- Open Access
Effect of bioeffectors and recycled P-fertiliser products on the growth of spring wheat
© The Author(s) 2016
- Received: 25 January 2016
- Accepted: 29 May 2016
- Published: 5 September 2016
The recycling of waste products into P fertilisers in agriculture is advisable from the perspective of sustainability. Bioeffectors (BEs), which have the ability to increase the plant uptake of P from recycled fertiliser products, may increase the fertiliser value of these products. This paper investigated the effect of a range of different recycled fertilisers on the growth and P uptake of wheat in pot experiments conducted at three different locations in Europe. Furthermore, investigations were undertaken as to whether the addition of a range of bioeffectors could significantly enhance P availability, P uptake and plant growth.
BE additions were found not to significantly increase the aboveground biomass of wheat plants or the uptake of P when plants were fertilised with recycled fertiliser products. This was shown across a range of pot experiments with soils of different P status. Only in the case of the positive control P fertiliser (TSP) was a positive effect of Proradix and RhizoVital on plant growth observed in one of the experiments, while in the same experiment RhizoVital and Biological fertiliser DC had a negative impact on plant biomass when the P fertiliser was Thomas phosphate. With regard to P uptake, there was only a slight positive effect of Proradix in plants not supplied with P fertiliser in this experiment. Clear differences were seen in the efficiency of P fertilisers. Generally, sewage sludge ash performed quite poorly (20–40 % of TSP), while sewage sludge, Thomas phosphate, P-enriched slag and the fibre fraction of pig manure all had a high availability of P (>74 % relative to TSP). Compost composed mainly of garden/park waste and sewage sludge was intermediate in availability (40–70 %). The elemental composition of the harvested wheat plants was significantly affected in all cases by the different P fertilisers added. The BE treatments significantly affected the elemental composition of the aboveground biomass in one of the experiments where the product Proradix had the greatest effect on elemental composition.
In conclusion, the experiments revealed a wide difference in the bioavailability of P in the different waste products, but the added microorganisms demonstrated a limited capacity to influence plant P uptake across a range of soils and waste products.
- Sewage Sludge
- Aboveground Biomass
- Wheat Plant
- Principal Component Analysis Plot
- Basic Oxygen Furnace
Phosphorus (P) is a non-renewable resource , and currently the majority of P added as fertiliser in agriculture is in the form of inorganic fertilisers. From a sustainability point of view, it is sensible to make better use of P resources that are discarded as waste from urban areas; hence, there is a need to improve the recycling of P from agricultural and urban wastes . Recycled fertiliser products are by no means homogenous and the availability of P for plant uptake from recycled fertiliser products may vary considerably, depending on the feedstock of the fertiliser and the subsequent type of processing . Sewage sludge as a fertiliser may contain a range of different P forms depending on the specific process used to recover P from the sewage water, but a precipitation reaction using Al, Fe, Mg or Ca to precipitate P is often employed . Sewage sludge may contain different types of organic contaminants . Although it is still used as a fertiliser in many countries across Europe , the use of sewage sludge has declined in a number of European countries, while sewage sludge application on agricultural land was banned in Switzerland in 2008 . A common practice that eliminates the organic contaminants in sludge is to incinerate the sewage sludge, thus producing sewage sludge ash. However, the availability of P in sewage sludge ash is quite low, but is also observed to be variable depending on the type of treatment in the sewage treatment plant . Techniques such as acid leaching and thermal treatment have been investigated for their potential to recover P from sewage sludge ash while separating it from the detrimental heavy metals with varying success [5, 7], but there is also a possibility of combining the upgrading of sewage sludge ash with the recycling of metallurgical slags. Slags from the metallurgical industry had been recycled as fertiliser in Germany in the form of Thomas phosphate for more than 100 years, but this is no longer produced . BOF (basic oxygen furnace) or LD (Linz–Donawitz) slag from steel production may be used as a liming agent in agriculture . It is also possible, however, to use sewage sludge ash as a means to enrich the hot liquid BOF slag with P, resulting in a fertiliser with a markedly higher P availability compared to the feedstock sewage sludge ash, due to a conversion of Ca3(PO4)2 (whitlockite) of low neutral ammonium citrate (nac) solubility into Ca-silico-phosphates with a higher nac solubility . P contained in animal manures can be used more sustainably if it is up-concentrated, and thereby more easily transported from areas with a P surplus to areas where soils have a P deficit . The majority of P (60–90 %) found in pig manure is inorganic  and only very little is in the form of phytate [11, 12]. Biomass ash (e.g. wood and straw ash) from bioenergy-plants could also potentially serve as a P fertiliser .
Different types of biostimulants or bioeffectors (BEs) have been investigated for their ability to increase plant productivity in agricultural systems . The concept of BEs covers quite a diverse group of natural products . In the present paper, the scope was limited to plant growth-promoting microorganisms (PGPM) focusing on plant growth-promoting rhizobacteria (PGPR)  and free-living fungi, such as species of the genus Trichoderma . PGPM may have the potential to enhance plant uptake of P from soil [14, 18, 19]. Improved growth under P-limiting soil conditions as a result of microbial inoculations has been observed in many different plant species, such as mung bean , bean , maize  and wheat . In soil, a large proportion of the total P pool is not directly available for plant uptake , and the ability to solubilise phosphates in the rhizosphere has been viewed as an important function of PGPM . The plant growth-promoting effect may, however, be overestimated due to a publication bias, and an observed positive plant growth response may be due to mechanisms other than an increased availability of P in the rhizosphere brought about by PGPM solubilisation of P, e.g. changes in root architecture and total root length .
Further research is therefore required into the potential of BEs to facilitate the plant uptake of nutrients from soil. Fungi of the ascomycete genera Trichoderma and Penicillium have been extensively studied for their potential as PGPM , and fungi of the genus Trichoderma may have an ability to increase plant nutrient uptake from soil [26, 27]. The specific T. harzianum strain Rifai 1295-22 (T22) has been observed to increase the solubilisation of sparingly soluble calcium phosphates  and to have a plant growth-promoting effect in willow , chickpea  and maize . Fungi of the genus Penicillium have been shown to have P-solubilising capabilities [31, 32], and members of this genus have been observed to have a positive effect on biomass and P uptake of wheat and bean . Wakelin et al.  found that a strain of P. bilaii is both capable of increasing the yield of medic and lentil in the field and of significantly increasing the level of HCO3-extractable P in soil microcosms, and more recently P. bilaii has been found to increase yield in maize in field trials . On the other hand, the positive effect of P. bilaii under P-limited conditions has also been linked in pea to an increase in the root adsorptive capacity under P-limited conditions rather than through increased P solubilisation . However, an investigation across a number of field studies involving wheat showed that P. bilaii does not significantly affect P uptake and yield . Gram-negative gammaproteobacteria of the genus Pseudomonas are ubiquitous bacteria in soil, are known to proliferate greatly in the rhizosphere  and have been studied for their plant growth-promoting activities for many years . Bacteria from this genus have been observed to increase plant productivity under P-limiting conditions [38, 39]. As a representative of Pseudomonas PGPR, the product Proradix was selected. This product has primarily been developed and investigated for its effects on plant resistance to pathogens [40–42], but there is also evidence that this product may improve plant growth under nutrient-limiting conditions . Low G + C Gram-positive bacteria of the genus Bacillus have been shown to solubilise calcium phosphates and increase the dry matter yield of wheat in a pot trial in which no P fertiliser or calcium phosphates were applied . A number of B. amyloliquefaciens strains have been investigated for their biocontrol capabilities  and the type strain (FZB42) for the subspecies plantarum of B. amyloliquefaciens  is reported to work as a biofertiliser and provide protection against various soil-borne diseases [46, 47].
The aim of the present paper was to investigate whether a variety of BE organisms could significantly enhance the availability and uptake of P from a range of recycled P-fertiliser products with very different P availability. The paper encompasses a number of pot experiments carried out in Denmark, Germany and the Czech Republic. The experiments included a negative control as well as a positive control (in two out of three studies) in which highly available triple superphosphate was added.
Sewage sludge ash in particular is an example of a product with quite low P solubility, offering considerable potential for improvement by BEs. The microorganisms were expected to have a positive effect on the solubilisation of fertiliser-derived P in the soil, as well as a direct effect on the plants through hormonal effects. The latter effects might occur in all P-addition treatments, whereas the positive effects on soil P availability are expected to be of greater significance in treatments with lower P availability, such as sewage sludge ash. It was therefore hypothesised that: (i) inoculation with the selected BE strains would increase the availability of P from the recycled fertilisers and (ii) inoculation with the selected BE strains would increase the uptake of P by wheat from soil, leading to a larger production of aboveground biomass.
Due to differences in the conditions between the individual experiments, it is not possible to compare the concentrations of elements or biomass produced per kg soil across experiments. We therefore only analyse the relative changes in, for instance, biomass compared to the negative and positive controls (where included) across experiments. We analyse the effect of BEs in the individual experiments. The fact that some of the BEs are tested using different soils and slightly different growing conditions serves as a stronger test of their performance than a single pot experiment would have.
The paper deals with the results of three separate pot trials. The pot trials were carried out at Arbeitsgemeinschaft Hüttenkalk e.V., Germany (HK Kalke experiment), the University of Copenhagen, Denmark (UCPH experiment), and the Czech University of Life Sciences Prague, Czech Republic (CULS experiment).
Sampling of soil and soil characterisation
P CAL (mg kg−1)
P TOT (mg kg−1)
Conventional farming system. Arable land food production. No P addition for more than 30 years
Continuous cropping. No addition of P fertiliser for more than 30 years
Continuous cropping (potato, wheat, barley). No addition of P fertilisers
Conventional farming system. Low P input
Bioeffector (BE) treatments
Bioeffector (BE) products applied in the experiments
Name of organism(s)
Type of organism
Application rate (cfu g−1 soil)
Koppert, The Netherlands
Trichoderma harzianum, strain T-22
Sourcon Padena, Germany
Pseudomonas sp., strain DSMZ 13134
Biological fertiliser DC
Terra Bioscience, Germany
T. harzianum and five species of Bacillus
Bacteria + fungi
P fertilisers and P-fertilisation treatments
P-fertilisation treatments applied in the experiments
Total P content in product (g kg−1)
(% of total P)
(g dry product kg−1 soil)
P app. rate
(mg P kg−1 soil)
Sewage sludge, HK Kalke
Sewage sludge, UCPH
Sewage sludge ash, HK Kalke
Sewage sludge ash, UCPH
Fibre fraction of pig manure
SSA-enriched LD slag
Compost mainly consisting of sewage sludge and garden/park waste
Ashes from cereal straw
Ashes from wood chips
For the majority of the fertilisers, the equivalent of 50 mg P kg−1 soil was added. There were, however, some deviations from this in the CULS experiment (Table 3). For both the HK Kalke experiment and the UCPH experiment, sewage sludge (SS) and sewage sludge ashes (SSA) were included. Furthermore, both TSP and a low-grade type of TSP, termed superphosphate (SP) here, were included in both these experiments as positive controls. In all three experiments, a negative control without the addition of P fertiliser (P0) was included. An overview of the BE and P-fertilisation treatments included in the three pot experiments is presented in Table 4.
Pot trial setup, growing conditions and harvest
Overview of the treatments applied in the different experiments (soils)
1. HK Kalke (Germany)
2. UCPH (Denmark)
3. CULS (Czech Republic)
Negative control (BE0)
Negative control (P0)
HK Kalke experiment
The air-dried and sieved soil (mesh size 5 mm) was mixed with water-washed quartz sand in a proportion of 2:1. This substrate was mixed with 0.843 g kg−1 Ca(NO3)2∙4H2O and 0.719 g kg−1 Patentkali (27.8 % K2O, 9.49 % MgO, 15.8 % S). Each pot was filled with 6 kg of the fertilised soil/sand mixture and watered to 70 % of WHC. Before watering, Bio-DC was mixed into the substrate of the Bio-DC treatment. Spring wheat (cultivar Aranka) was sown in 32 separate sowing holes (approximately, 2 cm deep). To each of the sowing holes, 2 ml of the Pro or RhVi suspensions were added in the corresponding BE treatments. After germination, plants were reduced to 24 wheat plants per pot. The pots were placed in a randomised design, with four replicates per treatment, in an outdoor roofed vegetation hall. Pots were irrigated with demineralised water to 60–70 % WHC (controlled gravimetrically once a week) during the whole vegetation period. The plants were supplemented with an additional 50 mg N kg−1 soil on day 40 in the form of Ca(NO3)2. The plants were harvested 8 weeks after sowing. The plants were at stage 59 (without P fertilisation) or stage 63 (with P fertilisation).
Growing conditions in pot experiments
Soil:sand ratio (mass)
Size of pots (L)
Mass of substrate (kg)
No of plants
No. of harvests
Final harvest (weeks)
App. of macronutrients at setup (mg kg−1 substrate)
Soil was air dried and sieved (mesh size 10 mm). No sand was added to the soil. For each pot, 5 kg (d.w.) of soil was used, which was mixed with 1.67 g of NH4NO3. 50 g of WoA or StA was thoroughly mixed with the soil prior to filling the pots (final dose 10 g of ash per kg soil). K2HPO4 was applied as a water solution and was also thoroughly mixed into the whole soil volume. The soil was watered to 40 % of WHC, 25 wheat seeds (cultivar Aranka) were sown in separate sowing holes (approximately, 2 cm deep) and 2 ml of BE suspension (or 0.25 mM CaSO4 in the BE0 controls) was applied to each hole prior to closing. After germination, the number of plants was reduced to 20 wheat plants per pot, and these were inoculated again by irrigation with 100 ml of BE suspension per pot. The pots were placed in an outdoor roofed vegetation hall. Pots were irrigated with demineralised water to 60–70 % WHC (controlled gravimetrically once a week) during the entire vegetation period. The experiment was undertaken in a randomised design with four randomisation procedures during the experiment. The plants were harvested after 16 weeks. At harvest, the plants were at full maturity.
Soil data recorded during the HK Kalke experiment
Soil (40 g) was sampled 27 days after sowing. The soil was air dried and completely passed through a 2 mm mesh sieve. For pH measurement, 10 g of soil was suspended in 25 ml of a 0.01 molar CaCl2 solution for 1 h, stirred twice and pH determined using a pH electrode (VDLUFA standard method A 5.1.1). For water extraction of soil phosphate according to Van der Paauw  and Murphy and Riley , 4.25 ml of soil was suspended in demineralised water for approximately 22 h. Thereafter 250 ml water was added; the mixture was mechanically shaken for 1 h and filtered. P determination was undertaken using a spectrophotometer and molybdenum blue method.
HK Kalke experiment
After harvest, the aboveground wheat plant material was dried at 60 °C. 400 mg of plant material was digested with 8 ml 69 % HNO3 supra and 1 ml 15 % H2O2 in high-pressure MARS express vessels in a MARS microwave digestion system. The element analyses of P, K, Mg, Ca, Mn and Na were performed by ICP-OES.
UCPH and CULS experiment
The plant material was dried at 65 °C and weighed to measure the dry aboveground biomass. For elemental analysis, the dry plant material was finely ground. Subsequently, 100 mg of dry plant material was mixed with 2.5 ml 70 % HNO3 and 1 ml 15 % H2O2, followed by digestion in a pressurised single-chamber microwave oven (UltraWAVE, Milestone Srl, BG, Italy). Samples were then diluted to 50 ml using Milli-Q water and analysed for their elemental content (B, Ca, Cu, Fe, K, Mg, Mn, P, S, Zn in UCPH and Ca, K, Mg, Mn, Na, P in CULS) by ICP-OES. For the samples from the final harvest in the UCPH experiment, only P was measured using flow injection analysis.
Data analyses and statistics
In the CULS experiment, the normalisation was undertaken separately for the two soils. Significance testing of differences between treatment means was performed using one- and two-way ANOVAs and Dunnett’s test (for comparisons versus the control only) or Tukey’s test (for all possible comparisons) for post hoc multiple comparisons. These were performed using the statistics module in Sigma Plot 13.0. In the UCPH experiment in which two separate samplings had been performed, the difference in normalised biomass between sampling days was tested using a paired t test. For the CULS experiment, all the P-fertiliser treatments were combined with the BE0 treatment only. The effect of different P substrates was therefore analysed by a two-way ANOVA, excluding data for the RhVi and BaPr BE treatments. The effect of BE treatments in the CULS experiment was tested in two separate two-way ANOVAs for the two soils, where only data from the straw and wood ash treatments were included.
The same model was used to express biomass as a function of the P concentration in the youngest fully developed leaf at day 25 in the UCPH experiment. These regressions and simple linear regressions were performed using the regression wizard in SigmaPlot 13.0 (Systat).
Principal component analysis (PCA) was performed on data for elemental concentrations. Beforehand, PCA data were standardised by subtracting the mean for each element and then dividing this by the standard deviation. PCA was performed in R version 3.1.1  using the ade4 package  with a chosen number of principal components of 10.
Aboveground biomass and P content
HK Kalke experiment
P content in aboveground biomass (mg P kg−1 soil) in the HK Kalke (a) , UCPH (b) and CULS (c) experiments
Tukey’s test (BE0 results)a
Sewage sludge ashes
Tukey’s test (BE0 results)
Sewage sludge ashes
Tukey’s test (BE0 results)a
In the UCPH experiment, plants were harvested after 32 (Fig. 1b) and 54 days (Fig. 1c). The normalised biomass across all treatments was significantly different between harvests (paired t test, P < 0.001); therefore, the normalised aboveground biomass data from the two harvests were analysed individually. The normalised biomass was significantly different between P-fertilisation treatments at both harvests (two-way ANOVA, P < 0.001). In contrast to this, the different BE inoculations did not affect the biomass obtained (two-way ANOVA, P > 0.05) and no interaction was observed between the two factors (two-way ANOVA, P > 0.05). At both harvests, all treatments in which a P fertiliser was applied resulted in a significantly higher aboveground biomass than the control without added P fertiliser (Dunnett’s test, P < 0.05). The absence of a significantly higher aboveground biomass when inoculating with the three BEs (TrP, Pro, RhVi) compared to the uninoculated control (BE0) was confirmed for sewage sludge, sewage sludge ash and compost as the P fertilisers in a follow-up experiment at the University of Copenhagen in which only five wheat plants were grown in each pot (Additional file 1: Fig. S1). P uptake was only evaluated for the P fertilisers SSA and FFPM at the second harvest (Table 6b). No effect was produced by either of the two main factors (P fertiliser and BE addition) and there was no interaction between the two factors in relation to the aboveground P content (two-way ANOVA, P > 0.05).
In the CULS experiment (Humpolec and Poděbrady soils), there was no significant effect of the soil type on the aboveground biomass in the control (P0/BE0) treatment (one-way ANOVA, P > 0.05, data not shown). For the Humpolec soil, the DKP treatment yielded a significantly lower normalised biomass compared to the control (two-way ANOVA on BE0 data with soil and P fertiliser as factors, Dunnett’s test P < 0.05), while in the Poděbrady soil the normalised aboveground biomass was not significantly different from the control when adding DKP (Dunnett’s test, P > 0.05). The addition of straw ash resulted in a significantly higher biomass at harvest in the Poděbrady soil (Dunnett’s test P < 0.05), while the biomass after adding straw ash was not significantly different from the control without the addition of P fertiliser in the Humpolec soil (Dunnett’s test P > 0.05). Finally, the addition of wood ash led to a significantly lower biomass compared to the control in the Humpolec soil (Dunnett’s test P < 0.05), while in contrast the addition of wood ash led to an increase in biomass compared to the control for the Poděbrady soil (Dunnett’s test P < 0.05). Overall, there was only a fairly limited difference in the harvested biomass in a comparison across P-fertilisation treatments (Fig. 1d, e). The maximum increase observed when looking across the BE0 treatments was 22 %. This increase was observed for both straw and wood ash in the Poděbrady soil. The effect of the addition of the two different ash types (straw and wood ash) in combination with the different BE inoculation treatments included here (BE0, RhVi, BaPr) was analysed in two separate two-way ANOVAs for the two soils (Humpolec & Poděbrady) included in this experiment. No significant effect was observed of ash type or BE addition or an interaction between the two factors for any of the two soils investigated (P > 0.05, two-way ANOVA). There was a significant effect of soil on P uptake in the P0/BE0 treatment (Table 6c, one-way ANOVA, P < 0.001). The data for P content (Table 6c) were subsequently analysed for the two soils independently, and a significantly higher P content was observed in the straw ash treatment compared to the control in both soils (Tukey’s test, P < 0.05), but the BE treatments did not result in a total P content that was significantly different from the control (Dunnett’s test, P > 0.05).
Soil-available P in the KALKE experiment and relationship with biomass
Correlation between plant P data and aboveground biomass
Fertiliser use efficiencies
Relative fertiliser efficiencies
HK Kalke (n = 4)
UCPH (n = 5)
UPCH_2a (n = 4)
FE (% of TSP)
PE (% of TSP)
FE (% of TSP), first harvest
FE (% of TSP), second harvest
FE (% of TSP)
Sewage sludge ash
Fibre fraction of pig manure
SSA-enriched LD slag
Compost of sewage sludge and garden/park waste
PCAs on plant compositional data
HK Kalke experiment
Was P the limiting factor in these experiments?
These pot experiments were undertaken on the assumption that P was the limiting factor in these trials. In the case of the UCPH experiment, the clear saturation-type relationship between P concentration in the youngest fully developed leaf during early growth and the subsequent biomass production (Fig. 3) served as validation that P limitation was in fact being studied in the UCPH experiment. Furthermore, the concentration of P recorded in leaves from the unfertilized treatment (Additional file 1: Table S3) was as low as 0.24 mg g−1 in one case and therefore probably within the deficiency range at this stage . Along the same lines, the clear relationships between soil P status and aboveground biomass in the HK Kalke experiment (Fig. 2) was validation that P was the limiting nutrient in the experiment. Here, we did not observe a positive response of P fertilisation on P concentration which shows that the P concentration of the whole shoot after 8 weeks of growth is not a robust measure of P deficiency. In contrast to the above, P could not be considered the sole limiting factor in the CULS experiment, since a positive growth response of adding readily soluble DKP (32 mg kg−1) as the P fertiliser was not observed in this experiment. In general, the concentration of P was probably in the deficient range across all treatments (below 0.1 % in stem and leaves, Additional file 1: Table S4). This may partly be explained by a nitrogen limitation in the Humpolec soil, since soil solution nitrate levels in the Humpolec soil during the pot experiment were three times lower than those recorded in the Podêbrady soil (data not shown).
Were other nutrients limiting or present in toxic concentrations?
In the UCPH experiment, the concentrations of Fe, K, S, Zn in the youngest fully developed leaf 25 days after sowing (Additional file 1: Table S3) were within the adequate range at this stage . For B, Ca, Cu, S and Zn this was also generally the case (Additional file 1: Table S3), but the leaf from one of the three control plants analysed showed concentrations (see minimum in Additional file 1: Table S3) in the deficiency range . For B, the highest concentration recorded (156 μg g−1) might be at the limit of toxicity at this stage . However, no clear symptoms were observed.
In the CULS experiment, the grain concentrations of K (Additional file 1: Table S4) indicated deficiency in this element across all treatments, while Mn (Additional file 1: Table S4) was in the adequate range for grain at maturity .
Did the added BEs enhance the availability of P from recycled fertiliser products?
As stated in the introduction, one possible mechanism for improving plant growth by a BE would be to increase the availability of P in the soil. When used in combination with recycled fertilisers, it is of interest whether or not the introduced organisms directly affect the solubilisation of the introduced P. In the HK Kalke experiment, no significant effect was observed for any of the tested BEs (Pro, RhVi, Bio-DC) on the level of available P in the soil (PH2O). Since we do not have soil data for the other experiments, we cannot make claims regarding the soil P availability in these experiments. This is in accordance with previous studies showing that although microbial inoculants may demonstrate potential for solubilisation of sparingly soluble P sources (such as Ca-phosphates) in vitro, this does not necessarily translate into increased plant availability of P in the soil . In the present study, there was no support for an increase of plant-available P in the soil as a result of inoculation with two bacterial products (Proradix and RhizoVital 42) and one fungal product (Biological fertiliser DC). There may be several possible explanations for the lack of a significant positive effect on P availability: (i) a limited proliferation of the introduced microorganisms in soil due to competition with native microorganisms, for example, (ii) the soil P level may not have been sufficiently low to promote the up-regulation of enzymes involved in P solubilisation, (iii) released P may have been taken up by the introduced microorganisms without subsequent release to the soil within the time frame of the experiments and finally (iv) the native microbial community of the soil and/or organic waste materials may have been optimal already in making P available from the introduced fertilizers.
Did the added bioeffectors affect the growth of plants and plant P uptake?
Despite previous reports that the tested organisms may enhance plant growth [30, 43, 46], only a small positive effect on aboveground biomass of Pro and RhVi in combination with TSP was found (Fig. 1a). The fact that there was only a positive effect in combination with TSP as a fertiliser may point towards a direct effect of the BEs on the plants rather than an effect on P availability in the soil. This interpretation was also supported by the fact that the uptake of P from TSP-fertilised soil was not significantly different between BE treatments (Table 6a). The direct effects of these microbial inoculants on the plants are in line with earlier work showing that Pro and RhVi may elicit defence responses in plants [41, 56], thus directly affecting the plant’s metabolism. In the P0 treatment, a positive effect of Pro and RhVi was observed on the total P content of the aboveground biomass, which seemed to indicate that under these P-limited conditions the two BEs did improve plant P uptake, even though a BE-mediated increase in PH2O was not observed.
As a prerequisite for an effect of BEs on the growth of wheat plants, the successful establishment of organisms in the rhizosphere may be required, and it has been stated that rhizosphere competence may be a key factor in the effectiveness of PGPM [57, 58]. On the other hand, there is also an example of a study where the supernatant of the culture medium in which T. harzianum T22 was grown resulted in a stronger effect on the growth of maize plants compared to inoculating with spores . This indicates that active growth in the rhizosphere may not always be a prerequisite for an effect of a PGPM and that a direct hormonal effect on the plants is a possible mode of action of these organisms. The present study did not measure whether the microorganisms established themselves in the rhizosphere of the wheat plants, meaning that it cannot be ruled out that the lack of a plant growth-promoting effect of the added BEs was due to an unsuccessful colonisation of the wheat rhizosphere. On the other hand, the fact that a significant BE effect was seen on the elemental composition of the aboveground biomass in the HK Kalke experiment may be an indication that the added microorganisms were in fact able to establish in the wheat rhizosphere in these pot experiments. In the CULS experiment, the plant elemental composition of the aboveground biomass did not give any indication of a BE effect.
Do the different recycled fertiliser products tested have potential as P fertilisers?
A low availability of P in the soil after fertilisation with sewage sludge ash was observed, which translated into a relative fertiliser efficiency based on biomass production of 24–41 % and P uptake of 31 %. This result was in line with earlier work, showing that phosphorus in sewage sludge ash is generally not readily taken up by plants . On the other hand, there may be considerable variations between different sewage sludge ashes, depending on the processing of sewage sludge in the water treatment plant . Sewage sludge, Thomas phosphate and sewage sludge-enriched BOF slag (LDS/SSA) all resulted in levels of available P similar to or higher than TSP. In fact, fertilisation with LDS/SSA resulted in a significantly higher level of PH2O compared to TSP. This was probably related to an increase in soil pH from ~5.6 in the TSP treatment to ~6.5 in the LDS/SSA treatment (Additional file 1: Table S1), since the availability of phosphates in soil is generally highest close to neutrality . Severin et al.  found that the LDS/SSA product had high efficiency as a P fertiliser  in accordance with this study’s results, yielding a P-fertilisation effect comparable to TSP. This shows the potential of this technology to produce a highly effective P fertiliser, partly based on sewage sludge devoid of any organic contaminants. However, the content of heavy metals could potentially be problematic. The content of Cr (1712 mg kg−1, data not shown) for instance is above the current Danish limits , while in Germany contents above 300 mg kg−1 have to be declared . An alternative to using sewage sludge ash could be to use sewage sludge as a fertiliser instead. Concerns may be raised regarding organic contaminants and problematic microorganisms, which are not relevant in the case of sewage sludge ash. However, organic contaminants probably do not pose a great threat here when the quality of present-day sewage sludge is taken into account . In the present study, sewage sludge was observed to possess high potential as a P fertiliser, resulting in responses that are 76–106 % of those observed when using TSP. This was in relatively good agreement with a pot trial using English ryegrass in which the efficiency of different sludges was 62–86 % of monocalcium phosphate . In the case of wood and straw ash, it was not possible to clearly evaluate their potential as P fertilisers based on the results presented here. This was due to the fact that (i) the CULS experiment lacked a positive control with the addition of a comparable level of total P and (ii) the input of P with the two different ash types was different. These problems aside, from the results presented here, it would not appear that wood ash and straw ash have great potential as P fertilisers, since the relative increase in biomass yield was not above 25 % in comparison to the HK Kalke and UCPH experiments showing yield increases of 50 % or more, even for sewage sludge ash. This result contradicted an earlier study in which a high P-fertilisation effect was found for rape meal, straw and cereal ashes . However, as observed from the PCA plot, a small effect was observed on the plant elemental composition due to the wood ash and DKP treatments and greater effect of the straw ash treatment, but these differences were not clearly associated with differences in the aboveground biomass. These effects were observed to be independent of soil type. The fibre fraction of pig manure (FFPM) prepared using a decanter centrifuge was shown to have a high fertiliser efficiency that was not significantly lower than the positive TSP control. This was in accordance with previous results showing a high P availability after application of this solid manure fraction to soil .
Based on the results from the HK Kalke experiment, we did not find evidence to support the hypothesis that BE products increase the availability of P in the soil. Furthermore, the BE products only had a very limited effect on the growth of wheat plants across all experiments. Further work is therefore needed to elucidate whether inoculation with BEs has agronomic potential in wheat production. A number of the tested recycled P-fertiliser products (sewage sludge, P-enriched BOF slag and fibre fraction of pig manure) were shown in the HK Kalke and UCPH experiments to have a high potential as P fertilisers without a requirement for further processing.
JDSL carried out the UCPH experiments, performed the majority of the data analysis in the paper and wrote the paper. MR carried out the HK Kalke experiment, contributed to data analysis and discussions of data. FM carried out the CULS experiment and performed the plant analyses for this experiment. MK supervised the analyses in the CULS experiment. PT supervised the experimental design in the CULS experiment. JM contributed to discussions regarding data interpretation. AN contributed to experimental design, data interpretation and the writing of the paper. All authors contributed to initial discussions of data. All authors read and approved the final manuscript.
This study was funded by the European Community’s Seventh Framework Programme 662 (FP7/2007–2013) under Grant Agreement no. 312117 (BIOFECTOR).
The authors declare that they have no competing interests.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
- Dawson CJ, Hilton J. Fertiliser availability in a resource-limited world: production and recycling of nitrogen and phosphorus. Food Policy. 2011;36:S14–22.View ArticleGoogle Scholar
- Hilton J. Johnston AE, Dawson CJ. The phosphate life-cycle: rethinking the options for a finite resource. In: Proceedings-International Fertiliser Society. 2010. International Fertiliser Society.Google Scholar
- Karunanithi R, et al. Phosphorus recovery and reuse from waste streams. Adv Agron. 2015;131:173–250.Google Scholar
- Smith SR. Organic contaminants in sewage sludge (biosolids) and their significance for agricultural recycling. Philos Trans A Math Phys Eng Sci. 2009;367(1904):4005–41.View ArticlePubMedGoogle Scholar
- Donatello S, Cheeseman CR. Recycling and recovery routes for incinerated sewage sludge ash (ISSA): a review. Waste Manag. 2013;33(11):2328–40.View ArticlePubMedGoogle Scholar
- Lamprecht H, et al. The trade-off between phosphorus recycling and health protection during the BSE crisis in Switzerland. A. Gaia Ecol Perspect Sci Soc. 2011;20(2):112–21.Google Scholar
- Nanzer S, et al. The molecular environment of phosphorus in sewage sludge ash: implications for bioavailability. J Environ Qual. 2014;43(3):1050–60.View ArticlePubMedGoogle Scholar
- Branca TA, et al. Investigation of (BOF) converter slag use for agriculture in europe. Metall Res Technol. 2014;111(3):155–67.View ArticleGoogle Scholar
- Severin M, et al. Phosphate fertilizer value of heat treated sewage sludge ash. Plant Soil Environ. 2014;60(12):555–61.Google Scholar
- Popovic O, Hjorth M, Jensen LS. Phosphorus, copper and zinc in solid and liquid fractions from full-scale and laboratory-separated pig slurry. Environ Technol. 2012;33(18):2119–31.View ArticlePubMedGoogle Scholar
- Leytem AB, Turner BL, Thacker PA. Phosphorus composition of manure from swine fed low-phytate grains: evidence for hydrolysis in the animal. J Environ Qual. 2004;33(6):2380–3.View ArticlePubMedGoogle Scholar
- Schlemmer U, et al. Degradation of phytate in the gut of pigs—pathway of gastrointestinal inositol phosphate hydrolysis and enzymes involved. Arch Tierernahr. 2001;55(4):255–80.View ArticlePubMedGoogle Scholar
- Brod E, et al. Waste products as alternative phosphorus fertilisers part I: inorganic P species affect fertilisation effects depending on soil pH. Nutr Cycl Agroecosyst. 2015;103(2):167–85.View ArticleGoogle Scholar
- Halpern M, et al. Chapter two-the use of biostimulants for enhancing nutrient uptake. Adv Agron. 2015;130:141–74.View ArticleGoogle Scholar
- du Jardin P. Plant biostimulants: definition, concept, main categories and regulation. Sci Hortic. 2015;196:3–14.View ArticleGoogle Scholar
- Vessey JK. Plant growth promoting rhizobacteria as biofertilizers. Plant Soil. 2003;255(2):571–86.View ArticleGoogle Scholar
- Rudresh DL, Shivaprakash MK, Prasad RD. Tricalcium phosphate solubilizing abilities of Trichoderma spp. in relation to P uptake and growth and yield parameters of chickpea (Cicer arietinum L.). Can J Microbiol. 2005;51(3):217–22.View ArticlePubMedGoogle Scholar
- Khan AA, et al. Phosphorus solubilizing bacteria: occurrence, mechanisms and their role in crop production. J Agric Biol Sci. 2009;1(1):48–58.Google Scholar
- Khan MS, et al. Plant growth promotion by phosphate solubilizing fungi—current perspective. Arch Agron Soil Sci. 2010;56(1):73–98.View ArticleGoogle Scholar
- Kucey R. Increased phosphorus uptake by wheat and field beans inoculated with a phosphorus-solubilizing Penicillium bilaji strain and with vesicular-arbuscular mycorrhizal fungi. Appl Environ Microbiol. 1987;53(12):2699–703.PubMedPubMed CentralGoogle Scholar
- Leggett M, et al. Maize yield response to a phosphorus-solubilizing microbial inoculant in field trials. J Agric Sci. 2014;153:1464–78.View ArticlePubMedPubMed CentralGoogle Scholar
- Singh S, Kapoor KK. Inoculation with phosphate-solubilizing microorganisms and a vesicular arbuscular mycorrhizal fungus improves dry matter yield and nutrient uptake by wheat grown in a sandy soil. Biol Fertil Soils. 1999;28(2):139–44.View ArticleGoogle Scholar
- Richardson AE, et al. Plant mechanisms to optimise access to soil phosphorus. Crop Pasture Sci. 2009;60(2):124–43.View ArticleGoogle Scholar
- Richardson AE. Prospects for using soil microorganisms to improve the acquisition of phosphorus by plants. Funct Plant Boil. 2001;28(9):897–906.View ArticleGoogle Scholar
- Harman GE, et al. Trichoderma species—opportunistic, avirulent plant symbionts. Nat Rev Microbiol. 2004;2(1):43–56.View ArticlePubMedGoogle Scholar
- Shoresh M, Harman GE, Mastouri F. Induced systemic resistance and plant responses to fungal biocontrol agents. Annu Rev Phytopathol. 2010;48:21–43.View ArticlePubMedGoogle Scholar
- Harman GE. Overview of mechanisms and uses of trichoderma spp. Phytopathology. 2006;96(2):190–4.View ArticlePubMedGoogle Scholar
- Altomare C, et al. Solubilization of phosphates and micronutrients by the plant-growth-promoting and biocontrol fungus Trichoderma harzianum Rifai 1295-22. Appl Environ Microbiol. 1999;65(7):2926–33.PubMedPubMed CentralGoogle Scholar
- Adams P, De-Leij F, Lynch J. Trichoderma harzianum Rifai 1295-22 mediates growth promotion of crack willow (Salix fragilis) saplings in both clean and metal-contaminated soil. Microb Ecol. 2007;54(2):306–13.View ArticlePubMedGoogle Scholar
- Akladious SA, Abbas SM. Application of Trichoderma harzianum T22 as a biofertilizer potential in maize growth. J Plant Nutr. 2014;37(1):30–49.View ArticleGoogle Scholar
- Takeda M, Knight JD. Enhanced solubilization of rock phosphate by Penicillium bilaiae in pH-buffered solution culture. Can J Microbiol. 2006;52(11):1121–9.View ArticlePubMedGoogle Scholar
- Wakelin SA, et al. Phosphate solubilization by Penicillium spp. closely associated with wheat roots. Biol Fertil Soils. 2004;40(1):36–43.View ArticleGoogle Scholar
- Wakelin SA, et al. The effect of Penicillium fungi on plant growth and phosphorus mobilization in neutral to alkaline soils from southern Australia. Can J Microbiol. 2007;53(1):106–15.View ArticlePubMedGoogle Scholar
- Vessey JK, Heisinger KG. Effect of Penicillium bilaii inoculation and phosphorus fertilisation on root and shoot parameters of field-grown pea. Can J Plant Sci. 2001;81(3):361–6.View ArticleGoogle Scholar
- Karamanos R, Flore N, Harapiak J. Re-visiting use of Penicillium bilaii with phosphorus fertilization of hard red spring wheat. Can J Plant Sci. 2010;90(3):265–77.View ArticleGoogle Scholar
- Grayston SJ, et al. Selective influence of plant species on microbial diversity in the rhizosphere. Soil Biol Biochem. 1998;30(3):369–78.View ArticleGoogle Scholar
- Buddrus-Schiemann K, et al. Root colonization by Pseudomonas sp. DSMZ 13134 and impact on the indigenous rhizosphere bacterial community of barley. Microb Ecol. 2010;60(2):381–93.View ArticlePubMedGoogle Scholar
- Mäder P, et al. Inoculation of root microorganisms for sustainable wheat–rice and wheat–black gram rotations in India. Soil Biol Biochem. 2011;43(3):609–19.View ArticleGoogle Scholar
- Haque M, Khan M. Effects of phosphatic biofertilizer with inorganic and organic sources of phosphorus on growth and yield of lentil. J Environ Sci Nat Resour. 2013;5(2):225–30.Google Scholar
- Moszcyńska E, Pytlarz-Kozicka M, Grzeszczuk J. The impact of applying biological treatment on the infection of potato tubers by the fungus rhizoctonia solani and the bacterium streptomyces scabiei. J Res Appl Agric Eng. 2015;60(4):46.Google Scholar
- Von Rad U, Mueller MJ, Durner J. Evaluation of natural and synthetic stimulants of plant immunity by microarray technology. New Phytol. 2005;165(1):191–202.Google Scholar
- Arndt W. Pseudomonas used for the treatment of plants and/or seeds and incubated in a culture containing phosphorus compounds, nitrogen compounds and succinic acid. 2005. Google Patents.Google Scholar
- Fröhlich A, et al. Response of barley to root colonization by Pseudomonas sp. DSMZ 13134 under laboratory, greenhouse, and field conditions. J Plant Interact. 2012;7(1):1–9.View ArticleGoogle Scholar
- Krebs B, et al. Use of Bacillus subtilis as biocontrol agent. I. Activities and characterization of Bacillus subtilis strains. Zeitschrift für Pflanzenkrankheiten und Pflanzenschutz. 1998;105(2):181–97.Google Scholar
- Borriss R, et al. Relationship of Bacillus amyloliquefaciens clades associated with strains DSM 7(T) and FZB42(T): a proposal for Bacillus amyloliquefaciens subsp amyloliquefaciens subsp nov and Bacillus amyloliquefaciens subsp plantarum subsp nov based on complete genome sequence comparisons. Int J Syst Evol Microbiol. 2011;61:1786–801.View ArticlePubMedGoogle Scholar
- Borriss R. Use of plant-associated Bacillus strains as biofertilizers and biocontrol agents in agriculture, in bacteria in agrobiology: plant growth responses. Berlin: Springer; 2011. p. 41–76.View ArticleGoogle Scholar
- Bákonyi N, et al. Comparison of effects of different biofertilisers on early development of cucumber and wheat seedlings. In Zbornik Radova 44. Hrvatski i 4 Medunarodni Simpozij Agronoma, Opatija, Hrvatska, 16–20 Veljače 2009. 2009. Poljoprivredni Fakultet Sveučilišta Josipa Jurja Strossmayera u Osijeku.Google Scholar
- VDLUFA M. Band 1. Die Untersuchung von Böden. Darmstadt: VDLUFA-Verlag; 1991.Google Scholar
- Van der Paauw F, Sissingh HA, Ris J. An improved method of water extraction for the assessment of soil phosphate supply: Pw value. Verslag. Landbollwk. Onderzoek. 1971. 749. (Dutch with an English summary).Google Scholar
- Murphy J, Riley JP. A modified single solution method for the determination of phosphate in natural waters. Anal Chim Acta. 1962;27:31–6.View ArticleGoogle Scholar
- Mason S, et al. Prediction of wheat response to an application of phosphorus under field conditions using diffusive gradients in thin-films (DGT) and extraction methods. Plant Soil. 2010;337(1–2):243–58.View ArticleGoogle Scholar
- Team RC. A language and environment for statistical computing. Vienna: R Foundation for Statistical Computing; 2014.Google Scholar
- Chessel D, Dufour AB, Thioulouse J. The ade4 package-I-One-table methods. R News. 2004;4(1):5–10.Google Scholar
- Reuter DJ. Plant analysis: an interpretation manual. Clayton: CSIRO Publishing; 1997.Google Scholar
- Khan MS, Zaidi A, Wani PA. Role of phosphate-solubilizing microorganisms in sustainable agriculture—a review. Agron Sustain Dev. 2007;27(1):29–43.View ArticleGoogle Scholar
- Qiao JQ, et al. Stimulation of plant growth and biocontrol by Bacillus amyloliquefaciens subsp. plantarum FZB42 engineered for improved action. Chem Biol Technol Agric. 2014;1(1):1–14.View ArticleGoogle Scholar
- Lugtenberg BJ, Dekkers LC. What makes Pseudomonas bacteria rhizosphere competent? Environ Microbiol. 1999;1(1):9–13.View ArticlePubMedGoogle Scholar
- Kucey R, Janzen H, Leggett M. Microbially mediated increases in plant-available phosphorus. Adv Agron. 1989;42(199228):60525–8.Google Scholar
- Brady NC, Weil RR. The nature and properties of soils. Upper Saddle River: Prentice-Hall Inc.; 1996.Google Scholar
- MST. Executive order on the use of waste for agricultural purposes (Bekendtgørelse om anvendelse af affald til jordbrugsformål, Slambekendtgørelsen). Danish environmental protection agency (Miljøstyrelsen): Copenhagen, Denmark. 2006. (In Danish).Google Scholar
- German Federal Ministry for food, a.a.c.p., Regulation on the placing of fertilisers, soil additives, growing media and plant additives (Verordnung über das Inverkehrbringen von Düngemitteln, Bodenhilfsstoffen, Kultursubstraten und Pflanzenhilfsmittel. Bundesgesetzblatt Teil I, 2012; 58. (In German).Google Scholar
- Frossard E, et al. The fate of sludge phosphorus in soil-plant systems. Soil Sci Soc Am J. 1996;60(4):1248–53.View ArticleGoogle Scholar
- Schiemenz K, et al. Phosphorus fertilizing effects of biomass ashes, in recycling of biomass ashes. Berlin: Springer; 2011. p. 17–31.View ArticleGoogle Scholar
- Christel W, et al. Pig slurry acidification, separation technology and thermal conversion affect phosphorus availability in soil amended with the derived solid fractions, chars or ashes. Plant Soil. 2016;401(1):93–107.Google Scholar