Short communication

Pelleting is a successful method to eliminate the presence of
Clostridium spp. from the digestate of biogas plants

Andrea Pulvirenti

a

,

1

, Domenico Ronga

a

,

*

,

1

, Massimo Zaghi

b

, Anna Rita Tomasselli

c

,

Lorenzo Mannella

a

, Nicola Pecchioni

a

,

d

a

Department of Life Sciences, University of Modena and Reggio Emilia, Via Amendola, n. 2, 42122 Reggio Emilia, RE, Italy

b

CAT, Cooperativa Agroenergetica Territoriale, Via Fossa Faiella, n. 6/A, 42015 Correggio, RE, Italy

c

SCAM Spa, Via Strada Bellaria, n. 164, 41126 Modena, MO, Italy

d

CRA-CER Cereal Research Centre, CREA e Council for Agricultural Research and Economics, S.S. 673 km 25,200, 71122 Foggia, Italy

a r t i c l e i n f o

Article history:
Received 7 March 2015
Received in revised form
3 August 2015
Accepted 4 August 2015
Available online 26 August 2015

Keywords:
Clostridium spp.
Biogas
Digestate
Pellet
Parmigiano Reggiano
Dairy farming

a b s t r a c t

Biogas production is increasing as a sustainable energy supply, with digestate resulting as a by-product of
biogas plants. As a result, the high concentration of Clostridium spp. in digestate became a concern in
dairy farming areas. Clostridium spores can contaminate soils and crops when digestate is used as fer-
tilizer, causing a con

flictual cohabitation of biogas with traditional cheese productions. In order to solve

the problem, this study aimed to search for a technical solution enabling either a drastic reduction or the
elimination of the content of Clostridium spp. within digestate. Results showed a complete elimination of
Clostridium spp. in pelleted stored solid digestate; in addition, pelleting caused a reduction of pH and
water mass fraction in terms of fresh weight, and a concentration of mineral nutrients compared to
stored solid digestate. Pellet can represent a possible sustainable solution both in reducing potential risks
linked to the presence of Clostridium spp. in digestate and in improving the transportation and distri-
bution of high-value fertilizer. Hence, pelleting of solid digestate could offer a simple and ef

ficient

method to allow cohesistence of biogas plants and dairy farming.

© 2015 Elsevier Ltd. All rights reserved.

1. Introduction

Digestate is either a solid or a liquid by-product of biogas plants.

The physical state of digestate mainly depends on biogas produc-
tion technologies implemented. Nowadays farmers use digestate
either as a fertilizer

[1]

or as a soil amendment in current crop

management

[2,3]

. Noteworthy, farmers cultivating forage crops for

the production of aged hard cheeses such as Parmigiano Reggiano
have become seriously concerned about the use of digestate in the
field, due to its high concentration of Clostridium spp.

[4,5]

. High

content of Clostridium spp. in the ratio of cows could pose serious
issues to cheese manufacturers as some species, such as Clostridium
tyrobutyricum, Clostridium butyricum and Clostridium sporogenes
are the main cause of alteration during the aging phase of cheese

[6]

. A diffused distribution of digestate in

fields might potentially

spread these ubiquitous spore-forming bacteria from one farm to
another, causing soil and crop contamination. Such potential eco-
nomic risk related to digestate obtained from biogas plants origi-
nates from substrates that were treated in biogas plants

[4]

. Some

studies reported that pathogenic bacteria such as Salmonellae,
Clostridiae and Listeria might survive after anaerobic digestion

[7,8]

,

as well as viable bacteria can grow after the application of digestate
to croplands

[8,9]

. Therefore, further treatment of the digestate was

advised to obtain a more ef

ficient reduction of pathogens

[10]

. High

temperature treatments can reduce the amount of various bacteria
within digestate, but anaerobic biogas plants work within a tem-
perature range (35e50

C) that does not allow the sanitation of

biomass and

final digestate. In fact, spores of Clostridium spp. were

not inactivated at 35

C or 53

C

[11]

. Moreover, in order to reduce

the risk of insurgence of undesired bacteria, waste material should
be treated before its use as fertilizer or amendment. On the other
hand, digestate contains a relatively high proportion of mineral
nutrients, which grants digestate so remarkable fertilizing value
that it could replace inorganic fertilizers

[12]

. Indeed, digestate can

be used as a useful source for crop nutrition, since nutrients from

* Corresponding author.

E-mail addresses:

[e-mail]

(A. Pulvirenti),

domenico.

[e-mail]

(D. Ronga),

[e-mail]

(M. Zaghi),

annarita.

[e-mail]

(A.R. Tomasselli),

[e-mail]

(L. Mannella),

[e-mail]

(N. Pecchioni).

1

These authors contributed equally to this work.

Contents lists available at

ScienceDirect

Biomass and Bioenergy

j o u r n a l h o me p a g e :

h t t p : / / w w w . e l s e v i e r . c o m/ l o ca t e / b i o m b i o e

http://dx.doi.org/10.1016/j.biombioe.2015.08.008

0961-9534/

© 2015 Elsevier Ltd. All rights reserved.

Biomass and Bioenergy 81 (2015) 479e482

ingestates used in digesters remain in digestate after the digestion
process

[13e15]

. Hence, the main objective of this study was to

find

a technical solution to either eliminate, or at least reduce, the
content of Clostridium spp. in the solid digestate, so that it can be
safely used in dairy-farming, hard cheese-producing areas as fer-
tilizer or amendment.

2. Material and methods

2.1. Anaerobic digestion and sampling

The present study was performed using samples of raw mate-

rials routinely used as input in the digester (ingestate) and diges-
tate

collected

from

the

CAT

(Cooperativa

Agroenergetica

Territoriale) biogas plant, located in Correggio, Reggio Emilia, Italy
(44

45

0

N; 10

48

0

E, altitude 28 m a.s.l.). Ingestates used in digestion

were maize (Zea mais L.) silage (43%), triticale (X Triticosecale
Wittmack) silage (22%), cow slurry (27%), and grape stalks (of Vitis
vinifera L.) (8%). Ingestate proportions were calculated according to
their fresh weight. Each ingestate was stored in concrete slabs
under a plastic tarpaulin. At sampling, maize silage had eight
months of storage, triticale silage ten, cow slurry three and grape
stalks six. Ingestates were collected right before their introduction
in the digestion process. For each ingestate, 20 samples were
collected from the pile at the height of 1 m above ground, the
diameter of cross-section was 20 cm, and the horizontal distance
was at least 1 m between each sample. The 20 samples were mixed
to extract three representative samples of 1.2 kg, which were
placed in sterile plastic bags and maintained cold during trans-
portation (5

C) to the laboratory, where they arrived within 2 h.

After a complete biogas production cycle, CAT made available six

different types of digestate for this study: fresh unseparated
digestate, fresh liquid digestate, fresh solid digestate, stored liquid
digestate, stored solid digestate and pelleted stored solid digestate.
The fresh unseparated digestate (90% of water mass fraction) was
separated into liquid and solid (94 and 82% of water mass fraction
respectively) by means of a helical compressor separator; liquid
digestate was then stored in a closed, underground concrete tank,
while solid digestate was stored in a concrete slab in open air until a
water mass fraction of about 54% was reached. Pellets were pro-
duced by a PP300 Kompakt pellet press that converted stored solid
digestate, dried at 15% of water mass fraction using the thermal
heat produced by cogeneration of biogas plant, into pellets of 8 mm
in diameter and 10 mm in length with

final water mass fraction of

8%. Pellet temperature was measured immediately after produc-
tion, using the thermal resistance sensor Pt100 (Type GE-PT100-
DINB/SC, Geass, Turin, Italy); pellet samples were immediately
put in sterile bags for further analyses.

Different forms of digestate were collected at the end of the

digestion process in sterile plastic bottles; fresh samples were
sampled soon after their production, before and after separation,
while stored samples were collected after three months of storage.

For each material (digestates and pellets), three samples were

collected following the same procedure used for the sampling of
the ingestate. Two subsamples of 100 g derived from each 1.2 kg
elementary sample of ingestate, digestate and pellet were used to
detect total Clostridium spp.

2.2. Microbiological analyses

Microbiological analyses were performed on each ingestate

used in digestion to characterize the total initial microbial
contamination of Clostridium spp. Furthermore, the different forms
of digestates were collected both at the end of the process (i.e. the
“fresh” samples) and after storage (i.e. the “stored” samples) in

order to be analyzed and evaluated for

final total microbiological

hazards compared to total Clostridium spp.

2.2.1. Sampling preparation

Microbiological sampling was performed by taking 10 g of

product from the subsample in absolute sterility and placing it in
90 cm

3

of sterile physiological (saline) solution. Samples were ho-

mogenized with a Lab Blender Stomacher 400 (Type BA
7021Seward, London) and the resulting solution was subjected to a
thermal shock for 10 min at 80

C, as reported in published pro-

tocols

[16e18]

. This treatment was necessary to induce spores

germination, thus eliminating all other vegetative forms. Subse-
quently, samples were used to perform serial dilutions.

2.2.2. Bacterial growth conditions

One (1) cm

3

of the last dilution was inoculated in triplicate

sterile plates previously marked with the indication of the dilution.
Each plate contained the reinforced clostridial agar (RCA) (Oxoid
S.p.a., Milan, Italy), consisting of tryptose 10 g, meat extract 10 g,
yeast extract 10 g, dextrose 5 g, sodium chloride 5 g, soluble starch
1 g, cysteine hydrochloride 0.5 g, sodium acetate 3 g, agar 15 g (total
volume

filled up to 1000 cm

3

with distilled water) to enumerate

Clostridium spp. After medium solidi

fication, a plug of agar at a

concentration of 20 g L

1

was placed around the plates to avoid

direct air contact with anaerobic cultures and to highlight the
metabolic gas production; furthermore, plates were placed in
anaerobic jars in order to prevent direct oxygen contact with cells.
Plates were incubated in anaerobic conditions, under 90% of N

2

and

10% CO

2

as volume fraction of these gases, in a thermostated

chamber at 30

C for 24/48 h as reported by Phillips and co-workers

[19]

. The detection of Clostridium spp. was only performed on

samples where spores had germinated.

2.2.3. Catalase test

Many cells synthesize different antioxidant enzymes; one of the

most important is catalase, which converts H

2

O

2

to H

2

O and

gaseous O

2

. Since Clostridia do not possess the enzyme catalase,

this test was considered a good discriminating factor during
isolation of studied strains, as reported by Jay and co-workers

[20]

.

2.2.4. PCR identi

fication of Clostridium spp.

DNA extraction and PCR were performed according to Klijn et al.

[6]

, in order to con

firm the identification of some of the isolated

strains. PCR for speci

fic amplification of part of the 16S rRNA gene

(nucleotides 41 to 1114) of 1070 bp was performed. Brie

fly, PCR

analysis was carried out in a

final volume of 50 mm

3

containing

1.57 kg m

3

TriseHCl (pH 8.8), 2.92 kg m

3

NaCl, 0.29 kg m

3

MgCl

2

, 1.21 kg m

3

deoxynucleoside triphosphates, 1 U of Taq po-

lymerase (Ampli-Taq; Perkin-Elmer, Waltham, MA, USA), and 15 ng
of primers P1 (5

0

-GCGGCGTGCCTAATACATGC-3

0

) and P2 (5

0

-

GGGTTGCGCTCGTTGCGGGA-3

0

). After being heated to 95

C to

eliminate all protease activity, 5 cm

3

of template DNA were added.

Ampli

fication was performed in 30 cycles of melting DNA at 94

C

for 1 min, annealing at 55

C for 1.5 min, and elongation at 72

C for

2.5 min. Fragments ampli

fied by PCR were separated by 1.5%

agarose gel electrophoresis and stained with ethidium bromide; a
100 bp DNA ladder (New England Biolabs, Hitchin, United
Kingdom) was used as size marker.

2.3. Chemical analyses

Stored solid and pelleted digestate were chemically and physi-

cally characterized: pH was measured with a pH meter (type Basic
20, Crison, Barcelona, Spain) using 3.0 g of homogenized fertilizer
added with 50 cm

3

of distilled water and shacked for 30 min at

A. Pulvirenti et al. / Biomass and Bioenergy 81 (2015) 479e482

480

room temperature (22

C); total nitrogen content was calculated

following UNI EN 15604

[21]

; organic nitrogen was calculated as

the difference between total nitrogen and the sum of ammonic
(UNI EN 15604 and UNI EN 15475)

[21,22]

plus ureic nitrogen (UNI

EN 15604)

[21]

; phosphorus as P

2

O

5

by AOAC 960.03

[23]

; potas-

sium as K

2

O using AOAC 983.02

[24]

; water mass fraction by AOAC

950.01

[25]

.

2.4. Statistical analysis

Analysis of variance (ANOVA) was performed by GenStat 12.0th

edition. Means were compared using Duncan's test at the 5% level.

3. Results and discussion

The persistence of speci

fic bacteria in digestates could be

explained by the presence of sporogenic species in ingestate

[26]

.

As reported in

Table 1

, the ingestate used in biogas plants showed

total Clostridium spp., CFU values ranging from 1.0

10^4 g

1

to

2.0

10^6 g

1

. Cow slurry was the ingestate with the higher

presence of total Clostridium spp., CFU value of 2.0

10^6 g

1

, while

maize silage showed the lowest value of 1

10^4 g

1

. The total

Clostridium spp. were found in the different fresh digestates, CFU
values ranging from 2.1

10^5 g

1

to 3.0

10^6 g

1

, the unsepa-

rated digestate reporting the higher value. The total Clostridium
spp. were also found in stored digestate samples, CFU values

ranging from 1.0

10^5 g

1

(stored solid digestate) to

2.8

10^6 g

1

(stored liquid digestate).

The presence of Clostridium spp. bacteria observed in the

digestate was already reported in earlier studies

[8,27e29]

. In

general, anaerobic digestion does not reduce Clostridium spp. con-
tent

[30]

. A similar behavior was observed in this research, con-

firming that the content of Clostridium spp. was not affected by
anaerobic digestion. In fact, the genus Clostridium survived in the
anaerobic digestion process

[31]

because only vegetative cells are

susceptible to temperatures above 50

C, while the elimination of

spores requires further and more intense heat-treatments

[32]

.

Bagge et al.

[30]

reported that pathogen regrowth during storage

was probably due to non-hygienic conditions of the storage tanks,
as showed for stored liquid digestate in present results (

Table 1

).

However, pelleting the stored solid digestate completely elimi-

nated the presence of actively growing cells of Clostridium spp.
Pellets were produced by a PP300 Kompakt pellet press, working at
high pressure and reaching after the process a pellet temperature
comprised between 75 and 95

C, empirically measured immedi-

ately after sampling as described in M

&M. Therefore, the synergic

effect of both pressure and temperature was the most probable
main cause of the drastic elimination of Clostridium spp. from the
pellet of stored solid digestate. In fact, sterilization of vegetative
microorganisms, as well as spores, is possible using high pressure
treatment at elevated temperatures ranging from 60 to 98

C

[33]

.

To con

firm the presence or absence of Clostridium spp. in samples,

all isolated strains were tested for catalase. Some of them, corre-
sponding to different form of digestate from the positive samples of

Table 1

, were PCR ampli

fied for a further validation (

Fig. 1

).

All strains, except pellet, were catalase negative and strains

ampli

fied with specific primers for the genus Clostridium produced

the characteristic amplicon between 1000 and 1100 bp, con

firming

their belonging to the genus. Both analyses con

firmed the presence

of bacteria bearing the characteristics of the genus Clostridium.

Some chemical and physical characteristics of pelleted vs.

normal stored solid digestate were also investigated. When
compared to stored solid digestate, pelleting showed a slight effect,
although signi

ficant, on pH, by reducing it from 9.09 to 8.90.

Pelleting showed a concentration effect on nutrients in the

fertilizer ready to be distributed due to the drastic reduction of the
water content from about 54% to about 8% (

Table 2

). Apparently, the

only parameter that did not increase in concentration, but
remained unchanged after the pelleting treatment was the organic

Table 1
Mean values of Clostridium spp. CFU per gram; results of catalase test and PCR
ampli

fications investigated in samples.

Material

CFU

Catalase test

PCR

Maize silage

1.0

10^4 e

negative

n.d.

Triticale silage

5.0

10^5 bc

negative

n.d.

Grape stalk

2.2

10^5 cd

negative

n.d.

Cow slurry

2.0

10^6 a

negative

n.d.

Fresh unseparated digestate

3.0

10^6 a

negative

positive

Fresh liquid digestate

7.0

10^5 b

negative

positive

Fresh solid digestate

2.1

10^5 cd

negative

positive

Stored liquid digestate

2.8

10^6 a

negative

positive

Stored solid digestate

1.0

10^5 d

negative

positive

Pelleted stored solid digestate

0 f

n.d.

n.d.

Mean values (n

¼ 18) in column followed by different lowercase letters are signif-

icantly different at P

< 0.05 according to Duncan's multiple range test. n.d. ¼ not

determined.

Fig. 1. Agarose gel (1.5%) electrophoretic separation of the amplicons obtained by using Clostridium genus speci

fic primers. The order of samples through lanes is as follows: 100 bp

DNA ladder, lanes M; STORED LIQUID DIGESTATE, lanes 1, 4, 6, 8, 10 and 13; FRESH UNSEPARATED DIGESTATE, lanes 2, 5, 7 and 15; FRESH SOLID DIGESTATE, lanes 3, 9, 12 and 14;
FRESH LIQUID DIGESTATE, lane 11; STORED SOLID DIGESTATE, lane 16.

A. Pulvirenti et al. / Biomass and Bioenergy 81 (2015) 479e482

481

nitrogen, equally present at 0.71% in both products. A possible
explanation for the observed value of the organic nitrogen could be
the mineralization of nitrogen, coupled to a partial volatilization of
ammonia, due to the thermal process of pelleting.

Together with the total elimination of Clostridium spp. these

values, although mainly due to the substantial dehydration,
undoubtfully increase the fertilizing value of digestate. Apart the
obvious advantage of nutrient concentration, the decrease of water
mass fraction allows a less expensive transportation and distribu-
tion, while the decrease of the normally high pH of digestate is of
particular value in agricultural areas of high soil pH, as in the region
of Parmigiano Reggiano production. This simple treatment allows a
safe and sustainable use of pelleted digestate as fertilizer or
amendment, also in dairy-farming agricultural areas where tradi-
tional hard cheeses like Parmigiano Reggiano are produced.
Moreover, since biogas plants produce renewable electrical and
thermal energy, these could and should be used for the industrial
process of pellet production.

4. Conclusions

In conclusion, this work evaluated the possible agricultural use

of the digestate in pellet in the dairy farming areas. Although it
should be speci

fically verified, its use could significantly reduce the

risk of alterations during the cheese aging, as the digestate pellets
obtained in the present study from stored solid digestate were free
of Clostridium spp. In addition, pellets showed reduced pH and
water mass fraction and an increase in macronutrient concentra-
tions. Therefore, the pelleting process performed thanks to the
energy produced in the biogas plant is proposed as the solution for
both eliminating any potential risk of digestate due to presence of
Clostridium spp. and increasing the digestate fertilizing value.

Acknowledgments

The authors wish to thank FONDAZIONE MANODORI of Reggio

Emilia, Italy, funder of the present study as part of the project:
“Digestato da impianti di produzione di Biogas: Valorizzazione di
una risorsa per l'ambiente e per il territorio reggiano

”.

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Table 2
Chemical and physical parameters investigated in stored solid digestate vs. pelleted
stored solid digestate on a fresh weight basis.

Parameter

Stored
solid digestate

Pelleted stored
solid digestate

pH

9.09 a

8.90 b

Total nitrogen

0.87 b

1.30 a

Organic nitrogen

0.71 n.s.

0.71 n.s.

Phosphorous (as P

2

O

5

)

0.96 b

2.43 a

Potassium (as K

2

O)

1.14 b

1.90 a

Water mass fraction

53.58 a

7.81 b

Mean values (n

¼ 3) in each row followed by different lowercase letters are

signi

ficantly different at P < 0.05 according to Duncan's multiple range test.

n.s.

¼ not significant.

A. Pulvirenti et al. / Biomass and Bioenergy 81 (2015) 479e482

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