Experiment with Surface Composting

BiosaHerb™ Environment increases biological activity in the soil!

In the 2022 growing season, Nordquist Ferment had a trial where we compared surface composting with and without BiosaHerb™ Environment

The experiment was partly funded by Forregion, and in collaboration with Norsøk, Vibhoda in Sunn Jord and field host and researcher Dag Molteberg.

The report below was prepared by Dag Molteberg, internal reporting in the project.

Experimental design

8 routes were laid out on the Traffic Change and followed throughout the season, 4 with biomass treatment (10 l/day) and 4 without treatment (0 l/day).

Each route was approximately 8.5 m wide and 25 m long. The spaces between the routes were approximately 3.5 m (a threshing floor width) wide and 25 m long.

Figure 1 shows how the routes were created.

Experiment, surface composting
Experiment, surface composting
Experiment, surface composting

A cumulative sum of daily degrees and precipitation for the 2022 growing season has been extracted from the Øsaker measuring station, see Figure 2 and https://lmt.nibio.no/stationinfo/118/.

From this it can be seen that the accumulated total for 20/4-22 was 16.5 degree days, which corresponds to 1% of the total for the entire season (1666 degree days). It was also on this day that the soil temperature exceeded 5 degrees at 1 and 10 cm depth. 20/4 was therefore defined as the start of the growing season.

All withdrawal dates have been converted to growth day numbers from the start date 4/20-22 in statistical analysis.

Experiment, surface composting

Material

Soil samples were taken from all 8 plots before surface composting (29/4-22), after surface composting (5/5-22), after germination and before fertilization (1/6-22), after rain and first fertilization (6 / 6-22) and after threshing (20/9-22). The soil sample depth was 5-10 cm for the first 3 samplings and 20 cm for the last two. Data are shown in Table 1 below.

Experiment, surface composting

Method

Microbial carbon is measured with a microbiometer, which is an analysis kit with equipment and chemicals (salts), and where you do the analysis using photography and image processing with your own mobile phone. The method reports the amount of carbon attached to microbial living material in a sample, divided into total amount, proportion attached to fungi and proportion attached to bacteria. Total amount is the sum of the amount for fungi and bacteria. The unit of measurement is µg/g soil or ppm. The report also provides the fungi:bacteria ratio. The analysis is carried out by passing a representative part of the soil sample through a small sieve (1-2 mm opening). Then 0.5 ml of soil is taken out of the sieved sample. In a test tube, a small bag of salt (NaCL + CaCl2) is stirred into 9.5 ml of water. The prepared soil sample (0.5 ml) is then added and stirred with the provided whisk for 30 seconds. The test tube is then left still for 5 minutes, then tapped lightly against a hard surface 3-4 times and left still for another 15 minutes. Soil particles have then sunk to the bottom, and only living fungi and bacteria remain in the water phase. A small amount of the water is taken out with a pipette and three drops are placed on a separate analysis card. With a separate app, a picture of the card is taken against a supplied background, and you get the report on your phone. The method is calibrated and validated against microscopy tests. See the website for more information https://microbiometer.com/.

The data in the experiment are fitted with a multiple regression model with response Y, where this can be affected by the time of testing and treatment with and without BiosaHerb™ Environment, as well as the interaction between these. An interaction means that the effect of added BiosaHerb™ Environment changes over time. A block effect (north and south fields) was also included to see if there are internal differences in the soil between routes 1-4 and 5-8. The residual error is deviation from the model. Using statistical software (JMP), all effects in the model were calculated, and statistical tests for significance were performed. Response Y is microbial carbon in fungi (Fungi), bacteria (Bact) and total (Total). The full model (1) for Y then looks like this:

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Model 1 Y = a0 + a1*Time Variable + a2*Treatment + b1*Time Variable * Treatment + a3*Block + Residual Error,

where a0, a1, a2, and a3 are parameters that affect the intercept (the intersection with the Y-axis), and b1 is a parameter that affects the slope of a straight line a+b*x. The residual error is the individual deviation of the model per observation.

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Results and discussion

Figures 1 and 2 show how microbial levels have changed with time and treatment. Trendlines are equipped with spline functions in JMP software.

Red symbols and lines are squares with BiosaHerb™ Environment (10 l/daa) and blue are without BiosaHerb™ Environment (0 l/daa). Solid lines (top) are for total microbial carbon, thick dashed lines are for bacterial carbon (middle) and fine dashed lines are for carbon in fungi (bottom). Values ​​for microbial carbon are shown along the Y-axis.

Figure 1 shows the sequence of withdrawals along the x-axis as a categorical variable (discrete values), while Figure 2 shows the date of withdrawal along the x-axis as a continuous variable. There are no clear differences between routes with BiosaHerb™ Environment and routes without BiosaHerb™ Environment for the first three withdrawal times, but it may appear that there is more microbial carbon in the soil after threshing. Exit point 4 shows the initial difference with and without BiosaHerb™ Environment. Figure 2 with time as a continuous variable (date) also shows that the first three times are quite close to each other, while withdrawals after threshing are further away from the others.

Experiment, surface composting

Figure 1. Total microbial carbon, for bacteria (Bact) and for fungi (Fungi) for all experimental routes at the different sampling times plotted as discrete values.

Experiment, surface composting

Figure 2. Total microbial carbon, for bacteria (Bact) and for fungi (Fungi) for all experimental routes at the different sampling times plotted as date (continuous value).

Statistical analysis

The statistical analysis is based on the general model 1, to investigate how the status of microbial carbon changes over time, and whether the level is affected by treatment with BiosaHerb™ Environment at any of the time points. From figures 1 and 2, it may appear that the effect of BiosaHerb™ Environment becomes greater over time, especially figure 2 indicates this. The curves indicating the development with and without BiosaHerb™ Environment in total, for fungi and for bacteria, also appear to be quite linear and can thus be described in the form of a straight line (a+b*x), where x is a continuous time variable.

If a good enough model description is found with as few model parameters as possible, it is easier to demonstrate whether the impact of BiosaHerb™ Environment is significant (probability P of error in claiming a difference is less than 5%). Different models were thus built based on both a discrete (time) and a continuous (growing day) time variable and a summary of these is summarized in Table 2. Simplified models were also made, where only the significant effects are included.

Experiment, surface composting

Table 2. Multiple regression models for microbial carbon Total, Fungi (fungi) and Bact (bacteria) with selected statistical values.

Models with all times

Two types of models were set up, where model 2-7 uses time as a discrete time variable, and model 8-13 uses growing days as a continuous variable. The interaction between time and BiosaHerb™ Environment treatment is also examined. Growing days is then fitted as a straight line with the possibility of different increments. For the discrete time variable in model 2-4, this means that a total of 10 parameters are required to describe both the main effect for the time variable (4 parameters) and the interaction effect against BiosaHerb™ Environment treatment (4 parameters), as well as BiosaHerb™ Environment alone. (1 parameter) and Block effect alone (1 parameter). For model 8-10, the number of parameters in the model could be reduced to 4.

For models 2-4, all effects are included in the model, even if they are not significant. For models 5-7, non-significant effects in models 2-4 are removed. Similarly, models 11-13 show the result where significant effects from models 8-10 have not been removed.

Experiment, surface composting

Figure 3 shows what the discrete model looks like (models 2-4) and Figure 4 shows what the continuous model looks like (models 8-10).

It is very clear for all models that microbial carbon (MBC) increases during the growing season. The level is statistically the same the first three times 29/4, 5/5 and 1/6, while it is increasing for the rest of the season. This applies both overall, for fungi and for bacteria. There is also a stronger increase for fungi than for bacteria. Typically, MBC for fungi starts at 100 µg/g soil in April-May and ends at about 210 µg/g in September. For bacteria, the increase is smaller, from about 185 to 235 µg/g. Total MBC starts at 285 µg/g and ends at about 450 µg/g.

The discrete models 2-7 do not show that there is a difference between microbial carbon with and without BiosaHerb™ Environment. Here there is also no clear block effect, although the P level is 7-9%, it is above the 5% limit and thus not significant.

The continuous models 8-13, on the other hand, show a clearly more pronounced effect also when treated with and without BiosaHerb™ Environment at the end of the season. It can be shown that the MBK for fungi is significantly improved during the growing season (P=3.6%). The level is exactly the same at the beginning of April-May (approximately 100 µg/g), but in September the BiosaHerb™ Environment-treated routes are at 237 µg/g and the untreated ones at 176 µg/g. No significant effect could be demonstrated for bacterial MBK and total MBK, but the P-values ​​were quite close to the significance requirement (P = 17% and 7%). Numerically, an increased MBK value could also be seen here for the BiosaHerb™ Environment-treated routes. Figure 4 shows that the BiosaHerb™ Environment-treated routes (blue, 10 l/daa) increase faster than the references (red, 0 l/daa). The block effect is also weak here, as for models 2-4.

Experiment, surface composting

From Table 2 it can be seen that models 2-7 have a higher degree of explanation (R2-Adj) and lower residual variation (RMSE) than models 8-13, but despite this, models 2-7 do not show that BiosaHerb™ Environment treatment is significant. This is because so many parameters are used to determine the time effect with interaction, and that the BiosaHerb™ Environment effect only becomes visible towards the end of the growing season. A linear model based on a continuous time variable based on growing days captures this better.

Models with reduced data volume

To improve the analysis of the BiosaHerb™ Environment effect, an additional set of models 14-19 was created, which is shown in Table 2. Here, all data for 29/4, 5/5 and 1/6 were combined into a common group called "Establishment". Data from 6/6 were omitted. Data for 20/9 were kept in the group "After threshing". These two groups were combined into a new time variable, Time 2. The analysis was then repeated. The advantage of this analysis is that you have far fewer parameters to adjust, and you focus more on the starting point and the end result without worrying so much about what the intermediate phases look like.

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To show that it makes sense to combine the first three samples, Figure 5 shows a one-way analysis of variance for MBK Total, for fungi (Fungi) and bacteria (Bact). There you can clearly see that the first three payouts are completely similar, while there is an increasing level for the last two.

Experiment, surface composting

The result of models 14-19 in table 2 is that the residual variation is reduced (RMSE decreases), and the degree of explanation for the models increases, compared to models 2-7 and 8-13. It is also clear that treatment with BiosaHerb™ Environment has an impact on both total MBK and fungi (Fungi). For bacteria, the effect is smaller and with P=9% it is not significant. The block effects are also not significant. However, with a new experiment, where one had focused more on the effect over the entire season and with slightly more data points, one could expect a significant effect here as well. In figure 6, the models are illustrated without block effects.

Experiment, surface composting

The models show that treatment with BiosaHerb™ Environment at 10 l/day increased total microbial carbon in the soil from 286 µg/g soil at spring establishment to 487 µg/g soil after threshing (September). This is an increase of 70%. The reference without treatment increased to 401 µg/g soil, an increase of 40%. The increase during the season was significant, as was the further increase with BiosaHerb™ Environment.

For microbial carbon attached to fungi, the starting level was 99 µg/g soil, this increased to 240 µg/g soil when BiosaHerb™ Environment was dosed at 10 l/daa, while the final level without BiosaHerb™ Environment was 174 µg/g soil. With BiosaHerb™ Environment the increase was 142% and without BiosaHerb™ Environment 76%. The increase during the season was significant, as was the further increase with BiosaHerb™ Environment.

For bacterial carbon, the initial level was 187 µg/g soil, the final level with BiosaHerb™ Environment 247 µg/g soil and without BiosaHerb™ Environment 227 µg/g soil. The increase was then 32% with BiosaHerb™ Environment and 21% without BiosaHerb™ Environment. The increase during the season was significant, but the further increase for BiosaHerb™ Environment was not statistically significant.

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The fact that the increase in microbial carbon is more obvious for fungi compared to bacteria may be due to the fact that BiosaHerb™ Environment helps to facilitate the fungi's transformation processes that take place in the soil, and that the biomass that is fermented improves the fungi's opportunities for establishment and residence. conditions.

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The modeling shows that it is important to choose the right type of model to compare this type of experiment, one should try to find a simple function that describes the change within the same treatment over time in the best possible way. From such a model, the effect of different treatments can then be investigated. A straight line can be a good approximation, but there may be other approximations that give even better models.

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In addition, it is also important to see the changes over a sufficiently long period of time. In this experiment, it is clear that the changes in the soil with the addition of BiosaHerb™ Environment have long-term effects. This needs to be investigated better, perhaps also over several growing seasons.

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BiosaHerb™ Environment is added as part of the surface composting. The amount of soil life measured with a microbiome clearly increases during the growing season after surface composting, both for fungi, bacteria and in total. But in addition, the analysis shows that BiosaHerb™ Environment enhances this effect. There is a need to investigate this effect more broadly, for more soil types and also under other weather conditions. The 2022 season was dry at the beginning, this may have delayed the effect of BiosaHerb™ Environment, but equally the addition of BiosaHerb™ Environment clearly produced more microbial carbon.

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Conclusion

Through the trial at Ormo, surface composting has provided an increased amount of microbial carbon throughout the 2022 season, both in total, for fungi and for bacteria.

Adding BiosaHerb™ Environment during surface composting has clearly enhanced the effect for fungi and overall, using BiosaHerb™ Environment at 10 l/day increased the amount of microbial carbon beyond what surface composting alone gave. For fungi, microbial carbon increased by 142% with BiosaHerb™ Environment and 76% without BiosaHerb™ Environment throughout the 2022 growing season. Total amount increased by 70% with BiosaHerb™ Environment and 40% without BiosaHerb™ Environment. For bacterial carbon, the increase was smaller and below the detection limit.

The effect of BiosaHerb™ Environment should be investigated in more detail for more soil types and for more growing seasons. It is clear that the change processes that BiosaHerb™ Environment helps to maintain continue for a long time after addition, so future studies should continue over a longer period of time and perhaps over more seasons.

Experiment, surface composting

Read the full report to NORSOEK below.