1. Introduction

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Soluble biopolymers (SBO) obtained from alkaline hydrolysis of bio-residues from municipal and agriculture source are multipurpose products for use in diversified fields (Montoneri, 2017). Examples are the fabrication and/or formulation of detergents, dyeing baths, flocculants, dispersants, binding agents for ceramics manufacture, emulsifiers, auxiliaries for soil/water remediation and enhanced oil recovery, nanostructured materials for chemical and biochemical catalysis, plastic materials, soil fertilizers and plant biostimulants for agriculture. The SBO are composed by a heterogeneous pool of macromolecules with 67–463 kg•mol−1 molecular weight and 6–53 polydispersity index. They contain aliphatic and aromatic C chains substituted by a wide variety of acid and basic functional groups of different strength (Tabasso et al., 2015). Their chemical nature bears chemical similarities with natural humic substances (HS).

The above findings prospect implementation of a bio-waste based circular economy, based on the following hypothetical scenario. The hydrolysis of municipal food wastes, private gardens and public parks trimmings, and post-harvest plant residues yields SBO for use in agriculture and for the manufacture of consumers’ chemical product in everyday life.

Renewable C is recycled repeatedly over the agriculture and urban environment. Biowaste treatment plants (Montoneri et al., 2011) become biorefineries, producing bio-based fuel and chemicals. SBO replace products from fossil sources. This reduces the depletion of fossil sources and the environmental impact of not renewable fuel and chemicals. The economy of biowaste treatment plants improves. The bio-waste based economy produces socio-economic benefits; such has reduction of citizens’ taxation for waste management and creation of new jobs (Montoneri, 2017).

The SBO are not commercial products yet. To contribute to the realization of the above perspectives, a hydrolysis prototype plant is under construction within the municipal biowaste (MBW) treatment plant of Acea Pinerolese in Pinerolo (TO), Italy. Acea collects MBW from 150,000 inhabitants of 47 municipalities (Montoneri et al. 2011). It process MBW through integrated anaerobic and aerobic fermentation. It produces biogas and compost, and thermal and electrical power from biogas for the nearby residential district. The prototype plant will be integrated into the existing biochemical facilities to hydrolyze the plant anaerobic fermentation digestate and/or the compost, produce 400-800 t/y SBO and validate the SBO performance and marketability in real operational conditions. In this scenario, it is essential assessing the compliance of SBO with countries’ regulatory requirements to ensure the quality, safety, and efficacy of the product and obtain its marketing authorization (Handoo et al., 2012).

Marketing approval of new products is quite complex process. It involves the assessment of critical parameters during product development. The regulatory requirements of various countries of the world vary from each other. Therefore, it is quite challenging to collect data, which is suitable to obtain approval in different countries. In the case of SBO, the authors have considered the Italian legislation on fertilizers, as starting point. The most recent Italian legislation on fertilizers (Presidente della Repubblica, 2010; Ministero delle Politiche Agricole e Forestali, 2017) allows marketing HS or HS-like products obtained by extraction of soil, peat or other fossil materials, or mixtures of vegetable materials after anaerobic and/or aerobic fermentation. Based on chemical composition, sourcing material and production process (see section 2.1), the SBO fall well into the above category of fertilizers.

The chemical similarity (Montoneri, 2017) of SBO with HS is a favorable feature, in relation to the perspective of successfully demonstrating the safety of SBO for commercial use. Humic substances isolated from soil or fossils are efficient for use as diet supplement in animal husbandry (Montoneri et al., 2013; Biagini et al., 2016). Use of SBO, in place of HS, appears highly desirable for reasons linked to the greater availability of biowastes and to the need to reduce landfill areas for biowaste disposal. Agriculture and animal husbandry constitute important markets for the potential allocation of large amounts of SBO. These facts offer worthwhile scope for investigating the full potential feasibility of SBO to enter the market.

Sortino et al. (2012), Montoneri et al. (2013) and Biagini et al. (2016) have dedicated studies to assessing the performance and safe use of SBO. Sortino et al. (2012) carried out an on field tomato cultivation study, adding SBO to the cultivation soil. They found that, compared to the no treatment and to the treatment using commercial fertilizers, the SBO enhance significantly plant growth and crop production, while not affecting chemical composition of soil and leaves. The authors concluded that the potential adverse environmental impact of periodically repeated applications of SBO to the same soil should raise no more concern than that contributed by conventional mineral and organic fertilizers.

Montoneri et al. (2013) performed an in vitro fermentation study to test SBO as diet supplements to modulate pig’s cecal fermentation of a protein feed. They added SBO at 0.05-0.2 % level in the fermentation slurry. The results showed that, during fermentation, ammonia production in the fermentation slurry containing SBO decreased by 17 %, compared to the control fermentation with no added SBO. The authors ascribed the SBO effect to reduced proteolysis and N mineralization. They expected favourable economics and lower environmental impact for the animal production industry, stemming from the use SBO as animal feed supplements.

Biagini et al. (2016) carried out an in vivo animal study. They evaluated the effect of different doses of SBO as feed additives in diets for fattening rabbits. Rabbits are herbivorous non-ruminants. They are able to utilize a wide range of feed resources. The rich microbial intestinal population (particularly at the caecum level) influences the post-gastric digestion of such animals. One of the leading causes of mortality is disease of the digestive apparatus, involving the microbiota. This feature makes the use of rabbits interesting to assess the influence of SBO on animal welfare and intestinal microflora. Rabbits serve to bridge the gap between the small animal models, which are perhaps more suitable for biomedical research, and larger animals used for animal production. The results of Biagini et al. (2016) study showed that diets containing 0.05–1 %w/w do not affect the live and slaughtering performances, except for the intestinal tract absorption area (higher in the intestines of rabbits fed with the high SBO content) or the apparent digestibility of the feed (lower in diets with low or no SBO content). The authors concluded that SBO might perform as feed additive for rabbits, without any adverse effects on animal productivity and health.

To find whether the encouraging results on the safe use of SBO in rabbits’ diet were general for other animals, the present work reports the in vivo study on pigs husbandry performed with diets containing similar SBO amounts as in the rabbits study. Pigs are large animals. Pigs and humans have anatomical and physiological similarities (Kobayashi et al., 2012). Pigs are models in biomedical research and toxicology testing (Swindle et al., 2012).

2. Methods And Materials

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2.1. SBO Preparation And Characterization
The SBO were obtained and characterized, as previously reported (Montoneri, 2017). Urban private gardening residues and public park trimmings collected in the province of Torino, Italy, were composted for 280 days. The compost was hydrolysed 4 h at 60 °C in aqueous KOH at pH 13 and 4 liquid/solid w/w ratio. Centrifugation of the recovered slurry separated the insoluble residue (IR) from the aqueous hydrolysate. Filtration of the recovered liquid phase through a 5 kDa cut off polysulphone membrane yielded the retentate and the permeate. Drying at 60 °C the retentate yielded the solid SBO in 30 % w/w amount, relative to the pristine compost (CP). The compost, and the IR and SBO products, were characterized by the data reported in Table 1 and 2.

The data in Table 1 were obtained by 13 C NMR spectroscopy, potentiometric titration, elemental C and N, and mineral elements,according to a protocol specifically developed for biowaste derived materials (Tabasso et al., 2015; Montoneri et al., 2013; Sortino et al.,2012). The data in Table 2 were obtained according to AOAC (2006).

2.2. Pigs Feeding Trials
The animal study was performed in 2011, in compliance with the Italian national legislation on the use of animals for research (Presidente della repubblica, 1992). The experimental work was performed according to the commonly accepted '3Rs' EU guidelines at the Cerzoo facilities (Università Cattolica del Sacro Cuore, 2017) in Piacenza, Italy. Before starting the work, the authors submitted the ethics committee of the local Italian authority (A.USL di Piacenza, 2017) the 2011 document (see supplementary material file), which contained all experimental details of the planned work. According to the above regulation, this document accomplished the legal requirements to start the work. The test animals were 54 castrated male and 54 female weaned piglets with average 7.74±0.46 kg weight, genetic type commercial hybrids. Agricola Serena di Lingiardi Sandro Fabrizio, Cascina Serena, Via Strada Provinciale, 104 – 24051, Antegnate (BG), Italy, AUSL code 007 LO 011 supplied the animals. These were housed in pens within a sun-lighted building, where temperature and humidity were controlled through water heaters and a computerized automatic ventilation system. The pens were equipped with mangers and automatic water nipple drinkers to dispense potable water analyzed for Na, K, Ca, Cl and E. coli.

Before starting the trials, pens were disinfected with non-toxic substances. Animals were subjected to veterinary examination health conditions and then placed in the pens, each containing three males or three females. The experimental plan comprised testing each feeding diet (Table 3) over 36 animals, corresponding to twelve replicates for each diet. Each treatment was equivalent for animal weight and sex. The diets did not contain zootechnical additives according to EU Reg.1831/03 (European Commission, 2017). They were iso-nutritive in each feeding period, according to the feed requirements as a function of age and weight (Fanimo et al., 2003). Mangimificio Rossana, Revere (Mantova), Italy prepared the two basal diets for the two different feeding periods. Cerzoo Piacenza, Italy, prepared the treatment diets by adding the required SBO amount to the basal diet and homogenizing through a horizontal mixer. Before the addition of SBO, examination of the basal diets guaranteed no cross contamination. Except for the added SBO, no other products were added to the basal diets. During the feeding periods, the trials were monitored twice a day, in the morning and in the afternoon, by examining the animals’ health conditions, and checking the availability of feed and water, and the correct functioning of the temperature and humidity control system. Animal growth was monitored by measuring the animal body weight at day zero, 14 and 42, and the feed consumption at day 14 and 42. Diets’ analyses were carried out on 250 g samples. Diets were analysed according to ISO methods, for the content of water (ISO, 2016a), ashes (ISO, 2013a), starch (ISO, 2013b), crude protein (ISO, 2014), ether extracts (ISO, 2016b), crude fibre (ISO, 2017), digestible (Morgan et al., 1987) and net (Noblet et al., 1994) energy. Values for these analytes, and for NDF, ADF, ADL and amino acids, were also calculated using Brill formulation feed management systems version 2.03, 2011, Brilliant Alternatives Inc., USA.

2.3. Statistical Analyses
Data were analysed by Anova using SAS, 2002-2008, version9.2 software. Averages over replicates were compared by Student t-test. The body weight data were analysed also for outliers. None was evident on day 14 and 42.

3. Results

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3.1. Chemical Composition Of SBO And Sourcing Compost
The SBO used in the pigs’ husbandry study is the same as the one used in the pigs in vitro study (Montoneri et al., 2013) and in the rabbits in vivo study (Biagini et al., 2016). They were obtained by hydrolysis in alkaline water of composted urban private gardening residues and public park trimmings collected in the province of Torino, Italy (see Section 2). The process yields SBO and an insoluble residue (IR). Table 1 and 2 report the collected chemical data for the pristine compost (CP) and the SBO and IR hydrolysates. Determination of the chemical structure of these materials is not as easy as for synthetic molecules. As anticipated in section 1, they contain molecules of different molecular weights and chemical composition. These materials are characterized at best for the content of the analytes reported in Table 1. It is not possible to identify a specific molecular structure. Nevertheless, the analytical data give some important information. The three materials contain organic and mineral matter. Compared to CP and IR, SBO contain more organic matter and less mineral matter, except for Ca, K, Cu, Zn and Pb.

The higher content of K, Ca, Cu, Zn and Pb in SBO stems from the different C types and functional groups distribution in the three materials (Table 1). 13 C NMR spectroscopy measurements yielded the functional groups data in Table 1. This analytical technique allows identifying the reported C types, but not obtaining their breakdown into more specific components. For example, the carboxyl (COX), phenoxy (PhOY), methoxy and amine (OMe + NR) groups cannot be broken down into free carboxylic acid, amide, ester, phenol, phenyl ethers, methoxy, and amine groups. Nevertheless, Table 1 shows the following significant differences among the three products. Compared to CP and IR, SBO contain about half alkoxy (RO) groups and twice more carboxyl (COX) groups. This is likely due to the hydrolysis of ester bonds in CP and formation of SBO carboxylate groups in the alkaline reaction medium. Potentiometric titration measurements yielded further details on the chemical nature of SBO. The authors did not carry the same measurements for CP and IR, due to their poor solubility properties.

Potentiometric titration allowed obtaining the breakdown of the SBO COX, PhOY and OMe and NR groups into carboxylates (COO-), amide (CON), phenol (PhOH), phenoxy (PhOR), and amine (NR) groups. The results of these measurements indicated that, in SBO, 92 % of the COX groups are in COOH form and 56 % of PHOY are in PhOH form. They also suggested the higher content of elemental N in SBO, compared to CP and IR, to arise from the presence of NR groups. These functional groups are the likely cause of the stronger capacity of SBO to complex and transport the K, Ca, Cu, Zn and Pb elements into the reaction soluble hydrolysate.

The data in Table 1 is not normally provided in as much detail for materials of biological sources, as those investigated in the present work. Montoneri et al. (2011) developed a specific analytical protocol to investigate the nature of the molecular pool representing these materials. For the specific use of SBO as diet supplement, Table 2 reports the conventional data on the product proximates’ composition. The data in Table 2 is consistent with the data in Table 1. The functional groups given in Table 1 are associated to the proximates listed in Table 2. For example, the OR and OCO groups may be associated to the polysaccharide fraction. The Ph and PhOY groups may be associated to the lignin fraction. The COX and NR groups may be associated to the protein fraction of the material.

3.2. Animal Diets And Live Performance
The animals were fed with the control diet (T1) and with the treatment diets containing SBO at 0.1 % (T2) and 0.2 % (T3) concentration, over two fattening periods, from 0 to 14 days and from 14 to 42 days. For the two periods, Table 3 reports the diets’ formulations and chemical compositions. The T1, T2 and T3 diets in each period have close nitrogen and energy content (see also Table 1S in supplementary material file).

In the present work testing a research product not yet known in the market, a first very important result was the animals’ mortality. This was generally very low. The animals’ death rate was zero for all groups in the first fattening period. In the second fattening period, the dead animals were two for the T1, one for the T2 and one for the T3 groups. Apparently, the mortality rate in each treated T2 and T3 groups was half that of the control T1 group. The dead animals were too few to run a statistical analysis and make a reasonable, reliable assessment of the significance level of this apparent result. It is a fact that, over the control and treated groups, the average death rate was 3.9 %. This value is in the normal range for pigs of the same age and weight, as those fed with the diets in Table 3. The anatomical and pathological examination of the dead animals did not evidence abnormalities possibly caused by the treatment diets.

Table 4 reports the feed average daily intake for the 54 males’ group and 54 females’ group, separately, in each fattening period, and the average intake calculated over the entire duration of the test. In each period, there are no significant differences in the intake of the control (T1) and of the treated diets (T2 and T3). Females tend to intake less feed than males, but this difference is not proven by the statistical analysis of the males’ and females’ pooled intake values.

Table 5 reports the animals’ growth performance. The data are averages of the data collected for male and female groups, in each control and treated group. For males, there are no significant effects of the diets with added SBO compared to the control diet. On the contrary, for females, the average growth of the group fed with the T2 diet in the first fattening 0-14 days period was significantly about 13 % lower than that of the T0 and T1 groups. This is true only for the first fattening period. At day 42, the performances of the females’ T0, T1 and T2 groups are not significant anymore.

Table 6 reports the feed conversion rate, calculated from the average daily feed intake in Table 4 divided by the average daily growth rate in Table 5. The data shows no significant differences between the control and the treated groups. Over the entire duration of the fattening trials (D0-D42), the females treated with the SBO T2 diet show apparently better-feed conversion rate than the female T1 and T3 groups. This results from the apparently higher daily growth of the T2, compared to T1 and T3 groups.

4. Discussion

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The lack of effects on pigs’ growth and mortality in the present work indirectly indicates that, at the tested concentration, SBO are not toxic. The animals exhibited no signs of toxicity, and no pathologies related to the SBO-supplemented diets. Biagini et al. (2016) reported similar conclusions in the study performed on rabbits fed with the same SBO and at the same concentrations in the feed. The greement between the two studies performed on different animal constitutes an encouraging data’s base line for focusing further studies to meet regulatory requirements and to market SBO as multipurpose bio-based chemicals. One important aspect for regulatory compliance is the presence of heavy metals in SBO (Table 1). The present study did not comprise any specific experiment to address this issue. The reason is that the experiments by Biagini et al (2016) and Sortino et al.(2012) performed with the same SBO have excluded potential toxicity by SBO’ s heavy metal accumulation in rabbits meat, soil and plants. On this basis, no risk for human health can reasonably be expected, even at the highest SBO 0.2 % concentration in feed and at 140 kg ha-1 dose applied in cultivation soil.

The data obtained in the present pig study and in the previous rabbit study (Biagini et al., 2016) do not confirm the economic expectations proposed in the vitro pigs’ cecal fermentation study by Montoneri et al. (2013). The reduced protein N mineralization during fermentation reported by these authors allowed hypothesizing enhanced meat production upon using protein diets added with SBO. This SBO effect on animal growth has not been evidenced in both the pigs’ and rabbits’ husbandry studies. However, the reduced protein mineralization is likely to have a positive environmental effect in reducing the ammonia content in manure and emission of GHG from the animal production industry. Ji et al. (2006) have reported that humic substances (HS) mined from different geological sites, and used as dietary supplements for weaning pigs, reduce ammonia emission from manure by 16 – 18 %. Montoneri et al. (2013) have measured similar ammonia reduction in the in-vitro pigs’ cecal fermentation in the presence of 0.05-0.2 % SBO. This shows that SBO are similar to HS not only for chemical nature (Tabasso et al., 2015), but also for properties. It is also a further argument supporting the compliance of SBO with the Italian regulation on fertilizers (Presidente della repubblica, 2010).

Two other studies have reported reduction of ammonia in SBO assisted anaerobic fermentation of municipal biowastes (Francavilla et al., 2016) and cow manure Riggio et al., 2017) carried out in bioreactor dedicated to biogas production. These findings have contributed in starting the construction of the hydrolysis prototype at the Acea Pinerolese waste treatment plant (see Section 1) under the EU cofunded LIFE16 ENV/IT/000179 LIFECAB project within the LIFE2016 project proposals. The project specific aim is to produce SBO in sufficient quantities, use it as additive in the Acea plant biowaste fermentation reactors, and validate in real operational environment the SBO assisted anaerobic fermentation process yielding biogas and digestate with reduced ammonia content.

Under the above circumstances and perspectives, it is worthwhile to build on the established research base line and to carry on further studies dedicated to assessing the SBO compliance with specific countries’ regulatory requirements, and the positive economic and/or environmental effects in the chemical industry, agriculture and animal production activities.

5. Conclusion

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The results of this in vivo pigs’ study and the previous in vivo rabbits’ study confirm that SBO supplemented diets do not affect negatively the growth and health status of animals. The similarity of chemical nature and properties of SBO and natural humic substances support the compliance of SBO with the Italian regulation on fertilizers. These facts encourage further studies dedicated to assessing SBO compliance with specific countries’ regulatory requirements, in order to scale up SBO production to commercial level and market the products for use in the chemical industry, agriculture, and animal husbandry.

Acknowledgments

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This work was carried partly with Regione Piemonte funds within the Biochemenergy project (www.biochemenergy.it) and with Ministero delle Politiche Agricole funds within the Agrienergia project. In addition, the authors acknowledge Polytechnic of Torino for contributing through the funding project Starting Grant RTD (project number: 54_RSG17NIR01).