Waste Management 104 (2020) 239–245

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Waste Management
journal homepage: www.elsevier.com/locate/wasman

Humic-like substances extracted from different digestates: First trials of

lettuce biostimulation in hydroponic culture
F. Guilayn b,⇑, M. Benbrahim c, M. Rouez b, M. Crest b, D. Patureau a, J. Jimenez a
a
LBE, Univ Montpellier, INRA, 102 avenue des Etangs, 11100 Narbonne, France
b
Suez, CIRSEE, 38 rue du Président Wilson, 78230 Le Pecq, France
c
RITTMO Agroenvironnement, ZA Biopôle, 37 rue de Herrlisheim, CS 80023, F-68025 Colmar, France

a r t i c l e i n f o a b s t r a c t

Article history: Digestate valorization is a key challenge for the feasibility of Anaerobic Digestion plants. In this study,
Received 16 October 2019 humic-like substances (HLS) extracted from two digestates (sewage sludge and manure) were used for
Revised 15 January 2020 the biostimulation of hydroponic cultures of Lactuca sativa (lettuce) at different doses and compared to
Accepted 17 January 2020
a commercial reference of leonardite HLS. Aerial biomass increase averages ranged from 7 to 30 %, but
the results presented a high coefficient of variation (around 20 %). The commercial reference did not pre-
sent statistically significant biomass yield improvement. The application of manure digestate extract at
Keywords:
the higher fulvic-like acids dose (4.6 mg/L of dissolved organic carbon) presented the best and most sig-
Anaerobic digestion
Biogas effluent
nificant results compared to the blank (Hoagland’s solution only). However, this result cannot be strictly
Digestate valorization dissociated from the supplementary amounts of nutrients brought by the extracts. Additionally, all the
Humates products presented low heavy metal content compared to the recent EU regulation for biostimulants
Biostimulation (2019/1009). This preliminary study confirmed the interest of extracting HLS from two digestates for
Soilless culture application as biostimulants, shedding light on a new perspective for digestate valorization.
Ó 2020 Elsevier Ltd. All rights reserved.

1. Introduction include a large range of organic substances aiming to boost biolog-

Valorization of organic residues by Anaerobic Digestion (AD)
has been considered as a standout option for bioenergy production
in terms of energy efficiency and environmental impact (The
Commission of the European Communities, 2009).
AD is still mostly seen as a biogas producing process, but the
liquid to slurry/solid effluent called digestate is the principal pro-
duct in terms of mass of any AD plant. Digestate management is
a key concern and it is still a challenge for the full-development
of AD. Digestate direct landspreading is a typical option, but it is
not always economically interesting. Especially in the case of cen-
tralized facilities with local nutrient surplus, transportation dis-
tances limit direct landspreading feasibility (Möller et al., 2010).
Additionally, it usually relies on heavy procedures imposed by
environmental authorities. Indeed, in some cases, it may raise con-
cerns on multiple environmental issues such as nutrient leaching
and greenhouse gas emissions (Nkoa, 2014).
In recent years, various products have been developed to
improve soil, plant development, and soil/plant interaction. Those
⇑ Corresponding author at: Suez, CIRSEE, 38 rue du Président Wilson, 78230 Le
Pecq, France.
E-mail address: felipe.guilayn@suez.com (F. Guilayn).
ical process at both soil or plant level (Calvo et al., 2014). One kind
of the so-called biostimulants is those leading to indirect positive
effects on plant growth, such as germination rates, root growth
and architecture, plant biomass, number of flowers, among other
(Calvo et al., 2014). Those include humic-like substances (HLS) that
might be extracted from natural fossil sources such as leonardite
and peat or organic residues such as compost, vermicompost,
anaerobic digestates and other anthropogenic materials (Atiyeh
et al., 2002; Calvo et al., 2014; Eyheraguibel et al., 2008; Fascella
et al., 2018, 2015). Nevertheless, the origin, the formation and
the structure of HLS are a matter of controversial scientific debate
over two centuries. This domain is still of a great interest pushed
by the recognized importance of humic substances for understand-
ing the fertility of natural soils but also due to its remarkable appli-
cations on agriculture or horticulture as soil conditioners and
biostimulants (Muscolo et al., 2013).
The application of digestates for hydroponics systems as a
source of nutrients and as a digestate treatment option have been
successfully reported (Krishnasamy et al., 2012). Moreover, dis-
solved organic matter from digestate has already been demon-
strated to present auxin-like effects in plants (Scaglia et al.,
2015). In parallel, digestates have already been used as substrates
for the extraction of valuable soluble organic compounds referred

https://doi.org/10.1016/j.wasman.2020.01.025
0956-053X/Ó 2020 Elsevier Ltd. All rights reserved.
240 F. Guilayn et al. / Waste Management 104 (2020) 239–245
to as HLS, soluble biopolymers, biobased organic substances, bio-
waste derived soluble substances and other variants (Fascella
et al., 2015; Montoneri, 2017; Prevot et al., 2015; Salati et al.,
2011).
The biostimulation of hydroponics systems by HLS has been
demonstrated either by the application of HLS from fossil sources
(Conselvan et al., 2017) and organic materials (Morard et al.,
2011; Palumbo et al., 2018). However, there are currently very
few studies testing digestate-extracted HLS for biostimulation
(Fascella et al., 2015; Massa et al., 2018, both in ornamental
plants). Moreover, despite the great diversity of digestates types
(Guilayn et al., 2019), no study could be found testing HLS
extracted from different digestates for plant growth biostimula-
tion. Adding to that, developing biostimulants from digestates is
now a concrete short-term valorization pathway with the recent
approval of a new European Union regulation on fertilizers (EC
2019/1009) that includes a product category of biostimulants.
Recommended doses of HLS and biostimulants, in general, are
still controversial (Lyons and Genc, 2016) and are usually based
on quantities per crop surface. The objective of this study was to
screen the dose-effect of HLS on root and aerial growth of lettuce
(Lactuca sativa var. Tourbillon) in a hydroponic (soilless) system.
These HLS were extracted from two different digestates and com-
pared to a commercial humic substance solution (extracted from
leonardite). Besides, the extraction method is performed in condi-
tions that are considered industrially scalable in the short-term.
2. Material and methods
2.1. Samples
Two digestates were used for HLS extraction, both from full-
scale facilities: the first was a wastewater treatment plant (WWTP)
sludge digestate. It was originated from a Continuous Stirred Tank
Reactor (CSTR) digester treating a thickened mixture of primary
and secondary sludge. It was operated with a retention time
25 days and under mesophilic conditions. The second was an agri-
cultural digestate originated from a dry-AD plug-flow digester
treating a mixture of cattle manure, chicken manure and a minor
amount of straw. It was operated under mesophilic conditions
and for a retention time of about 60 days. The HLS used as a refer-
ence consists of a commercial solution of humates produced from
an alkaline extraction of leonardite. This product is available in the
European market and it is indicated by the furnisher for agriculture
and horticulture biostimulation.
Macro and microelements of the commercial humates solution
and the digestate HLS-extracts are presented in Table 1.
2.2. Extraction of humic-like substances from digestates
For the extraction of HLS, digestates underwent an alkaline
reaction at pH 12 for 24 h at 150 RPM and 35 °C. pH was adjusted
with Sigma-Aldrich KOH dry pellets (85 % purity). The manure
digestate was previously diluted with distilled water for achieving
10 % TS. The reaction was followed by laboratory centrifuge at
4,000 g, 5 min and 20 °C. These conditions were optimized in pre-
viously unpublished studies. Slow centrifugation conditions were
preferred to simulate industrial conditions based on SUEZ exper-
tise. The supernatant containing a pool of various soluble organic
compounds was used as the HLS extract for hydroponics tests.
The extraction procedure is proposedly simple compared to several
HLS valorization articles usually including different kinds of purifi-
cation steps (Baccot et al., 2020; Massa et al., 2018; Palumbo et al.,
2018). Based on internal expertise, the above conditions are con-
sidered industrially scalable in the short-term.
2.3. Analytical methods
Dry matter and volatile solids were measured according to the
standard method of the American Public Health Association
(APHA, 2005). The analysis of macro and micro-elements was per-
formed by the external laboratory Aurea Agrosciences Ó (Ardon,
France). The total elements P, K, Mg, Ca, Na, B, Cu, Fe, Mn were ana-
lyzed according to the standard NF EN ISO 11885. Total Kjeldahl
Nitrogen (TKN) and Total Ammoniacal Nitrogen (TAN), were mea-
sured following the standard NF EN 13342.
Fractionation of the HLS extracts was performed as described in
detail by Baccot et al. (2017). In brief, the humic-like acids (HLA)
fraction was precipitated after filtration at 45 nm with cellulose
nitrate filters and precipitation at pH 2 with HCl 37 %. The super-
natant after HLA precipitation was again filtered at 45 nm. The fil-
trate was then passed through a DAX-8 resin column followed by a
XAD-4 resin column. The hydrophobic-like (HPO) molecules were
considered those retained by the DAX-8 resin. The transphilic-
like (TPH) molecules were considered those retained by the XAD-
4 resin. The fractioning mass balance was evaluated in terms of
dissolved organic carbon (DOC) measured with a Shimadzu carbon
analyzer model TOC-L. The permeating fraction after the two col-
umns were considered Hydrophilic-like compounds (HPI). The
HPO quantification is very similar to the procedure recommended
by the International Humic Substances Society for isolating fulvic-
like acids (FLA). The HPO fraction is hereafter identified simply as
FLA.
3D Fluorescence spectra were obtained with a Perkin Elmer
LS55 after filtration at 45 nm and dilution of liquid extracts. The
range of both excitation and emission wavelengths varied from
200 to 600 nm. With excitation wavelengths increments of
10 nm, the fluorescence values were recorded every 0.5 nm. As
described by Muller et al. (2014) and Jimenez et al. (2015), which
were based on Chen et al. (2003) and He et al. (2011). The fluores-
cence spectra were divided into 7 zones that can be associated
with different kinds of fluorescent organic matter. The total vol-
ume of intensity on each zone was calculated with double trape-
zoidal integration in R language version 4.4 with the function
‘‘trapz” from R package ‘‘pracma” version 1.99 (Borchers, 2018).
The own developed R package used for the regional integration
analysis is publicly available (Guilayn, 2018). A complexity ratio
was then calculated by dividing the total fluorescence intensity
volume of zones 4 to 7 (highly complex organic matter) by the vol-
ume of zones 1 to 3 (less complex protein-like organic matter).
Peaks associated with HLS are often reported in Zone 4 and 7
(Muller et al., 2014).
2.4. Hydroponics
For the hydroponic cultures, seeds of Lactuca sativa (var. Tour-
billon, Voltz, France) were left to germinate on peat (Klassmann
Deilmann, TS3, Germany) for 28 days in growth chamber with
16 h of light per day, air temperature between 25 °C, relative
humidity of 70 % and photon flux density of 150 mmol
m 2
s 1.
The dark period was at 20 °C and relative humidity of 70 %. Seed-
lings were transplanted to the hydroponic systems at BBCH 14 (4
full foliar). The system consisted of 21 trays of 20 L beakers con-
taining an aerated diluted (1/10) Hoagland’s solution (Hoagland
et al., 1950) with a density of 3 plants per beaker. For each product
and dose tested (modality), the hydroponic cultures were realized
in triplicates (3 trays) containing 3 plants of lettuce each (total of 9
plants per modality). The plants were grown in a greenhouse with
a daily air temperature between 21 and 28 °C and relative humid-
ity from 43 to 68 %. The air temperature of the dark period was
maintained at 20 °C and the relative humidity of the dark period
was maintained between 43 and 68 %. The nutrient solution had
 F. Guilayn et al. / Waste Management 104 (2020) 239–245 241

Table 1
Characterization of the digestate HLS-extracts and the commercial reference.

Charact. parameter Unit Sludge raw digestate Sludge HLS Manure raw digestate Manure HLS Commercial reference EU regulation limita
pH – 8.1 >12 8.6 >12 9.7 –
TS % 2.84 3.5 21.6 8.2 26.4 –
OM % 1.93 0.8 15.6 3.7 15.8 –
OM % TS 68.0 23 72.4 45 27 –
TAN g
kgTS 1 36.3 27.4 17.9 14.4 <0.45 –
Organic N g
kgTS 1 28.2 38.3 41.8 32.3 <8.0 –
TKN g
kgTS 1 64.4 65.7 59.7 46.7 8.0 –
P g
kgTS 1 35.3 14.9 9.72 5.5 2.9 –
K g
kgTS 1 12.6 250 66.7 249 181 –
Mg g
kgTS 1 4.93 <0.15 6.23 0.49 5.49 –
Ca g
kgTS 1 19.7 1.1 24.6 1.7 6.9 –
Na g
kgTS 1 2.89 4.00 5.28 5.00 1.67 –
S g
kgTS 1 NP 3.14 5.51 4.39 n.m. –
1
Cd mg
kgTS 2.30 <10 0.37 <0.067 0.23 1.5
1
Cr mg
kgTS 37.7 2.5 8.73 3.66 5.85 2 (CrVI)
1
Cu mg
kgTS 341 19 41.3 13.0 17.6 600
1
Hg mg
kgTS 2.50 0.2 <0.03 0.06 <0.03 1
1
Ni mg
kgTS 29.8 10 6.43 4.66 16.1 50
1
Pb mg
kgTS 64.6 <5.0 <5.0 <5.0 <5.0 120
1
Zn mg
kgTS 766 52 227 107 115 1500

AD: Anaerobic digestion. HLS: Humic-like substances extract. OM: Organic matter. TAN: Total ammoniacal nitrogen. TKN: Total Kjeldahl Nitrogen. TS: Total solids. WWTP:
wastewater treatment plant. Obs.: Sludge and Manure refer to digestate HLS-extracts.
a
EU regulation 2019/1009 for the product category PFC 6(B), which also includes requirements on pathogens and arsenic.

the following composition (mg
L 1): KNO3: 50.5; KH2PO4: 13.6; Ca
(NO3)2
4H2O: 118; MgSO4
7H2O: 49.2; H3BO3: 0.169; MnSO4
H2O:
0.168; ZnSO4
7H2O: 0.0088; (NH4)6Mo7O24
4H2O: 0.0036; CuSO4-

5H2O: 0.0078; Fe-EDDHA (6 % Fe): 18.6644. This composition
results in the following concentrations: 20.1 mg N
L 1, 3.10 mg
P
L 1, 23.4 mg K
L 1, 4.85 mg Mg
L 1, 20.0 mg Ca
L 1, 1.12 mg
Fe
L 1, 54.6 mg Mn
L 1, 29.5 mg B
L 1, 2.00 mg Zn
L 1, 1.99 mg Cu
L 1,
1.96 mg Mo
L 1. The solution was renewed 15 days after the trans-
plantation along with a second addition of biostimulants in the
same previous doses. The total duration of hydroponic culture
was 27 days, when the plants were in the development stage
41–43 as defined by Jenni and Bourgeois (2008) (adapted from
the BBCH scale).
The tested modalities consisted of a blank (no biostimulant), the
commercial reference at a recommended dose and the extracted
HLS from the two different digestates at different equivalents in
terms of dissolved organic carbon to the commercial reference:
WWTP sludge HLS-extract in total DOC equivalent and FLA equiv-
alent, manure digestate HLS extract in total DOC equivalent, FLA
equivalent, and HLA equivalent. Sludge digestate HLS-extract in
HLA equivalent was not performed because the low content of
HLA would imply an excessive dose of the extract. The experimen-
tal design with the applied doses for each modality is presented in
Table 2.
The dose for the commercial reference was stipulated consider-
ing a supplier recommended dose of 72 L/ha for a lettuce field with
an average of 60,000 plants/ha, which results in 1.2 mL by plant or
3.6 mL per tray. The products were applied two times: at the very
beginning of the test and after 15 days following the nutritive solu-
tion renewing. Results were evaluated in terms of weighted wet
(fresh) and dried aerial and root biomass. After weighting for wet
mass, biomass drying was performed at 40 °C until a constant
weight was observed.
2.5. Statistical tests
Statistics were performed by pooling results on the 9 plants for
each modality (3 replicate trails with 3 plants each) and consider-
ing them as independent replicates. Root and aerial biomasses
were treated separately and the different hydroponic modalities
were considered as groups for non-parametric analysis of variance.
A Kruskal-Wallis H test was performed to assess the significance of
different groups’ medians and the Conover-Iman test with
Benjamini-Hochberg p-value correction was used as post hoc anal-
ysis to identify the stochastic dominances. Statistics were per-
formed in R language version 3.5.1.
3. Results and discussion
3.1. General characterization
Macro and microelements of the HLS extracts and the commer-
cial reference are presented in Table 1, where they are compared to
the heavy metal limits of the recently approved European Union

Table 2
Experimental design for screening HLS doses.

Tested product Equivalency to commercial ref. Applied volume (mL/tray) Total DOCa HLAa FLAa (=HPO) TPHa HPIa
Blank NA NA – – – – –
Comm. ref. NA 3.6 20.0 13.7 2.0 0.9 3.4
Sludge Total DOC 96.8 20.0 0.8 3.3 3.3 12.6
Sludge DOC as FLA 59.4 12.3 0.5 2.0 2.0 7.7
Manure Total DOC 31.2 20.0 9.7 3.3 1.8 5.1
Manure DOC as FLA 19.1 12.2 6.0 2.0 1.1 3.1
Manure DOC as HLA 43.7 28.0 13.7 4.6 2.6 7.2

DOC: dissolved organic carbon. FLA: fulvic-like acids. HLA: humic-like acids. HLS: humic-like substances. HPI: hydrophilic compounds. HPO: hydrophobic-like compounds,
considered as FLA. NA: not applicable. Obs.: Sludge and Manure refer to digestate HLS-extracts. TPH: transphilic-like compounds.
a
Concentrations expressed in mg DOC.L 1.
242 F. Guilayn et al. / Waste Management 104 (2020) 239–245
(EU) regulation (2019/1009) including a product function category
of non-microbial plant biostimulants (PFC 6 - B).
The first glaring difference between the obtained products and
the commercial one is the product overall concentration. Indeed,
the HLS extracts produced from digestates present about 3 to 7
times inferior TS concentration (3,5 – 8,2 versus 26,4 % w/w),
which reflects the low initial TS of digestates. However, on a TS
basis, the digestate products present similar to superior OM con-
tent, 6 to 8 times more nitrogen, 2 to 5 times more phosphorus,
and about 1.5 times more K.
Regarding the latest EU regulation (2019/1009), all the three
samples were conforming to the heavy metal limits, except possi-
bly for a sewage sludge HLS-extract Cd concentration of
<10 mg
kgTS 1 that can be higher than the limit of 1.5 mg
kgTS 1.
It must be noticed that the regulation also includes limits of As
(40 mg
kgTS 1), Cr hexavalent (2 mg
kgTS 1) and pathogens,
which were not determined. Cr hexavalent should be conforming
since total Cr is next to the limit (about 2.5 and 3.7 mg
kgTS 1)
and ionic Cr tends to be readily converted to Cr trivalent in anaer-
obic conditions (Horst and Suthersan, 2004). Additionally, it must
be considered that sewage sludge digestates are not authorized
as component materials for this regulation, meaning that the
innocuity parameters are taken simply as a quality reference for
the sludge digestate HLS product.
3.2. HLS extracts organic matter characterization
Polarity fractionation and 3D fluorescence spectroscopy were
performed on the samples to confirm the presence and the quality
of HLS in the digestate extracts. First of all, both digestate extracts
present peaks and fluorescence regions that are typically observed
in anthropogenic organic waste materials containing HLS and aro-
matic soluble proteins (He et al., 2013; Huang et al., 2019). In the
HLS extract from sewage sludge, the high content of non-humic
hydrophilic substances (about 60 % of DOC, Fig. 1) hinders the
observation of HLA in its excitation–emission-matrix fluorescence
(EEM) (Fig. 2a). Indeed, in this extract, most of the DOC was present
as HPI. HLS were mainly present as FLA (16 %). The observed spec-
Fig. 1. Dissolved organic carbon (DOC), 3D Fluorescence (3DF) zones, and polarity fractionation (POL. FR.) of digestate humic-like substances (HLS) and the leonardite
commercial reference. FLA: Fulvic-like acids. HLA: humic-like acids. HPI: hydrophilic compounds. HPO: hydrophobic-like compounds. Obs.: Sludge and Manure refer to
digestate HLS-extracts. TPH: transphilic like compounds.
trum is very similar of the sludge DOM 3D fluorescence spectra
presented by Muller et al. (2014). The HLS from manure digestate
presented high content of HLA (about 50 % of DOC) and a similar
content of FLA to the sludge digestate extract (17 %). It presented
a quite similar spectrum to a compost DOM sample analyzed by
He et al. (2013). The commercial reference (HLS from leonardite)
presented a much higher content of HLA (68 %), lower content of
FLA (10 %) but also a relevant content of HPI (about 17 %). Globally,
the content of HLA was highly linearly correlated to the sum of
fluorescence in Zones IV, V, VI and VII and to the complexity
ratio from 3D fluorescence (R2 > 95 % in both cases), but the signif-
icance level of the regression slope is low (p-value < 0.10) due to a
small number of samples (3). The total content of fluorescence in
zones IV, V, VI and VII is better linearly correlated to the total
content of HLSs considering it as the sum of HLA, HPO and TPH
fractions (R2 > 99.9 % and p-value < 0.05). These results strongly
affirm the presence of both FLA and HLA in the three samples of
this study.
3.3. Hydroponic systems and plant biomass growth
By pooling biomasses from the 9 plants of each tested
modality (3 replicates with 3 plants each), a significant effect
of biostimulation is observed in roots biomass (p-value < 0.05
for wet and dry mass) and less significant for aerial biomass
(p-value < 0.05 and <0.10 for wet and dry mass, respectively).
Fig. 3 presents the results in terms of root and aerial biomasses
for both wet and dry weights. Differences in medians with 95 %
confidence interval are identified above the boxplots by the let-
ters ‘‘a”, ‘‘b” and ‘‘c”.
Regarding root biomass, the application of manure digestate
HLS-extract at the highest dose (HLA equivalent) resulted in posi-
tive significant results for wet and dry masses compared to the
blank and the commercial reference (p-value < 0.05, except wet
root biomass compared to the reference with p-value < 0.10).
The application of sewage sludge digestate HLS-extract at FLA
equivalent was also significantly positive compared to the
blank and the commercial reference (p-value < 0.05). The effects
 F. Guilayn et al. / Waste Management 104 (2020) 239–245
Fig. 2. Excitation - Emission Matrices of the HLS-extracts from WWTP sludge
digestate (a), manure digestate (b) and leonardite commercial reference (c).
were less apparent in the aerial parts. Only the manure digestate
HLS-extract at HLA equivalent presented a significant positive
result in terms of aerial biomass (p-value < 0.05, compared to the
blank).
The results are globally positive. Indeed, average biomass gains
range from 7 % (commercial reference) to 30 % (manure digestate
HLS-extract). However, there is in many cases a lack of significance
that can be explained by the overall high variability in results. Even
in the blank test, the aerial and root masses presented a coefficient
of variation exceeding 20 %. This high variability can be associated
with the short period of this preliminary test (3 weeks). Moreover,
only the root and aerial masses were measured. A full hydroponic
test is being carried out including measurements of root architec-
ture, chlorophyll index and a longer duration.
243
The best result of manure HLS-extract in HLA equivalent could
be associated with a higher concentration of FLA. In this modality,
the HLA dose was equivalent to the commercial reference, which
has not presented significant results. Better biostimulation of HLS
from digestates and composts compared to HLS from leonardite
have already been observed elsewhere (Fascella et al., 2018,
2015). In this modality, in order to ensure the same quantity of
HLA than the reference, the highest amount of FLA was added to
the system. FLA present smaller molecular weight allowing a better
absorption by roots and also a higher number of functional groups.
Thus FLA are usually more associated with the biostimulation of
plant/root growth than HLA. However, this trend might vary
depending on HLS structure and concentration (Muscolo et al.,
2013). For the biostimulation of plant growth, different results
have been reported in the scientific literature. Scaglia et al.
(2015) associated the auxin-like biostimulant effect of digestate
water extracts to the ‘‘neutral hydrophobic” fraction. Given the
fractionation procedure, this fraction would be close to the FLA
fraction in the current study. The authors have associated the hor-
mone effect to the presence of Indole-3 acetic acid, which is origi-
nated from a well-documented degradation of tryptophan under
anaerobic conditions (Scaglia et al., 2015). Similarly, Morard et al.
(2011) observed biostimulation of root growth in hydroponic sys-
tems associated with low molecular weight fractions of HLS
extracted from poplar sawdust. Tahiri et al. (2015) tested in vitro
biostimulation with HLS from landfill leachate and leonardite
and reported effective biostimulation when HLA were applied at
low concentrations but an inhibition of root growth associated
with FLA.
The Hoagland’s nutrient composition is considered optimal for
plant growth, meaning that the plants have grown under no nutri-
tional limitation. It must be considered, however, that in the pre-
sent study the effects of biostimulants cannot be strictly
dissociated from the supplementary addition of macro and trace
nutrients caused by the addition of the commercial solution or
by the digestate extracts. Table 3 summarizes the macronutrient
quantities for each modality (a) and the respective relative supple-
ments in comparison to the Hoagland’s solution only (b), caused by
the addition of biostimulants.
The additional quantities of N, P and K were less important
when adding the commercial reference (Table 3). The additional
quantities of N ranged from 17 to 50 %, K from 83 to 190 % and P
from 14 to 81 % when digestate extracts were added (Table 3). Any-
way, it must be considered, as previously stated, that Hoagland’s
solution composition is considered ideal for hydroponics. More-
over, even with greater nutrient supplementation than the best
modality, other modalities presented no significant results when
compared to the blank and the reference. It has been demonstrated
that HLS biostimulants can enhance plant nutrient uptake (Calvo
et al., 2014; Conselvan et al., 2017; Massa et al., 2018; Palumbo
et al., 2018), so a synergic effect of nutrient supplementation with
biostimulation can also explain positive results.
4. Conclusion
A pool of dissolved organic matter containing humic-like sub-
stances was extracted from two different digestates and compared
to a commercial product. The products presented low heavy metal
content compared to the latest EU regulation (2019/1009) for bios-
timulants. Biostimulation of Lactuca sativa hydroponics was tested
by adding the digestate products at different equivalency strate-
gies to the commercial reference under no nutrient limitation.
Depending on the dose, both digestate presented significant results
compared to the blank (from 20 up to 60 % mean root/aerial mass
increase), while the commercial product did not present significant
results.
244 F. Guilayn et al. / Waste Management 104 (2020) 239–245

Fig. 3. Doses and results of hydroponic cultures. DOC: Dissolved organic carbon. FLA: fulvic-like acids. HLA: humic-like acids. Obs.: Sludge and Manure refer to digestate HLS-
extract.

Table 3
Nutrient quantities in the hydroponic systems (a) and relative supplementary quantities compared to the blank (Hoagland’s solution only) (b).

(a) Nutrients quantities (g)

Tested product Equivalency to commercial ref. N P K Ca Mg
Blank NA 0.42 0.06 0.47 0.40 0.10
Comm. ref. NA 0.43 0.06 0.64 0.41 0.10
Sludge Total DOC 0.64 0.11 1.31 0.40 0.10
Sludge DOC as FLA 0.54 0.08 1.10 0.40 0.10
Manure Total DOC 0.56 0.09 0.99 0.40 0.10
Manure DOC as FLA 0.49 0.07 0.86 0.40 0.10
Manure DOC as HLA 0.59 0.08 1.36 0.41 0.10
(b) Supplementary quantities compared to blank (%)
Tested product Equivalency to commercial ref. N P K Ca Mg
Comm. ref. NA 1.8 4.5 36.6 1.6 0.6
Sludge Total DOC 52.5 80.5 180 0.5 0.1
Sludge DOC as FLA 28.3 22.7 136 0.9 1.2
Manure Total DOC 32.2 49.4 111 0.3 0.1
Manure DOC as FLA 17.3 13.9 83.1 0.6 0.7
Manure DOC as HLA 39.6 31.8 190 1.3 1.6

DOC: dissolved organic carbon. HLS: humic-like substances. NA: not applicable. HLA: humic-like acids. FLA: fulvic-like acids. Obs.: Sludge and Manure refer to digestate HLS-
extracts.

Acknowledgements
This work was supported by SUEZ, the French National Institute
for Agricultural Research (INRA) and the French National Associa-
tion for Research and Technology (ANRT) under the CIFRE grant
N° 2015/1499.
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