Use of Slaughterhouse Sludge in the Bioremediation of an Oxyfluorfen-Polluted Soil

The use of organic matter is a highly accepted environmental practice among scientists for the bioremediation of polluted soils. In this manuscript we study under laboratory conditions the bioremediation capacity of a new biostimulant obtained from slaughterhouse sludge in a soil polluted by the oxyfluorfen at a rate of 4 l ha−1 (manufacturer’s rate recommended) over a 90-day period. We determined its effects on dehydrogenase, urease, β-glucosidase and phosphatase activities, the soil microbial community structure and the evolution of the herbicide in soil. Possibly due to the high content of low molecular weight proteins in the biostimulant, the enzymatic activities were stimulated mainly at the beginning of the experiment. Soil biological parameters were inhibited in oxyfluorfen-polluted soil. At the end of the experiment and compared with the control soil, dehydrogenase, urease, β-glucosidase, and phosphatase activities significantly decreased by 47.8%, 50.5%, 36.4%, and 45.5% in the oxyfluorfen-polluted soil. At 5 days into the experiment, the use of the biostimulant in oxyfluorfen-polluted soils decreased soil enzymatic activities and microbial community inhibition. At the end of the incubation period the oxyfluorfen concentration had decreased by 60% in the polluted soil and amended with biostimulants. These results suggested that the use of this biostimulant with higher amounts of low molecular weight proteins and peptides had a positive effect on the remediating oxyfluorfen-polluted soils. Therefore, this study provides the use of a new biostimulant obtained from slaughterhouse sludge by enzymatic hydrolysis processes used in the bioremediation of a soil polluted by the oxyfluorfen herbicide. Oxyfuorfen herbicide caused a negative effect on soil biological properties The application of biostimulants obtained by enzymatic hydrolysis from slaughterhouse sludge decreased the toxic action of oxyfluorfen The low molecular weight protein of biostimulants increased the degradation of herbicide Oxyfuorfen herbicide caused a negative effect on soil biological properties The application of biostimulants obtained by enzymatic hydrolysis from slaughterhouse sludge decreased the toxic action of oxyfluorfen The low molecular weight protein of biostimulants increased the degradation of herbicide


Introduction
In the last decades there has been a notable increase in the amount of sludge resulting from slaughterhouse wastewater treatment, a consequence of the increase in meat production. Bouwman et al. (2013) suggest that by 2050 meat production will have doubled. Consequently, it is very likely that the increase in the number of slaughterhouse facilities to lead to an increase in the volume of slaughterhouse sludge (Bustillo-Lecompte and Mehrvar 2015).
Slaughterhouse wastewater has been classified by the United States Environmental Protection Agency as wastewater harmful to the environment (Alfonso-Muniozguren et al. 2021) mainly due to its high content of organic matter, suspended solids, oil and fats and nutrients (Aziz et al. 2019;Menegassi et al. 2020).
Currently, one of the most widely used bioremediation techniques among the scientific community is the application of organic matter to polluted soil, because it is a very cheap, effective technique that can be performed in situ (Davin et al. 2018). In these cases, organic matter reduces the concentration of contaminant in the soil via two different pathways. Firstly, this organic matter has the capacity to adsorb the toxins, decreasing their concentration in the soil solution and consequently reducing their toxicity. Second, the addition of this organic matter stimulates the growth of those microorganisms that are tolerant to the toxins presents in the soil. In doing so, the microorganisms increase the rate of pollutant degradation and so decrease their soil concentration Lipczynska-Kochany 2018;Ortiz-Botella et al. 2021). However, for this organic matter to be able to stimulate these soil microorganisms, there is a delay, while it is mineralized into simpler forms that are more easily available to the said microorganisms (Ortiz-Botella et al. 2021).
Using this slaughterhouse sludge to manufacture new edaphic biostimulant (BS) obtained by enzymatic hydrolysis processes would, therefore, have important socioeconomic and environmental impacts. On the one hand, there would be a solution to the accumulation of this organic waste and, on the other, the sludges could be of great use for bioremediating pesticide-polluted soils.
This study was prompted to obtain a new BS obtained from slaughterhouse sludge to bioremediate a soil contaminated with the oxyfluorfen herbicide, which exerts a persistent toxic effect on soil biological properties Rodríguez-Morgado et al. 2014;Franco-Andreu et al. 2016;Campos et al. 2019). Our hypothesis is supported by the existence of other BS obtained by enzymatic hydrolysis process from different organic wastes such as sewage sludge, chicken feathers, okara, rice bran, etc. used in the bioremediation of soils polluted by various pesticides Tejada et al. 2010Tejada et al. , 2011aTejada et al. , b, 2014Rodríguez-Morgado et al. 2014, 2015aOrts et al. 2017). These BS are characterized by presenting a high number of low molecular weight peptides, amino acids, etc. that are easily absorbed by soil microorganisms, thus accelerating the degradation of the pollutant in the contaminated soil.
There is an abundant bibliography that suggests understanding the behaviour of any xenobiotic in soil requires the study of biological parameters (Campos et al. 2019;Wołejko et al. 2020). This is because these biological parameters react much faster than physical and chemical ones (Kadian et al. 2012;Franco-Andreu et al. 2016;Orts et al. 2017). Therefore, the study of soil enzymatic activities could be very useful to understand the effect of slaughterhouse sludge on the bioremediation of oxyfluorfen-polluted soils.
Within the frame work of this hypothesis, the aims were: (1) to obtain a new biostimulant created from slaughterhouse sludge via enzymatic hydrolysis processes, and (2) to study the effectiveness of slaughterhouse sludge as a BS in the bioremediation of a soil polluted by oxyfluorfen, principally studying its repercussion on the target soil's biochemical properties.

Characteristics of Organic Wastes, Soil and Herbicide
The slaughterhouse sludge was supplied by the "Matadero del Sur" company, located in Salteras (Seville, Spain). This sludge was subjected to two different treatments to obtain two different organic products.
The first of the treatments consisted of concentrating the slaughterhouse sludge until reaching a dry matter value of 15%, since at this value, an easily manageable product is obtained. This concentration was carried out at 75 °C with a rotary evaporator. This first organic compound is called concentrated slaughterhouse sludge (SS).
The second organic compound was obtained by subjecting the slaughterhouse sludge to an enzymatic hydrolysis process. The hydrolysis process was performed in a bioreactor according to the pH-stat methodology (Adler-Nissen 1977). Figure 1 shows the conditions under which this biochemical process was performed. Once the soluble product was obtained, it was also concentrated to 15% at 75 °C with a rotary evaporator. This second organic compound is called BS obtained from slaughterhouse sludge. Table 1 shows the chemical composition of both organic compounds. The methodology measuring each chemical parameter is detailed in Rodríguez-Morgado et al. (2015b).
The experimental soil used was a Calcaric Regosol (FAO 1989). The soil characteristics are shown in Table 1. The analytical methods used in determining these soil parameters are detailed in Tejada et al. (2014).

Experimental Design
Three hundred grams of dried soil was mixed with oxyfluorfen and treated with SS at a random rate of 3% (9 g of product) and BS at a rate of 3.9% (11.7 g of product) to apply the same amount or organic matter to the soil (5.84 g organic matter per kg of soil). A non-amended and non-polluted soil was used as control. The incubation treatments were detailed in Table 2.
Both organic compounds were liquid and were solubilized in distilled water before applying.
Triplicate treatments were kept in microcosms at 25 ± 1 °C for 90 days. Distilled water was added to each soil to reach 30-40% of its water-holding capacity and the moisture losses were compensated by adding distilled water.

Statistical Analysis
To determine the differences between the results obtained for each parameter analysed, the data were submitted to two-way analysis of variance (ANOVA) using two factors (treatments and sampling time). This was then followed by the Tukey post-hoc test (HSD, p < 0.05). The ANOVA was performed using the Statgraphics Plus 2.1 software package.
For the statistical analysis, triplicate data were used for each treatment and each day of incubation.

Results and Discussion
After the enzymatic hydrolysis process and with respect to the SS, the BS showed a significant (p < 0.05) decrease in the Ca and micronutrients concentration (Table 1). It was also observed that significant changes occurred in the protein molecular weight distributions. In this respect, and compared with the SS, the proteins with the highest molecular weight (> 1000 KDa) in the new BS decreased by 35.4%. Also, and with respect to the SS, the 1000-300 Da and < 300 Da molecular weight proteins in the new BS increased significantly by 51.5% and 66.2%. The application of SS and BS significantly (p < 0.05) stimulated the soil enzymatic activities studied (Tables 3 and  4). Compared with the C treatment, the application of SS to the soil progressively increased the dehydrogenase, urease, β-glucosidase and phosphatase activities by 65.7%, 70.3%, 78.8% and 68%, respectively. However, the behavior of the enzymatic activities studied after applying the BS to the soils was very different. Dehydrogenase, β-glucosidase and phosphatase activities reached maximum stimulation 5 days after the start of the experiment, then gradually decreasing until 35 days after the experiment. From this date, the dehydrogenase, β-glucosidase and phosphatase activity began to increase again until the end of the incubation period, reaching a higher value than that obtained in the control treatment. In this sense, the stimulation of dehydrogenase, β-glucosidase and phosphatase activities was 54%, 68.6% and 59.8.
Compared with the C + SS treatment, after the application of the BS to the soil, the behavior of the urease activity was very similar throughout the incubation period (Table 3).
We think that the cause of the different behaviour in the two experimental organic wastes can be due to the difference in the molecular protein weight distribution percentage. Our results highlight that in both organic wastes the protein size distribution differs mainly in sizes of > 1000 Da and < 300 Da. The SS has a higher percentage of high molecular weight proteins, while BS showed a higher percentage of low molecular weight proteins. Since the microorganisms in the soil cannot directly absorb the high molecular weight proteins, to obtain energy these microorganisms need to degrade them. This mineralisation process is usually slow, and therefore, microbial stimulation in the SS-amended soil increased progressively throughout the experimental period. According to Rodríguez-Morgado et al. (2015b), when applying BS to the soil, microorganisms directly absorb these low molecular weight proteins, favouring their stimulation. The subsequent decrease in their enzymatic activities to values similar to the control at day 35 suggest that by then soil microorganisms had already consumed these low 5.5c ± 0.7 4.0c ± 0.7 3.7bc ± 0.6 3.7bc ± 0.7 4.1c ± 1.0 Urease activity (µg NH 4 + g −1 h −1 ) C 1.7b ± 0.2 1.7b ± 0.2 1.8b ± 0.1 1.7b ± 0.2 1.8b ± 0.3 1.9b ± 0.3 C + SS 2.7b ± 0.9 3.2bc ± 0.6 3.7c ± 0.8 4.8c ± 1.0 5.2c ± 0.9 6.4c ± 1.7 C + BS 2.9b ± 1.1 3.4bc ± 0.8 4.0c ± 1.0 4.7c ± 0.8 5.8c ± 0.6 6.9c ± 0.8 C + Ox 0.94a ± 0.13 0.98a ± 0.12 0.91a ± 0.13 0.83a ± 0.17 0.90a ± 0.09 0.94a ± 0.11 C + Ox + SS 1.7b ± 0.6 2.2b ± 0.6 2.9b ± 0.7 3.0b ± 0.8 3.8c ± 0.6 4.5c ± 1.0 C + Ox + BS 2.2b ± 0.4 2.5b ± 0.4 2.7b ± 0.5 2.7b ± 0.6 3.1bc ± 0.8 3.4c ± 0.7 molecular weight proteins. The progressive increase from day 35 on the soil biochemical activity is a consequence of the fact that the BS still showed a high number of high molecular weight proteins, implying that, as of this day, soil microorganisms began to excrete extracellular enzymes to obtain energy. This means that there was a progressive increase in the soil biochemical activity until the end of the incubation period. The soil behaviour of this new BS obtained from slaughterhouse sludge differs greatly from that obtained from other BSs also by enzymatic hydrolysis using the same enzyme from sewage sludge, chicken feathers, rice brain, condensed distillates of soluble wheat, and okara, which, after application, caused an increase in the soil biological activity during the first days of incubation. Subsequently this activity decreased and showed a biochemical activity similar to that of a non-polluted soil (Tejada et al. 2010(Tejada et al. , 2011bRodríguez-Morgado et al. 2015b;Orts et al. 2017). This is a consequence of the fact that in this type of BS, the amount of low molecular weight proteins (< 300 Da) was greater than that obtained with the BS of slaughterhouse sludge. Therefore, when the BS obtained from the slaughterhouse sludge was applied to the soil, the microorganisms quickly assimilated the low molecular weight peptides. Once these had been absorbed, the microorganisms had to obtain their energy from the high molecular weight proteins, excreting extracellular enzymes to degrade said proteins. Tejada and Benítez (2020) also observed that the biochemical activity was higher in soils amended with organic matter with a higher percentage of low molecular weight peptides. These authors also concluded that the higher soil microbial stimulation was possibly a consequence of a higher absorption of low molecular weight peptides by the microorganisms.
Applying oxyfluorfen to non-amended soil caused a significant (p < 0.05) inhibition in the soil enzymatic activities during the experiment (Tables 2 and 3). At the end of the experiment and compared with the C treatment, dehydrogenase, urease, β-glucosidase, and phosphatase activities significantly decreased by 47.8%, 50.5%, 36.4%, and 45.5%, respectively (Tables 3 and 4).
Applying oxyfluorfen to non-polluted soil caused an inhibitory effect on the soil biochemical activity and microbial population. These results are in accordance with Nadijer et al. (2013), Gómez et al. (2014), Franco-Andreu et al. (2016, Campos et al. (2019) and Wołejko et al. (2020), who highlighted the toxic effect of oxyfluorfen on soil biochemical activity.

PNP p-nitrophenol
The oxyfluorfen degradation was higher in the BSamended soil than with SS. The higher percentage of low molecular weight proteins (< 300 Da) in the BS enabled the herbicide-tolerant microorganisms to the said peptides more quickly. This stimulated the microbial population and consequently led to a greater degradation of the herbicide. The high percentage of high molecular weight protein in SS results in a lower stimulation in the herbicide-tolerant microbial population. Consequently, over time oxyfluorfen degradation is slower than when SS is applied.
Applying the herbicide to non-organic amended soil did not change the microorganisms population during the experimental period (Table 5). Similar results were found by Rodríguez-Morgado et al. (2014) and Gómez et al. (2014) in a soil with similar physicochemical characteristics and contaminated with the same dose of oxyfluorfen during a period of 120 days.
The application of organic matter to the soil caused an increase in total bacterial PLFA and total fungal PLFA population. Tian et al. (2017) and Zheng et al. (2021) also  found an increase in microbial biodiversity in soils with a high content of organic matter. These authors suggest that the application of organic matter to the soil provides substrates that can increase the total abundance of PLFA. When oxyfluorfen was applied to soils amended with both organic compounds, the bacteria and fungi population was not different from that observed for C + SS and C + BS treatments. This fact is possibly due to the fact that oxyfluorfen did not change the microbial biodiversity of the soil, as previously mentioned.

Conclusions
According to the authors' knowledge, the main novelty of this manuscript is the first study in which bioremediation tests have been carried out on soils polluted by the oxyfluorfen herbicide using slaughterhouse sludge, both concentrated and in the form of biostimulants after subjecting said sludge concentrated to an enzymatic hydrolysis process. The results obtained in this experiment indicated that slaughterhouse sludge decreased the inhibitory effect of the oxyfluorfen on the soil's biological properties. Consequently, slaughterhouse sludge could be considered as being very useful in bioremedying oxyfluorfen-polluted soils. However, the greatest bioremediation effects were obtained when the slaughterhouse sludge was transformed into a biostimulant by enzymatic processes rich in low molecular weight proteins. These proteins are easily assimilated by toxictolerant soil microorganisms which accelerated the degradation of the herbicide in soil.
However, the bioremediation effect of slaughterhouse sludge should be studied further. The type of contaminant in the soil, as well the soil properties, are characteristics to be considered for future studies to better understand the effect of these organic wastes on the bioremediation of polluted soils.
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