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Survival and phosphate solubilisation activity of desiccated formulations of Penicillium bilaiae and Aspergillus niger influenced by water activity
Journal of Microbiological Methods 150 (2018) 39–46
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Survival and phosphate solubilisation activity of desiccated formulations of Penicillium bilaiae and Aspergillus niger influenced by water activity
T
⁎
Nelly Sophie Raymonda, Dorette Müller Stövera, , Lars Stoumann Jensena, Sebastian Håkanssonb a
Plant and Soil Science Section, Department of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Denmark b Department of Molecular Sciences, Swedish University of Agricultural Sciences, P.O. Box 7015, 750 07 Uppsala, Sweden
A R T I C LE I N FO
A B S T R A C T
Keywords: Phosphate solubilising microorganisms Convective air drying Fluidized bed drying Sewage sludge ash Vermiculite Shelf-life
The impact of formulation and desiccation on the shelf life of phosphate (P)-solubilising microorganisms is often under-studied, particularly relating to their ability to recover P-solubilisation activity. Here, Penicilllium bilaiae and Aspergillus niger were formulated on vermiculite (V) alone, or with the addition of protectants (skimmed milk (V + SM) and trehalose (V + T)), and on sewage sludge ash with (A + N) and without nutrients (A), and dried in a convective air dryer. After drying, the spore viability of P. bilaiae was greater than that of A. niger. V formulations achieved the highest survival rates without being improved by the addition of protectants. P. bilaiae formulated on V was selected for desiccation in a fluidised bed dryer, in which several temperatures and final water activities (aw) were tested. The highest spore viability was achieved when the formulation was dried at 25 °C to a final aw > 0.3. During three months' storage, convective air dried formulations were stable for both strains, except in the presence of skimmed milk for P. bilaiae which saw a decrease in spore viability. In the fluidised bed-dried formulations, when aw > 0.3, the loss in viability was higher, especially when stored at 20 °C, than at aw < 0.1. P-solubilisation activity performed on ash was preserved in most of the formulations after desiccation and storage. Overall, a low drying temperature and high final aw positively affected P. bilaiae viability, however a trade-off between higher viability after desiccation and shelf life should be considered. Further research is needed to optimise viability over time and on more sustainable carriers.
1. Introduction Microbial inoculants are a promising tool for enhancing crop growth and productivity (Bhardwaj et al., 2014; Leggett and Gleddie, 1995; Singh et al., 2011). Phosphate-solubilising microorganisms (PSM) are among the beneficial microorganisms that may be inoculated to plants or soil with the aim of enhancing phosphorus (P) uptake by plants. In some agricultural soils, large amounts of P have been accumulated in past decades (Sattari et al., 2012). However, the accumulated P is often insufficiently available for plants, and PSMs have been suggested as a possibility for improving P bioavailability in soils or in different types of residual products with low P bioavailability (Adesemoye and Kloepper, 2009; Sharma et al., 2013). PSMs often show promising results in vitro in solubilising different forms of P, however when they are applied to soil/plant systems, their performances are inconsistent, as is the case with other microbial inoculants (Herrmann and Lesueur, 2013; Möller et al., 2017; Richardson and Simpson, 2011). Microorganisms are sensitive to environmental changes, and the different steps in the inoculum production can decrease cell survival and performance. An effective ⁎
Corresponding author. E-mail address: [email protected] (D. Müller Stöver).
https://doi.org/10.1016/j.mimet.2018.05.012 Received 23 April 2018; Received in revised form 16 May 2018; Accepted 16 May 2018 Available online 17 May 2018 0167-7012/ © 2018 Elsevier B.V. All rights reserved.
formulation is therefore often necessary to alleviate viability loss (Herrmann and Lesueur, 2013). Conventionally, dried formulations are preferred for microorganism preparation and storage (Morgan et al., 2006; Parnell et al., 2016). The desiccation allows cells to remain viable for long periods by inhibiting the metabolism and avoiding the growth of biological contaminants (Friesen et al., 2005, Friesen et al., 2006a, 2006b). Although drying is recognised as an efficient way of preserving microbial inoculants, the process inflicts stress on the microorganisms, and survival rates tend to be low (Friesen et al., 2005; Fu and Chen, 2011; Tadayyon et al., 1997). However, different drying techniques, carriers and protectants have been shown to significantly improve the survival rate of microorganisms (Friesen et al., 2004, 2005; Tadayyon et al., 1997). Shelf life is another important feature when developing a microbial formulation and appears to be highly dependent on the species and specific strain used (Antheunisse and Arkesteijn-Dijksman, 1979; Clerk and Madelin, 1965; Fu and Chen, 2011). Furthermore, most of the work on microorganism desiccation has focused on the formulation and survival rate, and often omitted to test the ability of the microorganisms to continue
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performing the desired activity after rehydration. Drying can affect survival, but can also increase the lag time before the beneficial effects of the inoculants become observable or suppress some metabolic activities (Moënne-Loccoz et al., 1999). In previous work, the possibility of using P-rich sewage sludge ash as an inoculation carrier and a P source to be solubilised by PSM was tested to develop a novel bio-based fertiliser (Raymond et al., 2018). PSM were able to grow on ash and solubilise P after the addition of specific carbon (C) and nitrogen (N) sources. With the goal of testing the bio-based fertiliser in large field experiments and eventually developing a commercial product, a formulation should be developed that is stable over time, inexpensive and environmentally friendly (Melin et al., 2011). Therefore different combinations of PSM, carriers and protectants were screened in a slow-drying convective air dryer and a fluidised bed dryer, representing a faster drying method often used in industrial applications. Two P-solubilising fungal strains were used: Penicillium bilaiae and Aspergillus niger. A different strain of P. bilaiae has previously been studied extensively by Tadayyon et al. (1997) and Friesen et al. (2004, 2005, 2006a, 2006b) for convective drying and fluidised bed drying on different carriers and under various desiccation conditions. Despite relatively high survival rates, the P-solubilisation recovery of the P. bilaiae strain after drying was not tested. However, to the best of the authors' knowledge, no work is currently available on drying methods for A. niger. Few studies are offered on the preservation of spores of P-solubilising fungi, however more work has been done on biocontrol fungi, many of them using the “Pesta” formulation (Elzein et al., 2004; Friesen et al., 2005; Shabana et al., 2003) or alginate encapsulation (Daigle and Cotty, 1995; Vassilev et al., 1997, 1998). Another desiccation carrier, micronised vermiculite, can be used for PSM. Vermiculite is a mineral possessing interesting characteristics for consideration as a desiccation carrier for a microbial inoculant because of its neutral pH and high porosity. Moreover when vermiculite is micronised, it also has the advantage of being relatively disperable in water and can therefore represent a practical way of inoculating seeds or different substrates, such as the insoluble form of P. Alternative carriers, such as recycled urban residues, are rarely used as desiccation and storage carriers and can be a inexpensive and renewable alternative to vermiculite. With the objective of inoculating sewage sludge ash with PSM to enhance P solubility, it is therefore of interest to also test ash as a desiccation and storage carrier, with and without the nutrient enrichment necessary for an optimal P-solubilisation. However, the ash exhibits an alkaline pH compared to conventional carriers and may jeopardise PSM survival. The hypotheses tested were as follows: (1) the ash carrier used for desiccation decreases the PSM survival rate due to a lower porosity and higher pH than the vermiculite, (2) the addition of a protectant or nutrients (skimmed milk, trehalose or nutrient mix) improves spore viability during the drying process and storage, (3) the survival rates of the spores are dependent on the drying conditions – low temperature, slow-drying kinetics and higher final water activity favour their survival – and P. bilaiae and A. niger have different optimal drying conditions, (4) low temperatures during storage reduce viability loss in all the formulations, and (5) the surviving spores have the same ability to solubilise P as fresh spores.
Table 1 pH and surface area of the two desiccation carriers. Properties
MEV vermiculitea
Ashb
pH Surface area (m2 g−1) Particles size (μm)
7–8 9.9 0.10–0.65
9.2 5.1 < 250
a b
MEV vermiculite characteristics from Dupré Minerals. Ash characteristics from Raymond et al. (2018).
from mycelium by filtering the suspension through sterile glass wool (Miracloth, EMD Millipore Corporation, Billerica, MA 01821 USA). The filtrate was centrifuged at 16 x g and 4 °C for 10 min. 2.2. Carriers and formulation Carrier A was prepared by mixing 25 g of dry sewage sludge incineration ash (Raymond et al., 2018; Thomsen et al., 2017) (Table 1) with 12.5 mL of inoculum suspension (ratio 2:1). The spore concentration of the inoculum was determined using a hemacytometer (Bürker, Brand, Germany) and adjusted to obtain 1 × 107 spores per gram of ash (based on the ash dry weight (dw)). For the nutrient-enriched formulation carrier A + N, P. bilaiae spores were suspended in a solution containing 107 g L−1 of fructose and 3.1 g L−1 (NH4)2SO4, and A. niger spores in a solution containing 107 g L−1 and 1.9 g L−1 NH4NO3. Spore concentration per g ash and moisture content were adjusted to obtain the same levels as in carrier A. Carrier V consisted of 25 g (dw) vermiculite (micronised vermiculite (MEV), Dupré Minerals Ltd., Staffordshire, England, Table 1), mixed with the inoculum at a moisture rate of 1:1.25. The spore concentration was adjusted to 1 × 108 spores per gram of vermiculite. Two protectants were also tested with the vermiculite: skimmed milk powder (10% w/w) (V + SM) and trehalose solution (inoculum added in 10% trehalose solution) (V + T), as these have earlier been shown to be appropriate desiccation carriers (Fu and Chen, 2011; Morgan et al., 2006). Carriers and nutrient solutions were sterilised individually by autoclaving for 20 min at 121 °C, whereas the trehalose solution was filtersterilised (0.2 μm, Nalgene, Thermo Fisher Scientific, Massachusetts, USA). All the formulations tested were produced in three independent replicates before being dried. 2.3. Convective air drying and storage of the dried materials The wet formulation mixtures were placed in Petri dishes in a convective air dryer, which consisted of a Plexiglas box connected to forced ventilation (1.6 m3 min−1). Samples were dried at room temperature (22 °C) with an average relative humidity (RH) of 60% until stabilisation of the sample weight, corresponding to about 36 h drying. Two sub-samples was kept to determine survival after drying, with the remaining of the sample divided into 2.5 g subsamples that were sealed air-tight in aluminium bags and stored at either 2 °C or 20 °C. Viability of the fungal strains in the stored products was tested after two weeks, after one month and after three months.
2. Materials and methods 2.4. Fluidised bed drying and storage of the dried materials 2.1. Phosphate solubulising microorganisms strains and inoculum preparation
From the convective air drying test, the formulation combining V and P. bilaiae was the most interesting due to its higher survival rate and lower formulation cost, and was therefore chosen for further investigations in a fluidised bed dryer (Fluid Bed Dryer, FBD 2000, Endecotts, London, UK). A first step was to evaluate the effect of different inlet temperatures. The drying process was monitored by measuring the outlet temperature and final water activity (Aqualab, METER Group, Inc. USA). The water activity (aw) targeted was aw < 0.1
Two PSM strains were tested: Penicillium bilaiae DBS-5, provided by Novozymes (Denmark), and Aspergillus niger (ATCC 9142). From frozen (−80 °C) spore stocks, fungal strains were propagated on potato dextrose agar (PDA) plates for about two weeks before being transferred to a new PDA plate. Spores from the second generation were then collected by washing the plate with sterile MilliQ water and then separated 40
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(RH < 10%). The airflow was adjusted to ensure appropriate fluidisation of the product and inlet temperatures were set to 25 °C for 25 min, 25 °C for 12.5 min then 40 °C for 5 min (25 °C + 40 °C), and 40 °C for 10 min, respectively. In a second round of tests, the effect of the final aw was tested with the same airflow and a 25 °C inlet temperature for 15 min in order to obtain an aw of 0.3, and for 14 min to obtain an aw of 0.5. For both desiccation experiments, three independent replicates were produced. After drying, two sub-samples were used to evaluate the spore survival after drying and the rest of the samples were processed for storage as described above for the convective air drying.
Table 2 Water activities (aw) recorded before and after desiccation with convective air drying. Values are the mean (n = 3) ± S.E. Formulations
aw before drying ( ± SE)
aw after drying ( ± SE)
A A+N V V + SM V+T
0.999 1.000 0.999 0.996 0.993
0.174 0.162 0.160 0.155 0.145
± ± ± ± ±
0.000 0.000 0.002 0.002 0.000
± ± ± ± ±
0.024 0.023 0.023 0.021 0.019
level set at p < 0.05). For the fluidised bed-drying assay, the effect of the different treatments (temperature, aw) was tested by a two-way Anova followed by an HSD post hoc test (Agricolae package, significance level set at p < 0.05). For the P-solubilisation biotest, the different treatments were compared to the performance of the control with fresh spores, including the effect of the additive and storage temperature, by a multivariate linear model including interactions, followed by an HSD post hoc test (Agricolae package, significance level set at p < 0.05).
2.5. Spore viability and phosphate solubilisation activity biotest Before and after drying, two sub-samples of 0.5 g (dw) of each inoculated carrier were shaken with 10 mL sterile distilled water (DI) amended with 0.05% Tween-80 for 20 min. The suspension was serially diluted and 100 μL of two different appropriate dilutions plated on PDA plates. Each dilution was plated in triplicate. The inoculated plates were incubated for about two days at 25 °C and the colonies counted. Plates with between 20 and 200 colonies were used to determine the viable cells. To establish the survival rate, the viable spore concentration after drying was divided by the viable spore concentration before drying. Spore viability in the stored samples was assessed accordingly. Phosphate solubilisation activity was tested by incubating the sewage sludge ash with the dried formulations. 1 g of dried and inoculated carrier A was weighed into a 15 mL tube and 0.420 mL of the same nutrient solution as described above added. The A + N treatment, which had already been amended with nutrients before drying, received only 0.420 mL sterile deionised water (DI) water. For the treatments containing vermiculite carriers (V, V + SM, V + T), a subsample of 0.6 g was suspended in 2.5 mL nutrient solution and thoroughly mixed. The suspension was then applied to 1 g sterile ash to obtain a spore concentration of 1 × 107 spores per gram of ash based on the original spore concentration before drying. After mixing the different carriers with the ash, the preparations were loaded into 1.2 mL deep 96 well plates (ABgene Storage, Thermo Fisher Scientific, Massachusetts, USA) using a MicroResp™ filling device. Weights of each treatment were recorded to determine the mass of ash per well (about 150 μg ash (dw) per well). Water content was adjusted to 45%. The plates were then sealed in a plastic bag and incubated at 25 °C in the dark. After 12 days' incubation for P. bilaiae and 10 days' for A. niger, three samples from each treatment were extracted to measure water-extractable P (WEP). Briefly, the ash samples from the well were shaken with DI water at a ratio of 1:60 (dwt:vol) at 150 rpm for 16 h. After centrifugation and filtration through Whatman® filter paper no. 5, the orthophosphate concentration of the filtered extract was measured with the molybdate blue-ascorbic acid assay, as described in Murphy and Riley (1962) and Watanabe and Olsen (1965). To compare the P-solubilisation to a fresh inoculum, ash was inoculated with either 1 × 106 or 1 × 107 spores per gram of ash and incubated under the same conditions as described above. These control treatments were repeated three times at the same time as after drying.
3. Results 3.1. Desiccation survival 3.1.1. Convective air drying Convective air drying was used to determine the impact of different desiccation carriers and formulations on the viability of two selected Psolubilising fungi after drying. Before drying, all the carriers had a water activity (aw) close to 1, and of between 0.145 and 0.175 after drying (Table 2). After the drying process, P. bilaiae showed a significantly higher survival on vermiculite carriers than on ash (Fig. 1) (p < 0.0001). The addition of skimmed milk or trehalose to vermiculite or nutrients to the ash did not affect their survival (p > 0.05). In contrast, the survival of A. niger spores was not affected by the type of carrier or by the addition of a protectant during the drying, and was generally lower than P. bilaiae (p > 0.05). In most cases for P. bilaiae, the vermiculite carriers supported a higher relative viability than the ash over time, but only because the survival after drying was higher. The storage temperature and the supplementation of the carriers with protectants or nutrients had inconsistent effects on spore survival; only the addition of skimmed milk significantly reduced spore viability, especially at 20 °C (p < 0.05). For A. niger, over the whole storage period, spore viability was greater on vermiculite carriers than on ash (p < 0.01). The highest viability was observed after three months' storage in most of the carriers. Spore viability on V, V + SM and A was affected by the storage temperature (p < 0.05), with a higher viability observed at 2 °C than at 20 °C. As also observed for P. bilaiae, skimmed milk as a protectant had a negative effect on the spore viability of A. niger on vermiculite. 3.1.2. Fluidised bed drying Fluidised bed drying was then used on the formulation combining P. bilaiae and V because the formulation was more promising due to its higher survival rate and lower cost with convective air drying. The water activities measured (Table 3) were within the targeted ranges. In the fluidised bed drying, survival was generally better than in the convective drying, and the highest spore survivals rates were recorded for the drying temperature of 25 °C (p < 0.0001) and final aw > 0.1 (p < 0.0001) (Fig. 2A). In most of the treatments, a significant decrease in spore viability was observed after storage of two weeks and of one month, except for the spores dried at 40 °C that remained stable (Fig. 2B and C). For the formulations including a final aw > 0.3, loss of viability was larger overall (p < 0.0001). This was particularly true for
2.6. Statistical analyses Statistical analysis was performed in R (R i386 3.3.2, GNU Project) using the RStudio development environment. The survival after convective air drying was analysed by a two-way Anova, including the effect of the carrier and the additives as factors, followed by an HSD post hoc test (Agricolae package (De Mendiburu, 2016), significance level set at p < 0.05). The effects of storage time and temperature on each carrier were tested. For each time point in the viability check, the effects of the carrier, storage temperature and protectant/nutrients were tested in a simple multivariate linear model including interactions, followed by an HSD post hoc test (Agricolae package, significance 41
Journal of Microbiological Methods 150 (2018) 39–46
40
a a a
30
b b
20
10
0 V
V+SM
V+T
A
V V+SM V+T A A+N
40
30
20
10
0
A+N
0,0
0,5
1,0
1,5
2,0
2,5
o
B
50
P. bilaiae viability (%) stored at 20 C
A
o
50
P. bilaiae viability (%) stored at 2 C
P. bilaiae viability (%) after drying
N.S. Raymond et al.
50
C
V V+SM V+T A A+N
40
30
20
10
0
3,0
0,0
0,5
30
20 a
a
10
a
a
a
A
A+N
V V+SM V+T A A+N
40
30
20
10
0
0 V
V+SM
V+T
0,0
0,5
1,0
1,5
2,0
2,5
o
o
40
E
50
1,0
1,5
2,0
2,5
3,0
Time (month) A. niger viability (%) stored at 20 C
D
50
A. niger viability (%) stored at 2 C
A. niger viability (%) after drying
Time (month) F
50
V V+SM V+T A A+N
40
30
20
10
0
3,0
0,0
0,5
Time (month)
1,0
1,5
2,0
2,5
3,0
Time (month)
Fig. 1. Spore viability after drying for P. bilaiae (A) and A. niger (D) and shelf life over time with storage at 2 °C (P. bilaiae (B), A. niger (E)) and 20 °C (P. bilaiae (C), A. niger (F)) (V: vermiculite; V + SM: vermiculite + skimmed milk; V + T: vermiculite + trehalose; A: ash; A + N: ash + nutrients). Values are the mean (n = 18) ± S.E. (bars)).
the formulation with aw = 0.5 stored at 20 °C. Between one and three months’ storage, the viability was stable in most of the formulations.
Table 3 Water activities (aw) recorded before and after desiccation of P. bilaiae spores on vermiculite with fluid bed drying. Values are the mean (n = 3) ± S.E. Drying treatment, target aw
aw before drying ( ± SE)
aw after drying ( ± SE)
25 °C aw < 0.1 25 °C aw = 0.3 25 °C aw = 0.5 25 °C + 40 °C aw < 0.1 40 °C aw < 0.1
0.998 0.998 0.999 0.999 1.001
± ± ± ± ±
0.114 0.292 0.543 0.053 0.068
a
a
80
60
b
40 c
20
d
3.2.1. Convective air drying Recovery of the P-solubilisation activity after desiccation and storage was required in the elaboration of the formulation and was compared to two controls with fresh spores. P. bilaiae showed a significant increase in P-solubilisation at a rate of 1 × 107 spores per gram of ash compared to 1 × 106 spores per gram (p < 0.0001), whereas similar amounts of solubilised P at both inoculation rates were observed for A. niger (p > 0.05) (Fig. 3). After drying, the P-solubilisation activity of P. bilaiae was between the levels of the two controls with freshly applied spores. A similar pattern was observed for A. niger, with the exception of the V + SM carrier, where less P was solubilised compared to the controls. Phosphate solubilisation levels of P. bilaiae remained relatively
0.012 0.015 0.038 0.004 0.007
80
B
25 oC aw 0.1 25 oC aw 0.3 25 oC aw 0.5 25 oC + 40 oC aw 0.1 40 oC aw 0.1
60
40
20
0
0
25C
aw
0.5 w 0.3 w 0.1 w 0.1 w 0.1 a a a a 25C 2 5 C + 40 C 40C 25C
P. bilaiae viability (%) stored at 20 oC
A
± ± ± ± ±
P. bilaiae viability (%) stored at 2 oC
P. bilaiae viability (%) after drying
80
0.001 0.000 0.001 0.001 0.001
3.2. Phosphate solubilisation activity
C
25 oC aw 0.3 25 oC aw 0.5 25 oC aw 0.1 25 oC + 40 oC aw 0.1 40 oC aw 0.1
60
40
20
0 0,0
0,5
1,0
1,5
2,0
Time (month)
2,5
3,0
0,0
0,5
1,0
1,5
2,0
2,5
3,0
Time (month)
Fig. 2. Spore viability after drying for P. bilaiae using the fluid bed dryer (A) and shelf life over time with storage at 2 °C (B) and 20 °C. (25C aw 0.1: vermiculite dried at 25 °C and final aw = 0.1; 25 °C aw 0.3: vermiculite dried at 25 °C and final aw = 0.3; 25C aw 0.5: vermiculite dried at 25 °C and final aw = 0.5; 25 °C + 40 °C aw 0.1: vermiculite dried at 25 °C + 40 °C and final aw = 0.1; 40C aw 0.1: vermiculite dried at 40 °C and final aw = 0.1). Values are the mean (n = 18) ± S.E. (bars)). 42
Journal of Microbiological Methods 150 (2018) 39–46
-1
P. bilaiae P-solubilisation activity (mg P g ash)
N.S. Raymond et al.
4
3
2
1
0 con
A. niger P-solubilisation activity (mg P g-1 ash)
1 month 20 oC 3 months 2 oC 3 months 20 oC
After drying 2 weeks 2 oC 2 weeks 20 oC 1 month 2 oC
Fresh inoculum 106 spores g-1 Fresh inoculum 107 spores g-1
V
V+SM
3
V+T
A
1 month 20 oC 3 months 2 oC 3 months 20 oC
After drying 2 weeks 2 oC 2 weeks 20 oC 1 month 2 oC
Fresh inoculum 106 spores g-1 Fresh inoculum 107 spores g-1
A+N
2
1
0 con
V
V+SM
V+T
A
A+N
Fig. 3. Phosphate solubilisation activity over time on the different desiccation carriers using the convective air drier (V: vermiculite; V + SM: vermiculite + skimmed milk; V + T: vermiculite + trehalose; A: ash; A + N: ash + nutrients). Values are the mean (n = 9) ± S.E. (bars)).
preparing a microbial formulation to ensure a certain number of viable cells in the final product. The carriers and their characteristics represent a real opportunity to improve cell viability during the desiccation process (Morgan et al., 2006). By using the convective air drying method, clear differences between vermiculite and ash carriers in spore viability were observed for P. bilaiae. Although A. niger did not display a higher viability on one of the two tested carriers after drying, the fungal strain's desiccation tolerance and carrier preference was clearly demonstrated. The viability of P. bilaiae spores was greater on vermiculite than on the ash carriers, and probably resulted from the physical structure of the two materials, such as their porosity (Vanek et al., 2016). The higher porosity of the vermiculite may have provided a superior protective environment against desiccation compared to the ash for the P. bilaiae spores, which at 1–3.5 μm (Buttner and Stetzenbach, 1993; Tadayyon et al., 1997) were smaller than the A. niger spores (3–5 μm (Silva et al., 2011)). The pH of the carrier or formulation is often mentioned as affecting cell viability during drying (Morgan et al., 2006). For bacteria, it is reported that the exposure to a low pH induces morphological changes in the cells and can generate a positive stress, especially if the cells are exposed to an acidic pH before desiccation (Fu and Chen, 2011; Morgan et al., 2006). The effect of an alkaline pH on spore viability has not so far been documented. If the alkalinity of the ash were a barrier providing optimal resistance to desiccation, it would be interesting to explore a pre-adaptation to
constant and similar to the controls over time. Storage temperature only affected A where higher P-solubilisation rates were observed when samples were stored at 2 °C after one and three months' storage (p < 0.0001). The different protectants/nutrients did not affect the ability of P. bilaiae to solubilise P from the ash, only trehalose tended to increase P-solubilisation activity. For A. niger, after two weeks' storage all the treatments had a Psolubilisation activity comparable to the controls with fresh spores. After one month of storage, treatments A, A + N and V + SM showed a reduced P-solubilisation activity. 3.2.2. Fluidised bed drying Phosphate solubilisation activity was also tested for the selected formulation, P. bilaiae and V. After fluidised bed drying, all the treatments had a P-solubilisation rate similar to that of the fresh spore controls (Fig. 4). Over time, only the final aw of the treatment impacted the ability to solubilise P from the ash (p < 0.0001) with an overall lower P-solubilisation for treatments with aw < 0.1. 4. Discussion 4.1. Desiccation survival Obtaining a high survival rate after drying is essential when 43
Journal of Microbiological Methods 150 (2018) 39–46
1 month 20 oC 3 months 2 oC 3 months 20 oC
After drying 2 weeks 2 oC 2 weeks 20 oC 1 month 2 oC
Fresh inoculum 106 spores g-1 Fresh inoculum 107 spores g-1 4
-1
P. bilaiae P-solubilisation (mg P g ash)
N.S. Raymond et al.
3
Fig. 4. Phosphate solubilisation activity over time using the fluid bed drier (25 °C aw 0.1: vermiculite dried at 25 °C and final aw = 0.1; 25 °C aw 0.3: vermiculite dried at 25 °C and final aw = 0.3; 25 °C aw 0.5: vermiculite dried at 25 °C and final aw = 0.5; 25 °C + 40C aw 0.1: vermiculite dried at 25 °C + 40 °C and final aw = 0.1; 40C aw 0.1: vermiculite dried at 40 °C and final aw = 0.1). Values are the mean (n = 9) ± S.E. (bars)).
2
1
0 con
25
w0 Ca
.5
0.3 aw 25C
C 25
0.1 aw C 25
0C +4
0 aw
.1 C 40
0 aw
.1
generating a large loss of viability during drying. This could also be the reason why the final survival rates after convective air drying were generally lower than in the fluidised bed dyer with a higher final aw. Friesen et al. (2005) found similar survival rates (60–80%) for P. bilaiae dried with a similar inlet temperature (20–30 °C) and at the same RH (60%) for aw > 0.3. In relation to the findings in the convective air drying experiment in which aw < 0.175 and lower viability were recorded, the authors also reported that drying the formulation below aw < 0.2 highly decreased spore viability. For a comparable final aw between the convective and fluidised bed drying, fluidised bed drying resulted in a higher survival rate, despite several studies previously concluding that a slow drying process is favourable for survival (Fu and Chen, 2011; Moënne-Loccoz et al., 1999). However, Fu and Chen (2011) also mentioned that a longer drying time exposes the microorganisms to stress for a longer period and can therefore generate viability loss. Thus a higher final aw generally led to higher spore survival, reinforcing the third hypothesis. However the slow drying kinetic did not lead to greater spore viability, in contrast to this same hypothesis in terms of kinetics.
alkaline conditions to determine whether a period of acclimatisation improves fitness to survive or causes damage to the spores (Fu and Chen, 2011; Melin et al., 2011; Morgan et al., 2006). Nevertheless, it would be interesting to pursue optimisation of sewage sludge ash or other thermally treated residue formulations as desiccation carriers as they represent a low-cost and renewable carrier. Recent studies have shown the positive effects of biochar as a microbial carrier (Hale et al., 2015; Vanek et al., 2016). To further enhance the viability of these alternatives carriers, different options can also be considered, such as a pre-adaptation or a pre-establishment of the fungal strain on the carrier, which in some cases has demonstrated a positive effect in terms of spore viability after desiccation for a biocontrol Pseudomonas fluorescens (Moënne-Loccoz et al., 1999). Thus, depending on the fungal strain, this study's first hypothesis regarding the effect of porosity could be confirmed, although the impact of carrier pH can only be speculated. The difference observed between the two types of carrier for P. bilaiae may also be the result of the different spore concentrations in the formulations leading to different recoveries. However this was not be shown with A. niger and it has also been found that for Fusarium oxysporum spore formulation, the concentration did not affect the viability after drying (Connick et al., 1998). This assumption must therefore be rejected. Although the addition of protectants during the drying process is frequently reported as resulting in the reduction of viability loss (Melin et al., 2011; Shabana et al., 2003), no clear effects were found in this study. In the formulation of fungal spores, the effects of protectants have been found to be fairly inconsistent given that spores by their nature are a natural preservation form (Shabana et al., 2003). The absence of an effect on spore viability during drying with the desiccation protectants allowed the second hypothesis to be partly rejected. Compared to the convective air drying system used first in this study, fluidised bed drying represented a faster method for air drying microorganisms and allowed more parameters to be controlled. Fluidised bed drying was only tested with P. bilaiae and vermiculite as this combination had shown the greatest tolerance to desiccation in the experiments on convective air drying. A drying temperature of 25 °C resulted in the highest survival rates, while increased temperatures greatly reduced spore survival, confirming the thermal sensitivity of the species (Friesen et al., 2005; Tadayyon et al., 1997) and supporting this study's third hypothesis. When dried at 25 °C in the fluidised bed dryer, a final aw at 0.3 and 0.5 induced a higher spore survival than a final aw at 0.1. Insufficient humidity in the formulation has been described by Beker et al. (1984) as creating damage to the yeast cell membrane,
4.2. Formulation shelf life During the first two weeks of storage, the spore viability generally decreased, except for the formulations with aw < 0.1 in which viability was stable and P. bilaiae formulated with V and V + T by convective air drying, which showed an increase in viability. The loss in spore viability during the first period (two weeks to one month of storage) was highest in the materials dried in the fluidised bed dryer to a final aw of 0.3 and 0.5 and stored at 20 °C. The effect of greater moisture content on spore viability loss during storage has been suggested by Tadayyon et al. (1997) as probably being linked to enzyme activity recovery or the start of germination followed by death due to a lack of sufficient moisture and nutrients. In the present study, it seemed that the lower final water activity in the carrier favoured spore viability over time, which has also been confirmed by Friesen et al. (2006a, 2006b) who found the least spore viability loss for aw between 0.1 and 0.2 over a six-month storage period. Between one and three months after drying, the spore viability was comparatively stable for P. bilaiae or even increased for A. niger. The increase in spore viability was rather surprising, but the heterogeneity of spores in formulations may also have interacted with spore recovery, contributing to the general variability in survival rates. Storage in a cool environment is widely recognised to reduce cell 44
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Declarations of interest
viability loss, however formulations with a long shelf life at room temperature offer a financial advantage and are more convenient for users (Melin et al., 2011; Parnell et al., 2016). For both strains, the viability of the formulations stored after convective air drying was not affected by the storage temperature, except in the V + SM treatment with P. bilaiae. However, in the fluidised bed drying, storage at 2 °C reduced the loss of viability, especially in the formulations with aw 0.3 and aw 0.5, emphasising a trade-off between survival after drying and storage stability and partially confirming this study's fourth hypothesis. The loss of viability during the first months of storage may be reduced by using other storage conditions such as vacuumed-sealed bags or ambient nitrogen atmosphere that have beneficial effects on some microorganisms (Melin et al., 2011; Önneby et al., 2014). The rehydration step is also often neglected, but can play an important role in the recovery and real survival rate (Fu and Chen, 2011; Poirier et al., 1999). It was not tested in this study.
None. Acknowledgments The authors would like to thank Armando Hernández for his support with the sample analysis, Tobias Thomsen for providing the ashes and biochars, and Paul Gregory from Dupré Minerals for providing the vermiculite. This study was supported by Innovation Foundation Denmark (grant number 1308-00016B to the project Microbial biofertilizers for enhanced crop availability of phosphorus pools in soil and waste, MiCroP). References Adesemoye, A.O., Kloepper, J.W., 2009. Plant–microbes interactions in enhanced fertilizer-use efficiency. Appl. Microbiol. Biotechnol. 85, 1–12. Antheunisse, J., Arkesteijn-Dijksman, L., 1979. Rate of drying and the survival of microorganisms. Antonie Van Leeuwenhoek 45, 177–184. Beker, M., Blumbergs, J., Ventina, E., Rapoport, A., 1984. Characteristics of cellular membranes at rehydration of dehydrated yeast Saccharomyces cerevisiae. Appl. Microbiol. Biotechnol. 19, 347–352. Bhardwaj, D., Ansari, M.W., Sahoo, R.K., Tuteja, N., 2014. Biofertilizers function as key player in sustainable agriculture by improving soil fertility, plant tolerance and crop productivity. Microb. Cell Factories 13, 66. Buttner, M.P., Stetzenbach, L.D., 1993. Monitoring airborne fungal spores in an experimental indoor environment to evaluate sampling methods and the effects of human activity on air sampling. Appl. Environ. Microbiol. 59, 219–226. Clerk, G., Madelin, M., 1965. The longevity of conidia of three insect-parasitizing hyphomycetes. Trans. Br. Mycol. Soc. 48, 193–209. Connick, W., Daigle, D., Pepperman, A., Hebbar, K., Lumsden, R., Anderson, T., Sands, D., 1998. Preparation of stable, granular formulations containing Fusarium oxysporum pathogenic to narcotic plants. Biol. Control 13, 79–84. Daigle, D., Cotty, P., 1995. Formulating atoxigenic Aspergillus flavus for field release. Biocontrol Sci. Tech. 5, 175–184. De Mendiburu, F.A., 2016. Statistical Procedures for Agricultural Research. Version 1.2–4. Elzein, A., Kroschel, J., Müller-stöver, D., 2004. Optimization of storage conditions for adequate shelf-life of ‘Pesta'formulation of Fusarium oxysporum ‘foxy 2′, a potential mycoherbicide for Striga: effects of temperature, granule size and water activity. Biocontrol Sci. Tech. 14, 545–559. Elzein, A., Kroschel, J., Cadisch, G., 2008. Efficacy of Pesta granular formulation of Strigamycoherbicide Fusarium oxysporum f. sp. strigae foxy 2 after 5-year of storage. J. Plant Dis. Protect. 259–262. Friesen, T., Hill, G., Pugsley, T., Holloway, G., 2004. Optimization of the convective air drying of Penicillium bilaii for improved efficiency. Dry. Technol. 22, 1153–1172. Friesen, T., Hill, G., Pugsley, T., Holloway, G., Zimmerman, D., 2005. Experimental determination of viability loss of Penicillium bilaiae conidia during convective air drying. Appl. Microbiol. Biotechnol. 68, 397–404. Friesen, T., Hill, G., Pugsley, T., Holloway, G., 2006a. A mortality model for Penicillium bilaiae subjected to convective air drying. Can. J. Chem. Eng. 84, 488–494. Friesen, T.J., Holloway, G., Hill, G.A., Pugsley, T.S., 2006b. Effect of conditions and protectants on the survival of Penicillium bilaiae during storage. Biocontrol Sci. Tech. 16, 89–98. Fu, N., Chen, X.D., 2011. Towards a maximal cell survival in convective thermal drying processes. Food Res. Int. 44, 1127–1149. Hale, L., Luth, M., Crowley, D., 2015. Biochar characteristics relate to its utility as an alternative soil inoculum carrier to peat and vermiculite. Soil Biol. Biochem. 81, 228–235. Herrmann, L., Lesueur, D., 2013. Challenges of formulation and quality of biofertilizers for successful inoculation. Appl. Microbiol. Biotechnol. 97, 8859–8873. Leggett, M., Gleddie, S., 1995. 4 Developing Biofertilizer and Biocontrol Agents that Meet farmers' Expectations. Advances in Plant Pathology. vol. 11. pp. 59–74. Melin, P., Schnürer, J., Håkansson, S., 2011. Formulation and stabilisation of the biocontrol yeast Pichia anomala. Antonie Van Leeuwenhoek 99, 107–112. Moënne-Loccoz, Y., Naughton, M., Higgins, P., Powell, J., O'connor, B., O'gara, F., 1999. Effect of inoculum preparation and formulation on survival and biocontrol efficacy of Pseudomonas fluorescens F113. J. Appl. Microbiol. 86, 108–116. Möller, K., Oberson, A., Bünemann, E.K., Cooper, J., Friedel, J.K., Glæsner, N., Hörtenhuber, S., Løes, A.-K., Mäder, P., Meyer, G., 2017. Improved phosphorus recycling in organic farming: navigating between constraints. Adv. Agron. 147, 159–237. Morgan, C.A., Herman, N., White, P., Vesey, G., 2006. Preservation of micro-organisms by drying; a review. J. Microbiol. Methods 66, 183–193. Murphy, J., Riley, J.P., 1962. A modified single solution method for the determination of phosphate in natural waters. Anal. Chim. Acta 27, 31–36. Önneby, K., Håkansson, S., Pizzul, L., Stenström, J., 2014. Reduced leaching of the herbicide MCPA after bioaugmentation with a formulated and stored Sphingobium sp. Biodegradation 25, 291–300. Parnell, J.J., Berka, R., Young, H.A., Sturino, J.M., Kang, Y., Barnhart, D., DiLeo, M.V.,
4.3. Phosphate solubilisation activity In previous studies on P. bilaiae, the activity of P-solubilisation was not tested after desiccation. For both drying methods and both strains, the P-solubilisation activity was preserved after drying and during the storage period with some exceptions, confirming the final hypothesis. For P. bilaiae P-solubilisation was maintained in all the treatments or even increased in the V + T. This is not surprising as trehalose is potentially a source of carbon that P. bilaiae is able to use for its growth and metabolism, thereby stimulating P-solubilisation. An increase in Psolubilisation activity was also observed on ash after two weeks and after one month of storage. This was rather surprising, but may also be the result of the spore heterogeneity in the formulation. After the fluidised bed drying, a lower P-solubilisation activity was observed in the treatment dried at 40 °C, probably due to a decrease in fitness and viability. For A. niger, a decrease in P-solubilisation activity was observed over time on ash carriers, although viability was constant, translating a probable loss of fitness of the spores. P-solubilisation was also generally lower in the V + SM treatments, which in addition to the general viability loss could be explained by the fact that skimmed milk contained some easily available forms of P that the fungus may have used as a nutrient source, thus reducing the need to solubilise P from ash. The storage time in this study was up to three months and showed promising activity recovery, however some studies have explored the viability and activity of microorganisms over a longer period. For example Elzein et al. (2008) stored the mycoherbicide Fusarium oxysporum for five years at 4 °C and found only a slight loss in viability, but 100% efficiency on the targeted weed compared to a fresh formulation. 5. Conclusion Overall, this study has provided information on the possibility of dry-formulating two PSM on vermiculite and sewage sludge ash and their P-solubilisation activity recovery after drying. In a convective air drying system, the addition of desiccation protectants or nutrients did not affect the viability of the two fungal strains tested. However, A. niger had a lower viability during drying than P. bilaiae, showing the strain's differential tolerance in terms of desiccation. The survival rate of P. bilaiae was enhanced on vermiculite, and faster drying rates with the fluidised bed dryer positively influenced the strain's survival. Nevertheless, despite higher viability after desiccation in formulations with aw > 0.3, the viability loss during storage was significantly increased, especially at 20 °C. In terms of P-solubilisation activity, the ability of the two strains was secured after drying and storage, except when skimmed milk was added and when the viability loss was too great, underlining the importance of testing the targeted activity when formulating microorganisms. 45
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taxonomy. Braz. J. Microbiol. 42, 761–773. Singh, J.S., Pandey, V.C., Singh, D., 2011. Efficient soil microorganisms: a new dimension for sustainable agriculture and environmental development. Agric. Ecosyst. Environ. 140, 339–353. Tadayyon, A., Hill, G.A., Mike Ingledew, W., Sokhansanj, S., 1997. Contact-sorption drying of Penicillium bilaii in a fluidized bed dryer. J. Chem. Technol. Biotechnol. 68, 277–282. Thomsen, T.P., Sárossy, Z., Ahrenfeldt, J., Henriksen, U.B., Frandsen, F.J., Müller-Stöver, D.S., 2017. Changes imposed by pyrolysis, thermal gasification and incineration on composition and phosphorus fertilizer quality of municipal sewage sludge. J. Environ. Manag. 198, 308–318. Vanek, S., Thies, J., Wang, B., Hanley, K., Lehmann, J., 2016. Pore-size and water activity effects on survival of Rhizobium tropici in biochar inoculant carriers. J. Microb. Biochem. Technol. 8, 296–306. Vassilev, N., Vassileva, M., Azcon, R., 1997. Solubilization of rock phosphate by immobilized Aspergillus niger. Bioresour. Technol. 59, 1–4. Vassileva, M., Azcon, R., Barea, J.-M., Vassilev, N., 1998. Application of an encapsulated filamentous fungus in solubilization of inorganic phosphate. J. Biotechnol. 63, 67–72. Watanabe, F., Olsen, S., 1965. Test of an ascorbic acid method for determining phosphorus in water and NaHCO3 extracts from soil. Soil Sci. Soc. Am. J. 29, 677–678.
2016. From the lab to the farm: an industrial perspective of plant beneficial microorganisms. Front. Plant Sci. 7, 1110. Poirier, I., Maréchal, P.A., Richard, S., Gervais, P., 1999. Saccharomyces cerevisiae viability is strongly dependant on rehydration kinetics and the temperature of dried cells. J. Appl. Microbiol. 86, 87–92. Raymond, N.S., Stöver, D.M., Peltre, C., Nielsen, H.H., Jensen, L.S., 2018. Use of Penicillium bilaiae to improve P-bioavailability of thermally treated sewage sludge− a potential novel biofertiliser. Process Biochem (in press). Richardson, A.E., Simpson, R.J., 2011. Soil microorganisms mediating phosphorus availability update on microbial phosphorus. Plant Physiol. 156, 989–996. Sattari, S.Z., Bouwman, A.F., Giller, K.E., van Ittersum, M.K., 2012. Residual soil phosphorus as the missing piece in the global phosphorus crisis puzzle. Proc. Natl. Acad. Sci. 109, 6348–6353. Shabana, Y.M., Müller-Stöver, D., Sauerborn, J., 2003. Granular Pesta formulation of Fusarium oxysporum f. sp. orthoceras for biological control of sunflower broomrape: efficacy and shelf-life. Biol. Control 26, 189–201. Sharma, S.B., Sayyed, R.Z., Trivedi, M.H., Gobi, T.A., 2013. Phosphate solubilizing microbes: sustainable approach for managing phosphorus deficiency in agricultural soils. Springerplus 2, 587. Silva, D.M., Batista, L.R., Rezende, E.F., Fungaro, M.H.P., Sartori, D., Alves, E., 2011. Identification of fungi of the genus Aspergillus section nigri using polyphasic
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Journal of Microbiological Methods journal homepage: www.elsevier.com/locate/jmicmeth
Survival and phosphate solubilisation activity of desiccated formulations of Penicillium bilaiae and Aspergillus niger influenced by water activity
T
⁎
Nelly Sophie Raymonda, Dorette Müller Stövera, , Lars Stoumann Jensena, Sebastian Håkanssonb a
Plant and Soil Science Section, Department of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Denmark b Department of Molecular Sciences, Swedish University of Agricultural Sciences, P.O. Box 7015, 750 07 Uppsala, Sweden
A R T I C LE I N FO
A B S T R A C T
Keywords: Phosphate solubilising microorganisms Convective air drying Fluidized bed drying Sewage sludge ash Vermiculite Shelf-life
The impact of formulation and desiccation on the shelf life of phosphate (P)-solubilising microorganisms is often under-studied, particularly relating to their ability to recover P-solubilisation activity. Here, Penicilllium bilaiae and Aspergillus niger were formulated on vermiculite (V) alone, or with the addition of protectants (skimmed milk (V + SM) and trehalose (V + T)), and on sewage sludge ash with (A + N) and without nutrients (A), and dried in a convective air dryer. After drying, the spore viability of P. bilaiae was greater than that of A. niger. V formulations achieved the highest survival rates without being improved by the addition of protectants. P. bilaiae formulated on V was selected for desiccation in a fluidised bed dryer, in which several temperatures and final water activities (aw) were tested. The highest spore viability was achieved when the formulation was dried at 25 °C to a final aw > 0.3. During three months' storage, convective air dried formulations were stable for both strains, except in the presence of skimmed milk for P. bilaiae which saw a decrease in spore viability. In the fluidised bed-dried formulations, when aw > 0.3, the loss in viability was higher, especially when stored at 20 °C, than at aw < 0.1. P-solubilisation activity performed on ash was preserved in most of the formulations after desiccation and storage. Overall, a low drying temperature and high final aw positively affected P. bilaiae viability, however a trade-off between higher viability after desiccation and shelf life should be considered. Further research is needed to optimise viability over time and on more sustainable carriers.
1. Introduction Microbial inoculants are a promising tool for enhancing crop growth and productivity (Bhardwaj et al., 2014; Leggett and Gleddie, 1995; Singh et al., 2011). Phosphate-solubilising microorganisms (PSM) are among the beneficial microorganisms that may be inoculated to plants or soil with the aim of enhancing phosphorus (P) uptake by plants. In some agricultural soils, large amounts of P have been accumulated in past decades (Sattari et al., 2012). However, the accumulated P is often insufficiently available for plants, and PSMs have been suggested as a possibility for improving P bioavailability in soils or in different types of residual products with low P bioavailability (Adesemoye and Kloepper, 2009; Sharma et al., 2013). PSMs often show promising results in vitro in solubilising different forms of P, however when they are applied to soil/plant systems, their performances are inconsistent, as is the case with other microbial inoculants (Herrmann and Lesueur, 2013; Möller et al., 2017; Richardson and Simpson, 2011). Microorganisms are sensitive to environmental changes, and the different steps in the inoculum production can decrease cell survival and performance. An effective ⁎
Corresponding author. E-mail address: [email protected] (D. Müller Stöver).
https://doi.org/10.1016/j.mimet.2018.05.012 Received 23 April 2018; Received in revised form 16 May 2018; Accepted 16 May 2018 Available online 17 May 2018 0167-7012/ © 2018 Elsevier B.V. All rights reserved.
formulation is therefore often necessary to alleviate viability loss (Herrmann and Lesueur, 2013). Conventionally, dried formulations are preferred for microorganism preparation and storage (Morgan et al., 2006; Parnell et al., 2016). The desiccation allows cells to remain viable for long periods by inhibiting the metabolism and avoiding the growth of biological contaminants (Friesen et al., 2005, Friesen et al., 2006a, 2006b). Although drying is recognised as an efficient way of preserving microbial inoculants, the process inflicts stress on the microorganisms, and survival rates tend to be low (Friesen et al., 2005; Fu and Chen, 2011; Tadayyon et al., 1997). However, different drying techniques, carriers and protectants have been shown to significantly improve the survival rate of microorganisms (Friesen et al., 2004, 2005; Tadayyon et al., 1997). Shelf life is another important feature when developing a microbial formulation and appears to be highly dependent on the species and specific strain used (Antheunisse and Arkesteijn-Dijksman, 1979; Clerk and Madelin, 1965; Fu and Chen, 2011). Furthermore, most of the work on microorganism desiccation has focused on the formulation and survival rate, and often omitted to test the ability of the microorganisms to continue
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performing the desired activity after rehydration. Drying can affect survival, but can also increase the lag time before the beneficial effects of the inoculants become observable or suppress some metabolic activities (Moënne-Loccoz et al., 1999). In previous work, the possibility of using P-rich sewage sludge ash as an inoculation carrier and a P source to be solubilised by PSM was tested to develop a novel bio-based fertiliser (Raymond et al., 2018). PSM were able to grow on ash and solubilise P after the addition of specific carbon (C) and nitrogen (N) sources. With the goal of testing the bio-based fertiliser in large field experiments and eventually developing a commercial product, a formulation should be developed that is stable over time, inexpensive and environmentally friendly (Melin et al., 2011). Therefore different combinations of PSM, carriers and protectants were screened in a slow-drying convective air dryer and a fluidised bed dryer, representing a faster drying method often used in industrial applications. Two P-solubilising fungal strains were used: Penicillium bilaiae and Aspergillus niger. A different strain of P. bilaiae has previously been studied extensively by Tadayyon et al. (1997) and Friesen et al. (2004, 2005, 2006a, 2006b) for convective drying and fluidised bed drying on different carriers and under various desiccation conditions. Despite relatively high survival rates, the P-solubilisation recovery of the P. bilaiae strain after drying was not tested. However, to the best of the authors' knowledge, no work is currently available on drying methods for A. niger. Few studies are offered on the preservation of spores of P-solubilising fungi, however more work has been done on biocontrol fungi, many of them using the “Pesta” formulation (Elzein et al., 2004; Friesen et al., 2005; Shabana et al., 2003) or alginate encapsulation (Daigle and Cotty, 1995; Vassilev et al., 1997, 1998). Another desiccation carrier, micronised vermiculite, can be used for PSM. Vermiculite is a mineral possessing interesting characteristics for consideration as a desiccation carrier for a microbial inoculant because of its neutral pH and high porosity. Moreover when vermiculite is micronised, it also has the advantage of being relatively disperable in water and can therefore represent a practical way of inoculating seeds or different substrates, such as the insoluble form of P. Alternative carriers, such as recycled urban residues, are rarely used as desiccation and storage carriers and can be a inexpensive and renewable alternative to vermiculite. With the objective of inoculating sewage sludge ash with PSM to enhance P solubility, it is therefore of interest to also test ash as a desiccation and storage carrier, with and without the nutrient enrichment necessary for an optimal P-solubilisation. However, the ash exhibits an alkaline pH compared to conventional carriers and may jeopardise PSM survival. The hypotheses tested were as follows: (1) the ash carrier used for desiccation decreases the PSM survival rate due to a lower porosity and higher pH than the vermiculite, (2) the addition of a protectant or nutrients (skimmed milk, trehalose or nutrient mix) improves spore viability during the drying process and storage, (3) the survival rates of the spores are dependent on the drying conditions – low temperature, slow-drying kinetics and higher final water activity favour their survival – and P. bilaiae and A. niger have different optimal drying conditions, (4) low temperatures during storage reduce viability loss in all the formulations, and (5) the surviving spores have the same ability to solubilise P as fresh spores.
Table 1 pH and surface area of the two desiccation carriers. Properties
MEV vermiculitea
Ashb
pH Surface area (m2 g−1) Particles size (μm)
7–8 9.9 0.10–0.65
9.2 5.1 < 250
a b
MEV vermiculite characteristics from Dupré Minerals. Ash characteristics from Raymond et al. (2018).
from mycelium by filtering the suspension through sterile glass wool (Miracloth, EMD Millipore Corporation, Billerica, MA 01821 USA). The filtrate was centrifuged at 16 x g and 4 °C for 10 min. 2.2. Carriers and formulation Carrier A was prepared by mixing 25 g of dry sewage sludge incineration ash (Raymond et al., 2018; Thomsen et al., 2017) (Table 1) with 12.5 mL of inoculum suspension (ratio 2:1). The spore concentration of the inoculum was determined using a hemacytometer (Bürker, Brand, Germany) and adjusted to obtain 1 × 107 spores per gram of ash (based on the ash dry weight (dw)). For the nutrient-enriched formulation carrier A + N, P. bilaiae spores were suspended in a solution containing 107 g L−1 of fructose and 3.1 g L−1 (NH4)2SO4, and A. niger spores in a solution containing 107 g L−1 and 1.9 g L−1 NH4NO3. Spore concentration per g ash and moisture content were adjusted to obtain the same levels as in carrier A. Carrier V consisted of 25 g (dw) vermiculite (micronised vermiculite (MEV), Dupré Minerals Ltd., Staffordshire, England, Table 1), mixed with the inoculum at a moisture rate of 1:1.25. The spore concentration was adjusted to 1 × 108 spores per gram of vermiculite. Two protectants were also tested with the vermiculite: skimmed milk powder (10% w/w) (V + SM) and trehalose solution (inoculum added in 10% trehalose solution) (V + T), as these have earlier been shown to be appropriate desiccation carriers (Fu and Chen, 2011; Morgan et al., 2006). Carriers and nutrient solutions were sterilised individually by autoclaving for 20 min at 121 °C, whereas the trehalose solution was filtersterilised (0.2 μm, Nalgene, Thermo Fisher Scientific, Massachusetts, USA). All the formulations tested were produced in three independent replicates before being dried. 2.3. Convective air drying and storage of the dried materials The wet formulation mixtures were placed in Petri dishes in a convective air dryer, which consisted of a Plexiglas box connected to forced ventilation (1.6 m3 min−1). Samples were dried at room temperature (22 °C) with an average relative humidity (RH) of 60% until stabilisation of the sample weight, corresponding to about 36 h drying. Two sub-samples was kept to determine survival after drying, with the remaining of the sample divided into 2.5 g subsamples that were sealed air-tight in aluminium bags and stored at either 2 °C or 20 °C. Viability of the fungal strains in the stored products was tested after two weeks, after one month and after three months.
2. Materials and methods 2.4. Fluidised bed drying and storage of the dried materials 2.1. Phosphate solubulising microorganisms strains and inoculum preparation
From the convective air drying test, the formulation combining V and P. bilaiae was the most interesting due to its higher survival rate and lower formulation cost, and was therefore chosen for further investigations in a fluidised bed dryer (Fluid Bed Dryer, FBD 2000, Endecotts, London, UK). A first step was to evaluate the effect of different inlet temperatures. The drying process was monitored by measuring the outlet temperature and final water activity (Aqualab, METER Group, Inc. USA). The water activity (aw) targeted was aw < 0.1
Two PSM strains were tested: Penicillium bilaiae DBS-5, provided by Novozymes (Denmark), and Aspergillus niger (ATCC 9142). From frozen (−80 °C) spore stocks, fungal strains were propagated on potato dextrose agar (PDA) plates for about two weeks before being transferred to a new PDA plate. Spores from the second generation were then collected by washing the plate with sterile MilliQ water and then separated 40
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(RH < 10%). The airflow was adjusted to ensure appropriate fluidisation of the product and inlet temperatures were set to 25 °C for 25 min, 25 °C for 12.5 min then 40 °C for 5 min (25 °C + 40 °C), and 40 °C for 10 min, respectively. In a second round of tests, the effect of the final aw was tested with the same airflow and a 25 °C inlet temperature for 15 min in order to obtain an aw of 0.3, and for 14 min to obtain an aw of 0.5. For both desiccation experiments, three independent replicates were produced. After drying, two sub-samples were used to evaluate the spore survival after drying and the rest of the samples were processed for storage as described above for the convective air drying.
Table 2 Water activities (aw) recorded before and after desiccation with convective air drying. Values are the mean (n = 3) ± S.E. Formulations
aw before drying ( ± SE)
aw after drying ( ± SE)
A A+N V V + SM V+T
0.999 1.000 0.999 0.996 0.993
0.174 0.162 0.160 0.155 0.145
± ± ± ± ±
0.000 0.000 0.002 0.002 0.000
± ± ± ± ±
0.024 0.023 0.023 0.021 0.019
level set at p < 0.05). For the fluidised bed-drying assay, the effect of the different treatments (temperature, aw) was tested by a two-way Anova followed by an HSD post hoc test (Agricolae package, significance level set at p < 0.05). For the P-solubilisation biotest, the different treatments were compared to the performance of the control with fresh spores, including the effect of the additive and storage temperature, by a multivariate linear model including interactions, followed by an HSD post hoc test (Agricolae package, significance level set at p < 0.05).
2.5. Spore viability and phosphate solubilisation activity biotest Before and after drying, two sub-samples of 0.5 g (dw) of each inoculated carrier were shaken with 10 mL sterile distilled water (DI) amended with 0.05% Tween-80 for 20 min. The suspension was serially diluted and 100 μL of two different appropriate dilutions plated on PDA plates. Each dilution was plated in triplicate. The inoculated plates were incubated for about two days at 25 °C and the colonies counted. Plates with between 20 and 200 colonies were used to determine the viable cells. To establish the survival rate, the viable spore concentration after drying was divided by the viable spore concentration before drying. Spore viability in the stored samples was assessed accordingly. Phosphate solubilisation activity was tested by incubating the sewage sludge ash with the dried formulations. 1 g of dried and inoculated carrier A was weighed into a 15 mL tube and 0.420 mL of the same nutrient solution as described above added. The A + N treatment, which had already been amended with nutrients before drying, received only 0.420 mL sterile deionised water (DI) water. For the treatments containing vermiculite carriers (V, V + SM, V + T), a subsample of 0.6 g was suspended in 2.5 mL nutrient solution and thoroughly mixed. The suspension was then applied to 1 g sterile ash to obtain a spore concentration of 1 × 107 spores per gram of ash based on the original spore concentration before drying. After mixing the different carriers with the ash, the preparations were loaded into 1.2 mL deep 96 well plates (ABgene Storage, Thermo Fisher Scientific, Massachusetts, USA) using a MicroResp™ filling device. Weights of each treatment were recorded to determine the mass of ash per well (about 150 μg ash (dw) per well). Water content was adjusted to 45%. The plates were then sealed in a plastic bag and incubated at 25 °C in the dark. After 12 days' incubation for P. bilaiae and 10 days' for A. niger, three samples from each treatment were extracted to measure water-extractable P (WEP). Briefly, the ash samples from the well were shaken with DI water at a ratio of 1:60 (dwt:vol) at 150 rpm for 16 h. After centrifugation and filtration through Whatman® filter paper no. 5, the orthophosphate concentration of the filtered extract was measured with the molybdate blue-ascorbic acid assay, as described in Murphy and Riley (1962) and Watanabe and Olsen (1965). To compare the P-solubilisation to a fresh inoculum, ash was inoculated with either 1 × 106 or 1 × 107 spores per gram of ash and incubated under the same conditions as described above. These control treatments were repeated three times at the same time as after drying.
3. Results 3.1. Desiccation survival 3.1.1. Convective air drying Convective air drying was used to determine the impact of different desiccation carriers and formulations on the viability of two selected Psolubilising fungi after drying. Before drying, all the carriers had a water activity (aw) close to 1, and of between 0.145 and 0.175 after drying (Table 2). After the drying process, P. bilaiae showed a significantly higher survival on vermiculite carriers than on ash (Fig. 1) (p < 0.0001). The addition of skimmed milk or trehalose to vermiculite or nutrients to the ash did not affect their survival (p > 0.05). In contrast, the survival of A. niger spores was not affected by the type of carrier or by the addition of a protectant during the drying, and was generally lower than P. bilaiae (p > 0.05). In most cases for P. bilaiae, the vermiculite carriers supported a higher relative viability than the ash over time, but only because the survival after drying was higher. The storage temperature and the supplementation of the carriers with protectants or nutrients had inconsistent effects on spore survival; only the addition of skimmed milk significantly reduced spore viability, especially at 20 °C (p < 0.05). For A. niger, over the whole storage period, spore viability was greater on vermiculite carriers than on ash (p < 0.01). The highest viability was observed after three months' storage in most of the carriers. Spore viability on V, V + SM and A was affected by the storage temperature (p < 0.05), with a higher viability observed at 2 °C than at 20 °C. As also observed for P. bilaiae, skimmed milk as a protectant had a negative effect on the spore viability of A. niger on vermiculite. 3.1.2. Fluidised bed drying Fluidised bed drying was then used on the formulation combining P. bilaiae and V because the formulation was more promising due to its higher survival rate and lower cost with convective air drying. The water activities measured (Table 3) were within the targeted ranges. In the fluidised bed drying, survival was generally better than in the convective drying, and the highest spore survivals rates were recorded for the drying temperature of 25 °C (p < 0.0001) and final aw > 0.1 (p < 0.0001) (Fig. 2A). In most of the treatments, a significant decrease in spore viability was observed after storage of two weeks and of one month, except for the spores dried at 40 °C that remained stable (Fig. 2B and C). For the formulations including a final aw > 0.3, loss of viability was larger overall (p < 0.0001). This was particularly true for
2.6. Statistical analyses Statistical analysis was performed in R (R i386 3.3.2, GNU Project) using the RStudio development environment. The survival after convective air drying was analysed by a two-way Anova, including the effect of the carrier and the additives as factors, followed by an HSD post hoc test (Agricolae package (De Mendiburu, 2016), significance level set at p < 0.05). The effects of storage time and temperature on each carrier were tested. For each time point in the viability check, the effects of the carrier, storage temperature and protectant/nutrients were tested in a simple multivariate linear model including interactions, followed by an HSD post hoc test (Agricolae package, significance 41
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40
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Fig. 1. Spore viability after drying for P. bilaiae (A) and A. niger (D) and shelf life over time with storage at 2 °C (P. bilaiae (B), A. niger (E)) and 20 °C (P. bilaiae (C), A. niger (F)) (V: vermiculite; V + SM: vermiculite + skimmed milk; V + T: vermiculite + trehalose; A: ash; A + N: ash + nutrients). Values are the mean (n = 18) ± S.E. (bars)).
the formulation with aw = 0.5 stored at 20 °C. Between one and three months’ storage, the viability was stable in most of the formulations.
Table 3 Water activities (aw) recorded before and after desiccation of P. bilaiae spores on vermiculite with fluid bed drying. Values are the mean (n = 3) ± S.E. Drying treatment, target aw
aw before drying ( ± SE)
aw after drying ( ± SE)
25 °C aw < 0.1 25 °C aw = 0.3 25 °C aw = 0.5 25 °C + 40 °C aw < 0.1 40 °C aw < 0.1
0.998 0.998 0.999 0.999 1.001
± ± ± ± ±
0.114 0.292 0.543 0.053 0.068
a
a
80
60
b
40 c
20
d
3.2.1. Convective air drying Recovery of the P-solubilisation activity after desiccation and storage was required in the elaboration of the formulation and was compared to two controls with fresh spores. P. bilaiae showed a significant increase in P-solubilisation at a rate of 1 × 107 spores per gram of ash compared to 1 × 106 spores per gram (p < 0.0001), whereas similar amounts of solubilised P at both inoculation rates were observed for A. niger (p > 0.05) (Fig. 3). After drying, the P-solubilisation activity of P. bilaiae was between the levels of the two controls with freshly applied spores. A similar pattern was observed for A. niger, with the exception of the V + SM carrier, where less P was solubilised compared to the controls. Phosphate solubilisation levels of P. bilaiae remained relatively
0.012 0.015 0.038 0.004 0.007
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P. bilaiae viability (%) stored at 20 oC
A
± ± ± ± ±
P. bilaiae viability (%) stored at 2 oC
P. bilaiae viability (%) after drying
80
0.001 0.000 0.001 0.001 0.001
3.2. Phosphate solubilisation activity
C
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Fig. 2. Spore viability after drying for P. bilaiae using the fluid bed dryer (A) and shelf life over time with storage at 2 °C (B) and 20 °C. (25C aw 0.1: vermiculite dried at 25 °C and final aw = 0.1; 25 °C aw 0.3: vermiculite dried at 25 °C and final aw = 0.3; 25C aw 0.5: vermiculite dried at 25 °C and final aw = 0.5; 25 °C + 40 °C aw 0.1: vermiculite dried at 25 °C + 40 °C and final aw = 0.1; 40C aw 0.1: vermiculite dried at 40 °C and final aw = 0.1). Values are the mean (n = 18) ± S.E. (bars)). 42
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0 con
A. niger P-solubilisation activity (mg P g-1 ash)
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Fresh inoculum 106 spores g-1 Fresh inoculum 107 spores g-1
V
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3
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0 con
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Fig. 3. Phosphate solubilisation activity over time on the different desiccation carriers using the convective air drier (V: vermiculite; V + SM: vermiculite + skimmed milk; V + T: vermiculite + trehalose; A: ash; A + N: ash + nutrients). Values are the mean (n = 9) ± S.E. (bars)).
preparing a microbial formulation to ensure a certain number of viable cells in the final product. The carriers and their characteristics represent a real opportunity to improve cell viability during the desiccation process (Morgan et al., 2006). By using the convective air drying method, clear differences between vermiculite and ash carriers in spore viability were observed for P. bilaiae. Although A. niger did not display a higher viability on one of the two tested carriers after drying, the fungal strain's desiccation tolerance and carrier preference was clearly demonstrated. The viability of P. bilaiae spores was greater on vermiculite than on the ash carriers, and probably resulted from the physical structure of the two materials, such as their porosity (Vanek et al., 2016). The higher porosity of the vermiculite may have provided a superior protective environment against desiccation compared to the ash for the P. bilaiae spores, which at 1–3.5 μm (Buttner and Stetzenbach, 1993; Tadayyon et al., 1997) were smaller than the A. niger spores (3–5 μm (Silva et al., 2011)). The pH of the carrier or formulation is often mentioned as affecting cell viability during drying (Morgan et al., 2006). For bacteria, it is reported that the exposure to a low pH induces morphological changes in the cells and can generate a positive stress, especially if the cells are exposed to an acidic pH before desiccation (Fu and Chen, 2011; Morgan et al., 2006). The effect of an alkaline pH on spore viability has not so far been documented. If the alkalinity of the ash were a barrier providing optimal resistance to desiccation, it would be interesting to explore a pre-adaptation to
constant and similar to the controls over time. Storage temperature only affected A where higher P-solubilisation rates were observed when samples were stored at 2 °C after one and three months' storage (p < 0.0001). The different protectants/nutrients did not affect the ability of P. bilaiae to solubilise P from the ash, only trehalose tended to increase P-solubilisation activity. For A. niger, after two weeks' storage all the treatments had a Psolubilisation activity comparable to the controls with fresh spores. After one month of storage, treatments A, A + N and V + SM showed a reduced P-solubilisation activity. 3.2.2. Fluidised bed drying Phosphate solubilisation activity was also tested for the selected formulation, P. bilaiae and V. After fluidised bed drying, all the treatments had a P-solubilisation rate similar to that of the fresh spore controls (Fig. 4). Over time, only the final aw of the treatment impacted the ability to solubilise P from the ash (p < 0.0001) with an overall lower P-solubilisation for treatments with aw < 0.1. 4. Discussion 4.1. Desiccation survival Obtaining a high survival rate after drying is essential when 43
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1 month 20 oC 3 months 2 oC 3 months 20 oC
After drying 2 weeks 2 oC 2 weeks 20 oC 1 month 2 oC
Fresh inoculum 106 spores g-1 Fresh inoculum 107 spores g-1 4
-1
P. bilaiae P-solubilisation (mg P g ash)
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Fig. 4. Phosphate solubilisation activity over time using the fluid bed drier (25 °C aw 0.1: vermiculite dried at 25 °C and final aw = 0.1; 25 °C aw 0.3: vermiculite dried at 25 °C and final aw = 0.3; 25 °C aw 0.5: vermiculite dried at 25 °C and final aw = 0.5; 25 °C + 40C aw 0.1: vermiculite dried at 25 °C + 40 °C and final aw = 0.1; 40C aw 0.1: vermiculite dried at 40 °C and final aw = 0.1). Values are the mean (n = 9) ± S.E. (bars)).
2
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generating a large loss of viability during drying. This could also be the reason why the final survival rates after convective air drying were generally lower than in the fluidised bed dyer with a higher final aw. Friesen et al. (2005) found similar survival rates (60–80%) for P. bilaiae dried with a similar inlet temperature (20–30 °C) and at the same RH (60%) for aw > 0.3. In relation to the findings in the convective air drying experiment in which aw < 0.175 and lower viability were recorded, the authors also reported that drying the formulation below aw < 0.2 highly decreased spore viability. For a comparable final aw between the convective and fluidised bed drying, fluidised bed drying resulted in a higher survival rate, despite several studies previously concluding that a slow drying process is favourable for survival (Fu and Chen, 2011; Moënne-Loccoz et al., 1999). However, Fu and Chen (2011) also mentioned that a longer drying time exposes the microorganisms to stress for a longer period and can therefore generate viability loss. Thus a higher final aw generally led to higher spore survival, reinforcing the third hypothesis. However the slow drying kinetic did not lead to greater spore viability, in contrast to this same hypothesis in terms of kinetics.
alkaline conditions to determine whether a period of acclimatisation improves fitness to survive or causes damage to the spores (Fu and Chen, 2011; Melin et al., 2011; Morgan et al., 2006). Nevertheless, it would be interesting to pursue optimisation of sewage sludge ash or other thermally treated residue formulations as desiccation carriers as they represent a low-cost and renewable carrier. Recent studies have shown the positive effects of biochar as a microbial carrier (Hale et al., 2015; Vanek et al., 2016). To further enhance the viability of these alternatives carriers, different options can also be considered, such as a pre-adaptation or a pre-establishment of the fungal strain on the carrier, which in some cases has demonstrated a positive effect in terms of spore viability after desiccation for a biocontrol Pseudomonas fluorescens (Moënne-Loccoz et al., 1999). Thus, depending on the fungal strain, this study's first hypothesis regarding the effect of porosity could be confirmed, although the impact of carrier pH can only be speculated. The difference observed between the two types of carrier for P. bilaiae may also be the result of the different spore concentrations in the formulations leading to different recoveries. However this was not be shown with A. niger and it has also been found that for Fusarium oxysporum spore formulation, the concentration did not affect the viability after drying (Connick et al., 1998). This assumption must therefore be rejected. Although the addition of protectants during the drying process is frequently reported as resulting in the reduction of viability loss (Melin et al., 2011; Shabana et al., 2003), no clear effects were found in this study. In the formulation of fungal spores, the effects of protectants have been found to be fairly inconsistent given that spores by their nature are a natural preservation form (Shabana et al., 2003). The absence of an effect on spore viability during drying with the desiccation protectants allowed the second hypothesis to be partly rejected. Compared to the convective air drying system used first in this study, fluidised bed drying represented a faster method for air drying microorganisms and allowed more parameters to be controlled. Fluidised bed drying was only tested with P. bilaiae and vermiculite as this combination had shown the greatest tolerance to desiccation in the experiments on convective air drying. A drying temperature of 25 °C resulted in the highest survival rates, while increased temperatures greatly reduced spore survival, confirming the thermal sensitivity of the species (Friesen et al., 2005; Tadayyon et al., 1997) and supporting this study's third hypothesis. When dried at 25 °C in the fluidised bed dryer, a final aw at 0.3 and 0.5 induced a higher spore survival than a final aw at 0.1. Insufficient humidity in the formulation has been described by Beker et al. (1984) as creating damage to the yeast cell membrane,
4.2. Formulation shelf life During the first two weeks of storage, the spore viability generally decreased, except for the formulations with aw < 0.1 in which viability was stable and P. bilaiae formulated with V and V + T by convective air drying, which showed an increase in viability. The loss in spore viability during the first period (two weeks to one month of storage) was highest in the materials dried in the fluidised bed dryer to a final aw of 0.3 and 0.5 and stored at 20 °C. The effect of greater moisture content on spore viability loss during storage has been suggested by Tadayyon et al. (1997) as probably being linked to enzyme activity recovery or the start of germination followed by death due to a lack of sufficient moisture and nutrients. In the present study, it seemed that the lower final water activity in the carrier favoured spore viability over time, which has also been confirmed by Friesen et al. (2006a, 2006b) who found the least spore viability loss for aw between 0.1 and 0.2 over a six-month storage period. Between one and three months after drying, the spore viability was comparatively stable for P. bilaiae or even increased for A. niger. The increase in spore viability was rather surprising, but the heterogeneity of spores in formulations may also have interacted with spore recovery, contributing to the general variability in survival rates. Storage in a cool environment is widely recognised to reduce cell 44
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Declarations of interest
viability loss, however formulations with a long shelf life at room temperature offer a financial advantage and are more convenient for users (Melin et al., 2011; Parnell et al., 2016). For both strains, the viability of the formulations stored after convective air drying was not affected by the storage temperature, except in the V + SM treatment with P. bilaiae. However, in the fluidised bed drying, storage at 2 °C reduced the loss of viability, especially in the formulations with aw 0.3 and aw 0.5, emphasising a trade-off between survival after drying and storage stability and partially confirming this study's fourth hypothesis. The loss of viability during the first months of storage may be reduced by using other storage conditions such as vacuumed-sealed bags or ambient nitrogen atmosphere that have beneficial effects on some microorganisms (Melin et al., 2011; Önneby et al., 2014). The rehydration step is also often neglected, but can play an important role in the recovery and real survival rate (Fu and Chen, 2011; Poirier et al., 1999). It was not tested in this study.
None. Acknowledgments The authors would like to thank Armando Hernández for his support with the sample analysis, Tobias Thomsen for providing the ashes and biochars, and Paul Gregory from Dupré Minerals for providing the vermiculite. This study was supported by Innovation Foundation Denmark (grant number 1308-00016B to the project Microbial biofertilizers for enhanced crop availability of phosphorus pools in soil and waste, MiCroP). References Adesemoye, A.O., Kloepper, J.W., 2009. Plant–microbes interactions in enhanced fertilizer-use efficiency. Appl. Microbiol. Biotechnol. 85, 1–12. Antheunisse, J., Arkesteijn-Dijksman, L., 1979. Rate of drying and the survival of microorganisms. Antonie Van Leeuwenhoek 45, 177–184. Beker, M., Blumbergs, J., Ventina, E., Rapoport, A., 1984. Characteristics of cellular membranes at rehydration of dehydrated yeast Saccharomyces cerevisiae. Appl. Microbiol. Biotechnol. 19, 347–352. Bhardwaj, D., Ansari, M.W., Sahoo, R.K., Tuteja, N., 2014. Biofertilizers function as key player in sustainable agriculture by improving soil fertility, plant tolerance and crop productivity. Microb. Cell Factories 13, 66. Buttner, M.P., Stetzenbach, L.D., 1993. Monitoring airborne fungal spores in an experimental indoor environment to evaluate sampling methods and the effects of human activity on air sampling. Appl. Environ. Microbiol. 59, 219–226. Clerk, G., Madelin, M., 1965. The longevity of conidia of three insect-parasitizing hyphomycetes. Trans. Br. Mycol. Soc. 48, 193–209. Connick, W., Daigle, D., Pepperman, A., Hebbar, K., Lumsden, R., Anderson, T., Sands, D., 1998. Preparation of stable, granular formulations containing Fusarium oxysporum pathogenic to narcotic plants. Biol. Control 13, 79–84. Daigle, D., Cotty, P., 1995. Formulating atoxigenic Aspergillus flavus for field release. Biocontrol Sci. Tech. 5, 175–184. De Mendiburu, F.A., 2016. Statistical Procedures for Agricultural Research. Version 1.2–4. Elzein, A., Kroschel, J., Müller-stöver, D., 2004. Optimization of storage conditions for adequate shelf-life of ‘Pesta'formulation of Fusarium oxysporum ‘foxy 2′, a potential mycoherbicide for Striga: effects of temperature, granule size and water activity. Biocontrol Sci. Tech. 14, 545–559. Elzein, A., Kroschel, J., Cadisch, G., 2008. Efficacy of Pesta granular formulation of Strigamycoherbicide Fusarium oxysporum f. sp. strigae foxy 2 after 5-year of storage. J. Plant Dis. Protect. 259–262. Friesen, T., Hill, G., Pugsley, T., Holloway, G., 2004. Optimization of the convective air drying of Penicillium bilaii for improved efficiency. Dry. Technol. 22, 1153–1172. Friesen, T., Hill, G., Pugsley, T., Holloway, G., Zimmerman, D., 2005. Experimental determination of viability loss of Penicillium bilaiae conidia during convective air drying. Appl. Microbiol. Biotechnol. 68, 397–404. Friesen, T., Hill, G., Pugsley, T., Holloway, G., 2006a. A mortality model for Penicillium bilaiae subjected to convective air drying. Can. J. Chem. Eng. 84, 488–494. Friesen, T.J., Holloway, G., Hill, G.A., Pugsley, T.S., 2006b. Effect of conditions and protectants on the survival of Penicillium bilaiae during storage. Biocontrol Sci. Tech. 16, 89–98. Fu, N., Chen, X.D., 2011. Towards a maximal cell survival in convective thermal drying processes. Food Res. Int. 44, 1127–1149. Hale, L., Luth, M., Crowley, D., 2015. Biochar characteristics relate to its utility as an alternative soil inoculum carrier to peat and vermiculite. Soil Biol. Biochem. 81, 228–235. Herrmann, L., Lesueur, D., 2013. Challenges of formulation and quality of biofertilizers for successful inoculation. Appl. Microbiol. Biotechnol. 97, 8859–8873. Leggett, M., Gleddie, S., 1995. 4 Developing Biofertilizer and Biocontrol Agents that Meet farmers' Expectations. Advances in Plant Pathology. vol. 11. pp. 59–74. Melin, P., Schnürer, J., Håkansson, S., 2011. Formulation and stabilisation of the biocontrol yeast Pichia anomala. Antonie Van Leeuwenhoek 99, 107–112. Moënne-Loccoz, Y., Naughton, M., Higgins, P., Powell, J., O'connor, B., O'gara, F., 1999. Effect of inoculum preparation and formulation on survival and biocontrol efficacy of Pseudomonas fluorescens F113. J. Appl. Microbiol. 86, 108–116. Möller, K., Oberson, A., Bünemann, E.K., Cooper, J., Friedel, J.K., Glæsner, N., Hörtenhuber, S., Løes, A.-K., Mäder, P., Meyer, G., 2017. Improved phosphorus recycling in organic farming: navigating between constraints. Adv. Agron. 147, 159–237. Morgan, C.A., Herman, N., White, P., Vesey, G., 2006. Preservation of micro-organisms by drying; a review. J. Microbiol. Methods 66, 183–193. Murphy, J., Riley, J.P., 1962. A modified single solution method for the determination of phosphate in natural waters. Anal. Chim. Acta 27, 31–36. Önneby, K., Håkansson, S., Pizzul, L., Stenström, J., 2014. Reduced leaching of the herbicide MCPA after bioaugmentation with a formulated and stored Sphingobium sp. Biodegradation 25, 291–300. Parnell, J.J., Berka, R., Young, H.A., Sturino, J.M., Kang, Y., Barnhart, D., DiLeo, M.V.,
4.3. Phosphate solubilisation activity In previous studies on P. bilaiae, the activity of P-solubilisation was not tested after desiccation. For both drying methods and both strains, the P-solubilisation activity was preserved after drying and during the storage period with some exceptions, confirming the final hypothesis. For P. bilaiae P-solubilisation was maintained in all the treatments or even increased in the V + T. This is not surprising as trehalose is potentially a source of carbon that P. bilaiae is able to use for its growth and metabolism, thereby stimulating P-solubilisation. An increase in Psolubilisation activity was also observed on ash after two weeks and after one month of storage. This was rather surprising, but may also be the result of the spore heterogeneity in the formulation. After the fluidised bed drying, a lower P-solubilisation activity was observed in the treatment dried at 40 °C, probably due to a decrease in fitness and viability. For A. niger, a decrease in P-solubilisation activity was observed over time on ash carriers, although viability was constant, translating a probable loss of fitness of the spores. P-solubilisation was also generally lower in the V + SM treatments, which in addition to the general viability loss could be explained by the fact that skimmed milk contained some easily available forms of P that the fungus may have used as a nutrient source, thus reducing the need to solubilise P from ash. The storage time in this study was up to three months and showed promising activity recovery, however some studies have explored the viability and activity of microorganisms over a longer period. For example Elzein et al. (2008) stored the mycoherbicide Fusarium oxysporum for five years at 4 °C and found only a slight loss in viability, but 100% efficiency on the targeted weed compared to a fresh formulation. 5. Conclusion Overall, this study has provided information on the possibility of dry-formulating two PSM on vermiculite and sewage sludge ash and their P-solubilisation activity recovery after drying. In a convective air drying system, the addition of desiccation protectants or nutrients did not affect the viability of the two fungal strains tested. However, A. niger had a lower viability during drying than P. bilaiae, showing the strain's differential tolerance in terms of desiccation. The survival rate of P. bilaiae was enhanced on vermiculite, and faster drying rates with the fluidised bed dryer positively influenced the strain's survival. Nevertheless, despite higher viability after desiccation in formulations with aw > 0.3, the viability loss during storage was significantly increased, especially at 20 °C. In terms of P-solubilisation activity, the ability of the two strains was secured after drying and storage, except when skimmed milk was added and when the viability loss was too great, underlining the importance of testing the targeted activity when formulating microorganisms. 45
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