and
slaughterhouse or energy crop residues (for PS). The diges-
tates were sampled directly after anaerobic digestion
processes (without post-treatments), stored at a temper-
ature

< 4

C and processed quickly to prevent chemical or

biological alterations.

2.2.

Analytical methods for digestate characterisation

The following parameters were determined in the fresh
digestate samples: electrical conductivity (EC) and pH; dry
matter content (DM) after drying the digestate sample at
105

C for 24 h; the volatile solids, which reflect the OM

content, by loss on ignition at 500

C for 24 h. The total organic

carbon (TOC) and total nitrogen (TN) were measured in freeze-
dried samples, by automatic microanalysis (EuroVector
elemental analyser). The dissolved organic carbon (DOC) was
measured after filtration of the fresh digestate (through
a synthetic filter with a pore diameter of 0.45

mm), using an

automatic analyser for liquid samples (TOC-V CSN Analyzer,
Shimadzu). Ammonium was determined by steam-distillation
from alkalised fresh samples with MgO, and chloride (Cl) by
potentiometry with silver nitrate. After HNO

/HClO

(2:1 v/v)

digestion, the following elements were determined by induc-
tively coupled plasma-optical emission spectrometry (ICP-
OES, Thermo Elemental Co. Iris Intrepid II XDL): P, K, S, Na, Ca,
Mg, Fe, Cu, Mn, Zn, B, Pb, Cd, Cr and Ni. The 5 d biochemical
oxygen demand (BOD

) was determined, with respirometric

Oxitop

IS 6 equipment (WTW, Germany), based on pressure

measurement, which is automatically computed as an oxygen
value with units of mg L

. In the Oxitop

equipment, the

cumulative oxygen consumption was recorded each day
during a period of 5 d. Salmonella spp. and Escherichia coli were
determined according to the modified method of the USEPA

Table 1

e The main technical aspects of the anaerobic co-

digestion processes which produced the 12 digestates
studied.

Digestate

Operation

Scale

Temperature (

HRT (d)

PS-EC1

CONT

PS-EC2

CONT

PS-EC3

CONT

PS-AB1

CONT

PS-AB2

CONT

PS-AB3

CONT

CS-G1

DISCONT

CS-G2

DISCONT

CS-G3

CONT

CS-AW1

DISCONT

CS-AW2

DISCONT

CS-AW3

CONT

38.5

CONT: continuous operation, DISCONT: discontinuous operation,
LS: laboratory-scale (2 L to 6 L digester), IS: industrial-scale (3000 m

digester), and HRT: hydraulic residence time.

182

[13]

and the most probable number (MPN) method

[14]

respectively.

2.3.

Potential phytotoxicity

To obtain homogeneous samples, the 12 digestate samples
were sieved (0.25 mm mesh-size) and then centrifuged. The
supernatants thus obtained were shaken and tested in
bioassays involving seed germination and seedling growth,
with both cress (Lepidium sativum ‘Alenois’) and lettuce (Lac-
tuca sativa ‘Bionda degli Ortelani’).

2.3.1.

Seed germination bioassays

In these bioassays, the Petri dishes used (8.5 cm diameter)
each contained 2 filter papers (base and cover), which were
moistened with 1 mL of the digestate. Digestate samples were
tested at concentrations of 100% (pure), 20%, 10%, 1% and 0.1%
(volume fraction), which were prepared with distilled water.
Ten seeds per dish were sown and each experimental treat-
ment was replicated 5 times. The dishes were transferred to
a germination chamber under controlled conditions of
temperature (17

C and 23

C for lettuce and cress, respec-

tively) and darkness for 3 d (cress) or 5 d (lettuce). After this
period, the number of germinated seeds was counted, the
radicle lengths of these seeds measured and the germination
index (GI) calculated as a percentage of the control (distilled
water), according to Zucconi et al.

[15]

2.3.2.

Plant growth bioassays

Plastic cell trays (10 cells/tray and 33 mL/cell), with a drainage
hole in the bottom of each cell, were filled with horticultural
perlite (1 mm

e2 mm in diameter) and used for these bioas-

says. Trays (in triplicate for each treatment tested) were
placed in a holder vessel (18 cm

6.5 cm 5 cm) - containing

400 mL of distilled water - for 24 h, until the perlite was
saturated by capillarity. Then, 2 seeds of cress or lettuce per
cell were sown and maintained under the same wetting
conditions for 9 d or 13 d, respectively, until seed germination
and seedling emergence occurred (and only 1 seedling/cell
was left). After this period, the water in the holder vessel was
replaced completely by 200 mL (equivalent to the perlite
container volume) of the digestate concentrations to be
examined: 20%, 10%, 1% and 0.1%, keeping trays without
digestate addition as a control. Pure digestate was not tested
for plant growth bioassays since GI was 0% for all the digestate
samples. In these bioassays, and due to the limited availability
of adequate volumes of the samples obtained from the raw
digestates, only 7 co-digestion mixtures were studied: PS-EC1,
PS-EC2, PS-EC3, PS-AB2, PS-AB3, CS-G3 and CS-AW3. Imme-
diately following the sowing, the trays and their holder vessels
were transferred to a growth chamber with controlled condi-
tions: 17

C (lettuce) and 23

C (cress) during the first

week, followed by 19

C (lettuce) and 21

C (cress)

during the remaining 3 weeks. During the bioassay, the daily
photoperiod was 16 h at a photosynthetic irradiance of
250

mmol m

provided by fluorescent tubes (Sylvania Cool

White VHO). Solution losses from the holder vessels (by plant
uptake

þ evaporation) were restored periodically using

distilled water. From the second week, a liquid, mineral
compound fertiliser (Hesi Coco

, The Netherlands; N-P

O 24:21:27, at 3 or 1.5 mL per L distilled water for cress and

lettuce, respectively) was added (in 3 successive applications)
to the digestate solutions to maintain seedling growth. At the
end of the experiment (4 weeks from sowing), 5 seedlings per
tray were harvested and their total dry mass (shoot

þ roots)

determined after drying at 105

2.4.

Statistical analyses

Basic statistical analyses of data, correlation coefficients and
regression models were produced using the SPSS 18.0 program
for Windows. The normal distribution of the data was checked
by the Shapiro-Wilk’s test, and failed data were adjusted to
a normal distribution through logarithmic transformation.

Results and discussion

3.1.

Physico-chemical characterisation

All digestate samples had low dry matter contents (

these

being

classified

liquid

products

(DM

mass

fraction

15%

). They were characterised by slightly-

alkaline pH values (

> 7.5), except in the case of CS-G1 and

CS-G3. The trend is for the pH to increase as anaerobic
digestion progresses, due to volatile fatty acid degradation
and ammonia production

[16]

. In addition, the pH is also

conditioned by the addition of strong bases or carbonates to
control pH and the buffer capacity of the system during
anaerobic digestion

[2]

. Generally, the pH of digestates derived

from animal slurries is in the alkaline range, around 8

[5,17]

The highest EC values were found in the PS digestates and

the CS-AW3 sample (

> 20 dS m

), which also had the

highest concentrations of Cl, while CS-G1, CS-G2 and PS-AB2
exhibited the highest Na concentration (

Table 3

). Therefore,

special care must be taken since excessive doses or continued
applications of digestates rich in Cl and Na to soils could lead
to an increase in soil salinity and inhibit plant growth.

Regarding the plant nutrient content and hence the fertil-

iser value, the most-significant property of the digestates was
that a large proportion of the N occurred as inorganic forms
(

), representing NH

-N more than 70% of the TN (on

a mass basis) in the PS mixtures and 39%

e61% in mixtures

with CS. This form of N can be easily lost by ammonia vola-
tilisation during storage and land spreading due to the alka-
line pH of the digestates

[18]

. In addition, NH

-N is nitrified

rapidly in soil under favourable conditions, this form being
highly available to crops but also subjected to leaching
through the soil profile, which may result in groundwater
pollution. Therefore, storage and land spreading operations
with digestates must be carefully controlled to avoid negative
environmental impacts.

The TOC/TN ratio was highest for CS-AW3, CS-G1, CS-G2

and CS-G3 (

> 8), clearly higher than the mean value of 4

reported by Siebert et al.

b i o m a s s a n d b i o e n e r g y 4 0 ( 2 0 1 2 ) 1 8 1

for digestates from different

sources. Unbalanced C/N ratios in digestates can limit their
use in agriculture, as an excess of degradable organic
substrate for microorganisms with respect to N leads to
immobilisation of this nutrient in the microbial biomass and

183

to crop deficiency, while excess N causes losses through
ammonia volatilisation, nitrate leaching, etc.

[19]

The nutrient concentrations of the digested materials were

within the range found by other authors

[5,17]

. As a whole, the

major elements were N and K, followed by P and Ca (

Tables 2

and 3

). PS-EC1, PS-EC2 and PS-EC3 together with PS-AB2 and

CS-AW3 showed the highest concentrations of P, K, Ca and
Mg, whereas the S concentration was generally higher in PS
samples than in those from CS mixtures (

Table 3

). From the

standpoint of quality criteria for digested materials

b i o m a s s a n d b i o e n e r g y 4 0 ( 2 0 1 2 ) 1 8 1

, no

limits have been established for their nutrient contents,
although a number of parameters should be declared,

Table 3

e The concentrations (mg L

) of elements in the 12 digestate samples (mean value, expressed on a fresh mass

basis). CV: coefficient of variation.

Digestate

Pig slurry
PS-EC1

401

1993

633

666

1574

155

22.9

49.2

8.4

3.2

PS-EC2

367

1970

721

699

1495

143

23.4

45.9

7.0

3.2

PS-EC3

417

1863

698

697

1613

224

31.0

62.5

7.8

2.7

PS-AB1

219

799

324

696

1598

11.4

84.4

14.3

2.2

PS-AB2

302

828

365

995

2120

15.4

140.2

15.1

3.1

PS-AB3

680

218

726

1993

2.9

34.7

4.0

2.3

Median

384

1345

499

698

1606

103

19.1

55.8

8.1

2.9

(CV)

(39.3)

(59.4)

(55.4)

(16.5)

(14.9)

(69.9)

(56.1)

(55.6)

(46.2)

(16.5)

Cattle slurry
CS-G1

180

1550

267

1164

685

117

13.7

18.1

10.8

1.8

CS-G2

265

1753

333

1842

665

165

17.1

28.3

13.0

4.8

CS-G3

192

448

3.2

10.6

1.4

1.3

CS-AW1

113

1008

257

276

366

6.0

7.7

2.8

1.7

CS-AW2

131

1035

314

303

452

6.9

8.0

3.1

3.5

CS-AW3

457

4026

698

746

1418

301

27.5

27.7

10.8

3.4

Median

155

1293

290

525

558

106

10.3

14.4

6.9

2.6

(CV)

(73.2)

(82.1)

(62.8)

(91.6)

(57.6)

(80.4)

(72.8)

(56.8)

(72.8)

(49.4)

ANOVA

NS: not significant. * and **: significant at probability level P

< 0.05 and P < 0.01, respectively.

a considering the following grouped mixtures: pig slurry

þ energy crop residues (PS-EC1, -2 and -3), pig slurry þ animal by-product (PS-AB1, -2

and -3), cattle slurry

þ glyceryne (CS-G1, -2 and -3) and cattle slurry þ agroindustrial residues (CS-AW1, -2 and -3).

Table 2

e The main characteristics and composition of the 12 digestate samples (mean value, expressed on a fresh mass

basis). EC: electrical conductivity, DM: dry matter, TOC: total organic carbon, DOC: dissolved organic carbon, BOD

: 5

d biochemical oxygen demand, TN: total nitrogen and CV: coefficient of variation.

Digestate

(dS m

)

(g L

)

TOC

(g L

)

DOC

(g L

)

BOD

(g L

)

(g L

)

-N

(g L

)

(g L

)

(g L

)

TOC/TN

Pig slurry
PS-EC1

7.82

26.0

43.9

14.7

4.3

6.5

3.6

2.9

1.1

3.1

4.1

PS-EC2

7.92

24.1

38.3

12.2

3.7

4.0

3.5

2.6

1.1

3.1

3.5

PS-EC3

7.90

23.3

28.3

8.3

3.7

4.7

3.4

2.7

1.2

2.7

2.4

PS-AB1

7.95

21.1

21.0

5.8

1.2

2.3

2.9

2.2

0.5

2.2

2.0

PS-AB2

7.86

30.8

29.5

8.4

3.5

6.2

4.9

3.4

0.8

3.1

1.7

PS-AB3

8.20

30.3

19.5

5.9

2.4

2.2

4.0

3.5

0.2

2.0

1.5

Median

7.91

25.0

28.9

8.4

3.6

4.4

3.6

2.8

0.9

2.9

2.2

(CV)

(1.7)

(15.2)

(31.8)

(38.6)

(35.6)

(43.7)

(18.2)

(16.9)

(50.9)

(18.2)

(40.9)

Cattle slurry
CS-G1

5.64

14.5

38.3

17.8

10.6

37.5

1.9

1.0

0.5

1.8

9.5

CS-G2

7.35

11.7

72.9

42.8

27.6

52.5

2.3

0.9

0.4

1.6

18.5

CS-G3

6.35

5.2

17.6

8.3

8.2

10.6

0.6

0.4

0.1

0.8

13.6

CS-AW1

7.86

8.7

24.4

9.4

1.2

1.3

1.4

0.8

0.2

1.1

6.6

CS-AW2

7.90

10.0

17.6

5.8

1.0

1.2

1.5

0.9

0.2

1.2

3.8

CS-AW3

7.50

25.7

90.1

33.7

5.4

5.9

4.0

2.4

0.8

3.1

8.5

Median

7.42

10.9

31.4

13.6

6.8

8.3

1.7

0.9

0.3

1.4

9.0

(CV)

(12.8)

(56.2)

(71.0)

(77.5)

(109.6)

(118.9)

(58.0)

(67.1)

(71.9)

(49.8)

(52.2)

ANOVA

***

NS: not significant. *, ** and ***: significant at probability level P

< 0.05, P < 0.01 and P < 0.001, respectively.

a considering the following grouped mixtures: pig slurry

þ energy crop residues (PS-EC1, -2 and -3), pig slurry þ animal by-product (PS-AB1, -2

and -3), cattle slurry

þ glyceryne (CS-G1, -2 and -3) and cattle slurry þ agroindustrial residues (CS-AW1, -2 and -3).

184

including: bulk density, DM, OM, pH, salt content, TN, P

O, CaO, MgO, S, NH

-N, NO

-N, micronutrients, Cl and Na.

In the current Spanish legislation for fertilisers

[10,20]

minimum limits for N, P and K contents are specified for
marketable

liquid

organo-mineral

fertilisers.

However,

according to the legal requirements, digestates cannot be
considered balanced fertiliser products and they must be
complemented with other inorganic fertilisers. Another
significant limitation is the digestate TOC content, generally
lower than the required concentration (

> 4% on a mass basis).

Therefore, the inclusion of a new fertiliser category in the
current Spanish legislation, specifically considering digestate
composition, may be advisable.

The most-abundant micronutrient in the digestates was Fe

(

Table 3

), CS-AW3 having the highest value due to the use of Fe

salts during its anaerobic digestion. High concentrations of Cu
and Zn were found in the digestates due to the use of pig and
cattle slurry as the major co-digestion substrates, since these
two elements are frequently used as additives - to prevent pig
and cattle diseases and to stimulate livestock growth. The Cu
and Zn concentrations were especially high in digestates
arising from PS mixtures (on a dry mass basis): (76

e682)

mg kg

, with a median value of 173 mg kg

for Cu, and

(222

e4757) mg kg

with a median value of 446 mg kg

for Zn.

Some values were higher than the established limits

, so

special care must be taken with respect to Cu and Zn
concentrations, especially for digestate produced from PS.

With respect to heavy metals, the concentrations on a dry

mass basis in some digestate samples for Ni (n

¼ 7, 6 mg kg

to 36 mg kg

), Pb (n

¼ 7, 11 mg kg

to 46 mg kg

), Cr (n

¼ 2,

3 mg kg

and 55 mg kg

) and Cd (n

¼ 6, 0.1 mg kg

1.0 mg kg

) were lower than both the limits established by the

cited Spanish legislation and the quality protocols for the
production and use of digestates

. The concentrations of

heavy metals found in this study were similar to those
reported previously for digested materials from substrates of
different origin

[6,11,21]

Our results reveal the influence of the raw materials on the

digestate characteristics. The coefficients of variation (

Tables

2 and 3

) of most of the parameters analysed reveal, in general,

a high degree of variability. Thus, digestates should be ana-
lysed fully before use. However, highly-significant correla-
tions between the DM and parameters such as TOC, Ca and Fe
were found, while TN, NH

-N, K, S, Zn and Cl were correlated

significantly with EC (

Table 4

), indicating their preferential

association with the solid or liquid digestate fraction,
respectively.

The use of regression equations based on highly-

significant correlations between EC and ionic species (NH

, etc.) resulted in a reliable tool for providing a quick esti-

mation of nutrient contents in animal slurries

[22,23]

. As

a result, the EC and DM of digested materials can be used
successfully to estimate other valuable and useful parameters
which are much-more difficult and time-consuming to
analyse (

Table 5

) and which, together with BOD or DOC data,

characterise the quality of digested materials, as discussed in
the following section.

3.2.

Digestate biodegradability

The digestate samples showed a great variability in their
degree of stability, according to the BOD

values (

These results indicate the great influence on digestate
stability not only of the raw materials used as co-digestion
substrates but also of the co-digestion process, as shown by
the clear differences in the organic load and its biodegrad-
ability among digestate samples from cattle slurry

þ glycerine

mixtures (CS-G). The CS-G digestates showed BOD

values

that were clearly higher than those of the other digestates
tested (

b i o m a s s a n d b i o e n e r g y 4 0 ( 2 0 1 2 ) 1 8 1

), indicating that they had the lowest degree of

microbial stability, associated with the presence of easily-
degradable compounds. The industrial digestate sample
(CS-AW3) had the highest DM content of all the digestates
produced from CS mixtures, but only 16% of the TOC occurred
as DOC, which led to a higher degree of stability (less biode-
gradable material) than for the digestates from CS-G mixtures.
This is related to the use of cattle manure and, especially,
silage (solid lignocellulosic materials) as co-substrates for

Table 4

e Correlation matrix between selected parameters related to the digestate composition (n [ 12).

Parameters

TOC

-N

TOC

0.946***

0.973***

-N

0.984***

0.951***

0.607*

0.644*

0.599*

0.875***

0.900***

0.837**

0.878***

0.856***

0.725**

0.612*

0.577*

0.584*

0.923***

0.780**

0.800**

0.789**

0.705*

0.655*

0.886***

0.752**

0.684*

0.932***

0.787**

0.806**

0.941***

0.859***

0.814**

0.836**

0.813**

0.649*

0.793**

0.968***

0.936***

0.979***

0.814**

0.888***

NS: not significant. *, ** and ***: significant at probability level P

< 0.05, 0.01 and 0.001, respectively.

EC: electrical conductivity (dS m

). DM: dry matter, TOC: total organic carbon, TN: total nitrogen. NH

-N, P, K, Ca, and Mg in g L

on a fresh

mass basis. Fe, Mn, Zn and Cl in mg L

on a fresh mass basis. The DM, TOC and Zn data were adjusted to a normal distribution (Shapiro-Wilk’s

test) through logarithmic transformation.

185

anaerobic digestion, which enriched the digestate in OM of
low biodegradability.

The CS-AW1 and CS-AW2 samples had the lowest BOD

values and percentages of DOC with respect to TOC (

<16%),

indicating a high stability degree. By contrast, digested
materials where PS was the main substrate showed an inter-
mediate behaviour, with BOD

values between 2 g L

and

7 g L

and DOC values between 1 g L

and 4 g L

, repre-

senting from 20% to 40% of the TOC in the digestates.

Therefore, differences in stability degree between diges-

tates were related directly to the concentration of DOC, readily
available for microorganisms. The BOD

was correlated

significantly with both TOC (r

¼ 0.722; P < 0.01) and, especially,

DOC (r

¼ 0.960; P < 0.001), which demonstrates the strong

influence of DOC on digestate biodegradability characteristics.
A significant regression equation was obtained for BOD

and

DOC (g L

fresh digestate mass): lgBOD

¼ 1.172lgDOC þ 0.057,

which allows cautious prediction of BOD

from DOC data.

Considering the BOD data after 24 h, which corresponds to

the period of maximum biological activity, and expressing
them as the average oxygen uptake rate (on an organic
matter basis), only the CS-AW3 sample satisfied the respira-
tion index (

<1 mg g

) proposed for stabilised biowastes

[9]

. On the other hand, using the criterion established by

Ponsa´ et al.

[24]

, the digestates can be classified as follows

(on a dry mass basis): 1) low biodegradability (

<2 mg g

PS-AB3, CS-AW1, CS-AW2 and CS-AW3 (1.4, 0.9, 1.0
and 0.8 mg g

, respectively); 2) moderate biodegradability

(2 mg g

e5 mg g

): PS-AB1, PS-AB2, PS-EC1, PS-EC2

and PS-EC3 (2.1, 3.2, 2.4, 2.2 and 2.6 mg g

, respectively);

and 3) high biodegradability (

> 5 mg g

): digestates from

CS-G mixtures (8.2, 20.0 and 11.8 mg g

for CS-G1, CS-G2

and CS-G3, respectively). The procedure for certifying the
quality of digestates in the United Kingdom

[12]

establishes

a dissolved chemical oxygen demand (COD) lower than
0.43 g g

OM as a stability requirement. In our study, the CS-G

digestates did not meet this requirement, assuming that DOC
contributes greatly to COD, thus underlining the high biode-
gradability of the CS-G digestates. In such cases, a longer
residence time during anaerobic co-digestion, further pro-
cessing or post-treatment stabilisation is recommendable
before digestate application to soil, in order to obtain the
maximum agricultural and environmental benefits.

Among the quality criteria defined for digestates, biode-

gradability or the degree of stability is the parameter for
which there is least agreement. As a result, different
threshold values have been established as quality criteria,
based on respiration indices

[9]

, volatile fatty acid contents

b i o m a s s a n d b i o e n e r g y 4 0 ( 2 0 1 2 ) 1 8 1

or chemical oxygen demand

[12]

. In our previous work

[25]

, digestate composition parameters such as DOC, BOD

and DOC/TN were related to the C and N dynamics in a soil
amended with digestates. In this case, a digestate suitable
for use as a fertiliser was defined based on DOC

< 1.5 g L

BOD

< 2.5 g L

and DOC/TN

< 1, while digestates showing

characteristics such as DOC

< 5.5 g L

, BOD

< 6.0 g L

and

DOC/TN

< 1.5 were established as less-appropriate for fer-

tilisation purposes, with a curing or maturation period being
required in order to increase digestate stability. Highly-
biodegradable

digested

materials,

such

the

CS-G

samples, were not suitable for agricultural use as they

caused a high CO

-C production and led to N-immobilisation

in soil, thus greatly limiting their N-fertilising potential in
the short-term.

3.3.

Evaluation of the potential phytotoxicity of digestates

and their hygiene

The effects of the 12 digestates and 5 digestate concentrations
studied (100% -pure-, 20%, 10%, 1% and 0.1%) on the GI,
expressed as a percentage of the control (distilled water), of
cress and lettuce seeds (germination bioassays) are shown in

Fig. 1

(a) and (b), respectively. Notable and highly-significant

effects of both digestates and concentrations were found.
Among the 12 digestates examined, the average GI ranged
from 34% (CS-G3) to 75% of the control (CS-AW1) for cress and
from 34% (CS-G3) to 82% (CS-AW2) for lettuce and, with regard
to the concentration tested, from 0% (100% digestate concen-
tration, both cress and lettuce) to 102% (1% digestate
concentration, cress) or 110% (1% digestate concentration,
lettuce). At a digestate concentration of 1%, various digestates,
particularly PS-AB1 and CS-AW3, exceeded the threshold
value for GI of 125% (of the control), in both the cress and
lettuce bioassays; thus, these two digestate solutions could be
considered to have plant nutrient or plant growth stimulant
attributes, as suggested by Emino and Warman

[26]

and

Moldes et al.

[27]

The GI of both cress and lettuce at the digestate concen-

trations of 20% and 10% were inversely correlated with the EC
values (P

< 0.01) of the digestates (an indicator of their salt

content) and also with the concentrations of TN (P

< 0.01),

-N (P

< 0.05), K (P < 0.01), S (P < 0.05), Zn (P < 0.01) and Cl

< 0.01). McLachlan et al.

[28]

obtained similar results when

they studied the potential phytotoxicity of digestates
produced from municipal solid wastes, high soluble salt
concentrations being the major reason for phytotoxicity. Tam
and Tiquia

[29]

related the phytotoxic effects of spent pig litter

to its salt, NH

-N, Cu and Zn contents.

For concentrations of 1% and, especially, 0.1%, TN, NH

-N,

K, S, Zn and Cl were not correlated significantly with the GI for
cress, but the latter was correlated positively with EC, TN,

Table 5

e The linear regression equations (y [ Ax D B)

calculated for selected parameters determined in the
digestates (

n [ 12).

lgTOC

lgDM

1.176***

0.708**

0.946***

3.807***

4.286**

0.856***

289.6**

318.4*

0.789**

0.141***

0.117

0.973***

-N

0.124***

0.431*

0.984***

0.084***

0.543

0.875***

lgZn

0.037**

0.764**

0.814**

0.070***

0.144

0.968***

NS: not significant, *, ** and ***: significant at P

< 0.05, 0.01 and 0.001,

respectively.
TOC: total organic carbon, TN: total nitrogen, DM: dry matter, NH

N, K, Ca, and Cl in g L

(fresh mass basis). Fe and Zn in mg L

(fresh mass basis). EC: electrical conductivity in dS m

b i o m a s s a n d b i o e n e r g y 4 0 ( 2 0 1 2 ) 1 8 1

186

Fig. 1

e The effects of digestates and concentrations on the germination index (GI, expressed as percentage of the control) of

cress (a) and lettuce (b) seeds. Each value is the mean of five replications ± the standard error of the mean. GI was 0% for all
the pure (100%) digestate samples studied.

Fig. 2

e The effects of digestates and concentrations on the biomass accumulation (expressed as total dry mass, shoot D roots,

in mg) of cress (a) and lettuce (b) seedlings. Each value is the mean of three replications ± the standard error of the mean. Control
(without digestate addition): CO.

187

-N and S for lettuce at 1% concentration (P

< 0.05). Indeed,

parameters related to digestate stability (DOC, DOC/TN and
BOD) correlated negatively with GI, especially for cress at both
concentrations of 1% and 0.1% (P

< 0.05). Several authors

[7,30]

related decreases in the phytotoxic effects of digestates to
reductions in the amount of easily-biodegradable organic
compounds.

The influence of digestates PS-EC1, PS-EC2, PS-EC3, PS-AB2,

PS-AB3, CS-G3 and CS-AW3, at the concentrations of 20%, 10%,
1% and 0.1%, and of the control (without digestate addition), on
biomass accumulation in cress and lettuce plants - expressed
as total dry mass (shoot

þ roots) per seedling - is presented in

Fig. 2

(a) and (b), respectively. Significant differences in seedling

dry mass among both digestates and concentrations were
found, the best results (on average) being obtained with PS-
AB3, PS-EC1, PS-EC2, PS-EC3 and CS-AW3 in cress and with
PS-EC1, PS-EC2 and PS-EC3 in lettuce; the concentrations of 1%
(cress and lettuce) or 0.1% (cress) yielded the greatest seedling
biomass. In addition, most of the best treatments indicated
above were as efficient as the untreated controls, or even more
so, with regard to seedling biomass accumulation. This indi-
cates - as do the seed germination results - plant nutrient,
growth stimulant or even phytohormone-like effects of these
digestate solutions.

Therefore, agricultural uses of stable digestates can be

conditioned by their phytotoxic effects during early growth
(germination), due mainly to salinity. Thus, digestate appli-
cation rates must consider the concentrations of Na and Cl,
and also heavy metals (especially Cu and Zn), in order to
avoid any risk of metal accumulation in soil as well as sali-
nisation or phytotoxic effects, already detected following
excessive application of animal manure and slurries

[31,32]

In order to avoid phytotoxicity, digestate application to soil
should be done well in advance of sowing, avoiding direct
contact with young plants or germinating seed. The
maximum benefit from the nutrients provided can be
obtained if digestate is applied together with the irrigation
water, acting as a dilutor.

Another significant biological aspect of digestate quality

is hygiene. In Spain, the RD 824/2005

[10]

states the

requirements for fertilisers prepared from certain organic
residues, which must not exceed the following maximum
levels of micro-organisms: Salmonella spp., absent in 25 g of
product, and E. coli,

< 1000 MPN g

fresh product. In the

present study, E. coli was detected in most digestate samples
at levels lower than 1000 MPN g

fresh digestate, but

Salmonella spp. was present in 25 g fresh digestate in some
samples (data not shown). This must be related to the fact
that most digestate samples were derived from mesophilic
processes (with the exception of CS-G3) without post-
treatment, where hygiene is not guaranteed. So, further
treatment for inactivation of pathogens is required, to avoid
health risks. Both the European legislation

[33

e35]

and

quality standards for certification of digestates

b i o m a s s a n d b i o e n e r g y 4 0 ( 2 0 1 2 ) 1 8 1

are

severe, focusing on three aspects in the case of anaerobic
digestion: process management (thermophilic temperature,
retention time, etc.), pre- or post-treatments (pasteurisation,
steaming, sterilisation, etc.) and end-product requirements
that ensure the sanitation of the digested materials accord-
ing to the source of the feedstocks.

Conclusions

The digestates had a high potential fertiliser value due to their
contents of N, P, K and micronutrients. However, their high
variability and unbalanced contents necessitate their analysis
before they can be integrated into fertilisation programmes.
Together with the sanitary quality of the digestates,
a minimum degree of stability of their OM is required to obtain
the maximum benefits of digestate recycling in agriculture,
which must be harmonised at an international level. There-
fore, digestate characterisation is an unavoidable task, to
determine the application rates, phytotoxicity risk and need
for a safety period, before sowing, or a stabilisation treatment,
before soil application.

Acknowledgements

This research was funded by the “Ministerio de Ciencia e
Innovacio´n, Plan Nacional I

þDþI 2008-2011” and EU through

FEDER Funds “Fondo Europeo de Desarrollo Regional, una
manera de hacer Europa”, in the framework of the project
“singular estrate´gico PROBIOGAS” (Refs.: PSS-120000-2008-58;
PSS-120000-2008-62). The authors thank all the research
groups involved in the project PROBIOGAS (

http://www.

probiogas.es

), especially the GIRO and AINIA Technological

Centres, the University of Oviedo, San Ramo´n Group and
Treatments of Juneda Society (Tracjusa), for providing the
digested materials used in this work. The authors also thank
Dr. D.J. Walker for the English revision.

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