The effect of sewage sludge and BAF inoculant on plant condition and yield as well as biochemical and microbial activity of soil in willow (Salix viminalis L.) culture as an energy crop

Experimental design and sample collection

Analyses of the residual effect of sewage sludge from the wastewater treatment plant in Szamotuły and the BAF inoculant on selected plant physiological parameters and soil microbial activity were performed in the years 2013–2015 on fields of the Experimental and Teaching Station in Gorzyń, branch in Złotniki (N: 52° 29′ 0″, E: 16° 49′ 53″), belonging to the Poznań University of Life Sciences (PULS). In the wastewater treatment plant in Szamotuły according to the Directive (91/271/EWG) of the purification process is used biological treatment of secondary sludge. In addition, the chemical process is used—the simultaneous removal of phosphorus with the help of PIX-113 coagulant. The wastewater treatment plant is not additionally supplied with industrial sewage. People Equivalent is on the level 28,880.

Two types of set of parameters were tested simultaneously during the experiment. One concerning the transformation of carbon in the soil after the application of sludge (presented here) and the second concerning the transformation of nitrogen. The latter one regarding nitrogen transformation was published by Wolna-Maruwka et al., 2018. Field trials were conducted as unifactorial experiments in four replications. Four variants were applied in the experiment: (1)—control (soil with no fertiliser application); (2)—soil fertilised with sewage sludge; (3)—soil fertilised with sewage sludge and inoculated with BAF; (4)—soil inoculated with BAF.

Willow (Salix viminalis L.) cv. Start was grown after winter rape on lessive soil formed from light loamy sands, classified in quality classes IVa and IVb, of the very good and good rye complex According to the World Reference Base this soil is classified among Albic Luvisols. In the autumn before the experiment was established a single mineral phosphorus–potassium fertilisation was applied by broadcasting phosphorus fertiliser (triple superphosphate, 46% P₂O₅) at 80 kg P₂O₅·ha⁻¹ and potassium fertiliser (60% potassium salt) at 100 kg K₂O·ha⁻¹. Both macronutrients were used to provide plant nutrition during the first year of experiment and in subsequent years. In the spring prior to willow planting nitrogen fertilisation was applied using 34% ammonium nitrate at 90 kg N·ha⁻¹. Stem cuttings 20 cm in length were planted on 25 April 2013 at a 70-cm row spacing and plant spacing of 35 cm within the row. Weeds were controlled mechanically when willow plants reached a height of 10–15 cm. No disease or pests were reported on plants during the vegetation period. The harvested plots were 15 m² in size. The other cultivation measures were performed following the principles of good cultivation practice for that species.

In each year of culture after completion of the vegetation period plant number per unit area was determined; it did not change significantly in the period of the study. Moreover, shoot diameter at a height of 30 cm from the ground, plant height (PH) and the number of shoots (NSs) from the rootstock were determined on five successive plants. The yield of biomass was assessed each time after cutting all shoots from the plot using a rotating disc mower. Moisture content of the harvested material was determined by the dry-weight method.

Characterization of sewage sludge

In the above-mentioned variants sewage sludge was applied to the soil once in the spring of 2013 (March), and then analyses of the application effects were performed three times in each of the years of analysis (May, July, September). The sewage sludge application rate per one m² was 0.00451 Mg d.m., and it did not exceed the dose of municipal sewage sludge for reclaimed areas and those cropped for non-food or non-feed purposes admissible under the Regulation of the Minister of Environmental of 13 July 2010, specifying it at a total 45 Mg d.m.·ha·3 years⁻¹. Characteristics of the basic chemical composition of soil prior to sludge application and after its application are given in Table 1. Sludge used in the experiment met the requirements of respective standards in relation to contents of heavy metals and biological contamination.

Characterization of microbial consortium

In turn, the applied BAF, applied directly to soil in experimental variants three and four, was developed at the Department of General and Environmental Microbiology, PULS.

The biopreparation consisted of 15 strains of bacteria (Bacillus subtilis, B. amyloliquefaciens), five of actinomycetes (Streptomyces spp.) isolated from mature compost prepared from plant residues and sewage sludge as well as four strains of Trichoderma harzianum fungus derived from the collection of the Institute of Plant Genetics in Poznań.

The above-mentioned strains were examined, among others, from the point of view of their proteolytic and cellulolytic activities. The inoculant was tested in terms of its biochemical activity. The bacterial count in one ml of inoculant was 1.76·10⁶ cfu, the count of Actinobacteria was 2.3·10³ cfu, while that of fungi was 1.89 10² cfu.

Photosynthesis activity

The C_i 340aa handheld photosynthesis system (CID BIOSCIENCE Inc., Camas, WA, USA) was used to evaluate net photosynthetic rate (P_N), stomatal conductance (g_s), transpiration rate (E) and intercellular CO₂ (C_i) concentration. For these purposes constant conditions were maintained for leaf chamber measurements: CO₂ inflow concentration (390 μmol CO₂) mol⁻¹, photosynthetic photon flux density 1,000 μmol (photon) m⁻²·s⁻¹, chamber temperature 25 °C, relative humidity 40 ± 3%. These measurements were performed three times during the experiment in the 3rd, 15th and 27th month, respectively, 1st, 2nd, 3rd year. For this purpose five plants of each combination were selected.

Microbiological and biochemical analyses

Soil samples for biochemical and microbiological analyses in each vegetation season were collected at three dates (May, July and September). In soil samples collected from a depth of 0–20 cm under plants microbial counts were determined.

Microbiological analysis was performed on the basis of serial dilution method and involved determining (using selective substrate in five replications) the numbers of colony forming units (CFU g⁻¹ d.m. of soil) of the total number bacteria, oligotrophic and copiotrophic bacteria, Actinobacteria, and fungi. Estimation of CFU number of the above-mentioned microorganisms is a measure of the intensity of their current metabolic activity.

The total bacterial number was determined on Merck standard agar (three g yeast extract; 5.0 g peptone from casein (free from fermentable carbohydrates); five g sodium chloride; 12 g agar, one l H₂O), after 5–6 days of incubation at 28 °C. Oligotrophic bacteria were counted on diluted nutritive broth (0.1 g peptone, 0.1 g beef extract, 0.05 g sodium chloride, 20 g agar, one l H₂O) at 28 °C after 21 incubation days (Ohta & Hattori, 1980). Copiotrophic bacteria were determined on nutritive broth (10 g peptone, 10 g beef extract, five g sodium chloride, 20 g agar, one l H₂O) at 28 °C after 7 days of incubation (Ohta & Hattori, 1980). Fungi were determined on Martin substrate (one g KH₂PO₄, 0.5 g MgSO₄, five g peptone, 10 g glucose, 3.3 ml Bengal, 0.1 g chlortetracycline, 25 g agar, one l H₂O) for 5 days at 24 °C (Martin, 1950), and numbers of Actinobacteria were assessed on a selective Pochon substrate (0.05 g asparagine, 0.1 g nystatin, two g starch, five g K₂HPO₄, 2.5 g MgSO₄·7H₂O, 2.5 g NaCl, 0.05 g MnSO₄·5H₂O, 0.05 g Fe₂(SO₄)₂·5H₂O, 25 g agar, one l H₂O) following plate incubation for 7 days at 26 °C (Grabińska-Łoniewska, 1999).

The analyses of soil enzymatic activity in different variants were based on the spectophotometric method applied to measure dehydrogenases activity (DHA), where 1% TTC (triphenyltetrazolium chloride) was used as a substrate. The measurement took place after 24-h incubation at a temperature of 30 °C and a wavelength of 485 nm and it was expressed in μmol TPF (triphenyl formazan)·24 h⁻¹ g⁻¹dm of soil (Thalmann, 1968).

Additionally, biochemical analyses of soil involved the determination of activities of acid phosphomonoesterase (PAC) (EC 3.1.3.2) and alkaline phosphomonoesterase (PAL) (EC 3.1.3.2.) according to Tabatabai & Bremner (1969). The activities were determined using disodium p-nitrophenyl phosphate tetrahydrate as a substrate, after 1 h incubation at 37 °C at a wavelength of 400 nm. Results were converted into μmol (p-nitrophenol) PNP h⁻¹ g⁻¹ d.m. of soil. A pH-appropriate buffer was used to determine each of the phosphatases.

Weather conditions

It is generally accepted that climatic conditions found throughout Poland are suitable for willow cultivation. This species needs approx. 500 mm annual precipitation to grow. Weather conditions in the years of the study during the vegetation period of willow are presented using the hydrothermal index according to Sielianinov (Table 2). The hydrothermal index Sielianinov expressed as a quotient of the amount of the monthly sum atmospheric precipitation and sum of average daily air temperatures in a given month for a period of time. In all the years mean air temperature ranged from 8.6 to 10 °C and it was comparable to the multiannual mean. A crucial element of optimal growth and biomass yielding conditions is also total precipitation, particularly its distribution. Variability of weather conditions in the years of the study was reflected in the values of the Sielianinov index. More advantageous moisture conditions for willow plants were found in the years 2013 and 2014 than in the drier 2015 (K = 0.9), which was reflected in the volume of produced biomass.

Interpretation of the Sielianinov hydrothermal coefficient:

• K > 1.5—excessive moisture for all plants

• K = 1.0–1.5—sufficient moisture

• K = 0.5–1.0—insufficient moisture

• K < 0.5—moisture level below the requirement for most plants—drought.

Statistical analysis

Statistical evaluation were performed using the Microsoft Excel and Statistica 12.0 (StatSoft Inc., Krakow, Poland) software packages. In order to compare mean values of the biological parameters at individual dates of analysis and the effect of sewage sludge the two-way analysis of variance was used (α = 0.05). It was followed by detailed Tukey’s test (post-hoc Tukey HSD).

To present results of microbiological analyses in order to select the multiple regression model, stepwise regression with the backward sequence procedure from the 5th degree was applied. Independent variables were successive powers of the variable X as time from the application of sewage sludge and supplemented with the BAF, while the counts of selected microbial groups and soil enzymatic activity were the dependent variables. Moreover, the coefficient of determination R² was used to define the goodness of fit of the model.

The data were subjected to a principal component analysis (PCA). The results of PCA are presented in graphic form. Pearson correlation analysis was carried out for all analysed variables. These analyses were performed for dry matter yield (DMY), shoot thickness (ST), PH, and NS.

Two heatmaps and cluster analyses were here performed—the first one analysed similarities between the microbial soil activity, gas exchange parameters and terms of analysis and experimental variants. The second one investigated the correlations between variables. The first one presents mean values of measured parameters at a certain term of analysis. Afterwards a cluster analysis was performed to group similar terms and variants in regard to all analysed parameters. Euclidean distance measures and Ward hierarchical clustering were used to determine the dendrogram. The Euclidean distance measure can designate a similar structure in interactions between analysed parameters.

In the second heatmap, Pearson’s correlation matrix was designated for the soil microbial activity and gas exchange parameters. Afterwards, a cluster analysis was performed, in order to arrange analysed parameters in groups, with the highest degree of association within each group and the lowest degree of association between different groups.

Soil microorganisms

Statistical analyses were conducted for the results of 3-year studies on the residual effect of sewage sludge on counts of selected groups of soil microorganisms under energy willow culture. The two-way analysis of variance showed a highly significant effect (α = 0.001) of the investigated factors on the dynamics of changes in the counts of selected groups of soil microorganisms, such as the total bacterial count, counts of Actinobacteria, oligotrophic and copiotrophic bacteria (Table 3). Multiple regression analysis showed that best models for each tested microbiological parameter (counts of selected microbial groups and microbial activity) were never higher than 2nd degree models. No model of a higher degree showed a better goodness of fit, while determination of coefficient R² for the 2nd degree model was the highest, ranging from approx. 0.2 to 0.8.

The analysis of mean results from a given year for individual experimental variants showed that the application of sewage sludge and sewage sludge combined with the BAF modified the total bacterial counts in the soil both in the year of application and in the next year (Fig. 1A). The highest total number of microorganisms was recorded in the second year of sludge influence in the variant in which sludge was applied in combination with the BAF. In the third year of analysis, counts of the microbial group decreased in all experimental variants; nevertheless, the highest count was recorded in the same treatment as in the second year. Analogously as in the case of the total number of bacteria, analyses of mean counts of Actinobacteria from a given year in individual treatments indicate a significant stimulatory effect of applied municipal sludge combined with the BAF (Fig. 1B). In the other experimental variants when interpreting the results for the first, second and third year, the number of that microbial group was comparable to that in the control.

Data presented in our results show that municipal sewage sludge and the application of the BAF, as well as the time from their application, had a significant effect on the fluctuations in counts of fungi in the soil. Analyses of mean results from a given year for individual experimental treatments (Fig. 1C) showed significant stimulation of fungal growth by sewage sludge in the second year of field experiments in the treatment in which sludge and the BAF were applied simultaneously.

Analysis of mean counts of oligotrophic bacteria in a given year for individual experimental treatments showed that the growth of investigated microorganisms was stimulated by sewage sludge introduced to the soil only in the second year of this waste impact (Fig. 1D). In turn, growth of another microbial group, that is, copiotrophs, was stimulated by sewage sludge introduced to the soil in all the years of impact of this waste. Similarly as in the case of other microbial groups, this effect was most markedly reflected also in the second year of sludge impact in the variant in which sludge was applied together with the BAF, while it was weakest in the third year, as presented in Fig. 1E.

Biochemical activity

The two-way analysis of variance showed a highly significant effect (α = 0.001) of investigated factors on the activity of selected soil enzymes: dehydrogenases and acid and alkaline phosphatases (Table 4).

Dehydrogenase activity is considered as an indirect indicator of microbial counts and activity in the soil. The presented mean values of dehydrogenase activity from a given year over the period of three years following the application of sewage sludge and the BAF (Fig. 2A) indicated that the applied factors had a significant effect modifying levels of these enzymes in lessive soil. Similarly as in the case of counts of selected microbial groups, the highest level of dehydrogenase activity was recorded in the second year after the application of sewage sludge together with the BAF and sludge alone. Moreover, a strong stimulatory impact of sludge and the BAF was found not only in the second year of analysis, but also in the year of application and in the third year after application, with this activity in individual years exceeding that in the control by 63, 42 and 54%, respectively.

Phosphatases PAC (acid phosphatase) and PAL (alkaline phosphatase) were other enzymes used as indicators of soil quality in this experiment. The activity of acid phosphatase differed from that of alkaline phosphatase. In the variants in which sewage sludge and the BAF were applied, lower values were recorded for the activity of PAC in comparison to those in the control (Fig. 2B).

Results obtained in the field trials indicate the highest values of PAL in all the variants in which sewage sludge and the BAF were applied (Fig. 2C). Additionally, a stimulatory effect of sewage sludge on the activity of this enzyme was also recorded in the third year after its application.

Estimation of photosynthesis activity

A highly significant (α ≤ 0.05) effect of date and variant of application on all analysed gas exchange parameters was found. An increase of net photosynthesis rate was noted for each variant in the 15th month of the experiment. However, the highest values were each time observed in the variant for used alone BAF, while the lowest level was recorded for control plants. At the 27th month of the experiment a decrease was noted to a similar level as at the beginning of the experiment. However, where sewage sludge and BAF and alone BAF were used, the values were highest at this measurement. A high level of net photosynthesis rate (P_N) was related to stomatal conductance (g_s) and transpiration rate, while intercellular CO₂ concentration was the lowest in plants in the 15th month for plants with BAF application. There was a similar level of CO₂ intercellular concentration (C_i) in plants cultivated in the variants with sewage sludge and BAF and alone sewage sludge (Fig. 3).

The heatmap of interactions between mean values of microbial soil activity and gas exchange parameters revealed similarities between variants of sewage sludge, BAF and sewage sludge and BAF in the 15th month of experiment. The control was combined into one group for the 3rd and 27th month after application. The rest of variants and terms were grouped together. Based on this heatmap we can also find the lowest and highest values of the parameters. The highest mean values of bacteria, Actinobacteria, fungi and copiotrophic were recorded in the variant of sewage sludge and BAF in the 15th month after application, and in DHA and PAC in the 27th month after application of sewage sludge and BAF. PAL revealed the highest level in the 27th month in variant where alone sewage sludge, while P_N and g_s was noted for variant with BAF in the 15th month after application. We can also find that the highest parameters of soil microbial activities were observed together with high levels of P_N, g_s and E, while C_i was recorded at the lowest level (Fig. 4).

A high negative correlation between C_i and all microbial parameters (excluding PAC and PAL) and P_N, g_s and E was found. A negative relation of PAC and PAL with the groups of microorganisms and gas exchange parameters (excluding C_i) was recorded. A positive relation of P_N, g_s and C_i to the groups of microorganisms and DHA was found (Fig. 5).

Biomass yield and biometric measurements

The analysis of variance for recorded results showed a significant effect of the years of analysis, the experimental factor as well as the interaction of the year of analysis and the variant of sludge application on the DMY, ST, and additionally a significant effect of the years of analysis on the NS and height of willow plants (PH) (Table 5).

The yield of willow dry mass significantly varied in the years of the study (Table 6). A lower biomass was produced in the first year of culture, which is a characteristic trait of this species. Among the three analysed years the greatest DMY was recorded in the second year of the experiments. In the first year of culture the highest DMY was recorded in the treatment with sludge application (14.6 t·ha⁻¹), while it was lowest in the control (11.3 t·ha⁻¹). In 2014 the lowest yield was also found in the control (19.3 t·ha⁻¹) and following the application of the BAF (19.2 t·ha⁻¹), while it was highest after the combined application of sludge and the BAF (25.8 t·ha⁻¹). In the third analysed year of the study, similarly as it was in the earlier years, the lowest yield of biomass was obtained in the control plot, with the highest yield harvested in the treatment with the combined application of sludge and the BAF.

In comparison to the control in the treatment with the combined application of sewage sludge and the BAF an increase in yield of willow dry mass was recorded, amounting to 3.7 t·ha⁻¹. In contrast, no significant difference was found between treatments in which either sludge (19.4 t·ha⁻¹) or sludge together with the BAF was used (19.9 t·ha⁻¹). The smallest increment in the DMY in relation to the control, amounting to 1.4 t·ha⁻¹, was recorded following the application of the BAF.

A significant effect on the years of the study and sludge application was also observed in the case of ST (Table 6). The thickest rods, with the diameter of 17.0 mm, were obtained in the second year of the study, while they were thinnest in the third, the driest year of culture (12.3 mm). In relation to the control the greatest increment in rod thickness (2.1 mm) was recorded in the sewage sludge treatment. In the first year of the experiments the thickest rods were obtained in the treatments in which the BAF was introduced to the soil, while in the following 2 years it was in treatments with sewage sludge application. The number of willow shoots recorded in this study was significantly modified by the year of culture (Table 6). The greatest NSs from a rootstock was obtained in the second year of the experiments, while no difference was observed in this respect between 2014 and 2015. The greatest, although not confirmed statistically, NSs was recorded in treatments with the application of the BAF. Analogously, year of culture had a significant effect on shoot height analysed in this study. The tallest plants were reported in the third year of analysis (Table 6). The tested combinations showed no effect on the value of this trait, with an upwards trend for height observed following each application.

Biometric characteristics and dry mass of willow plants were subjected to PCA to underline differences between the tested treatments. The analysis of our results shows that the components PC 1 and PC 2 explained 71% (A), 69% (B), 60% (C) and 88% (D) of the data set variation. In the first year, positive relations were noted in the pairs NS and ST, and DMY and PH. The control plants were negatively related to examined biometrical parameters (Fig. 6A). In the second year a high positive association between DMY and PH was recorded, furthermore variants sludge sewage and BAF and alone BAF were positively related to all biometrical parameters (Fig. 6B). In the third year a positive relation between almost all analysed biometrical parameters and variant with sludge sewage and BAF was found (Fig. 6C). The PCA throughout all years revealed a positive relation between NS and DMY, as well as between all analysed treatments and biometrical parameters. This was also valid for the third year (excluding ST) (Fig. 6D).