1. Introduction
Biochar is a carbon-rich, porous material derived from the thermal decomposition of organic biomass at high temperatures (350 to 1000 °C) in low-oxygen conditions [
1]. Traditionally, biochar has attracted interest for its applications in agriculture and environmental management, particularly in soil management and carbon sequestration. Biochar has also gained interest in applications such as environmental remediation, construction materials, horticulture, and climate change mitigation [
2,
3].
Since 2011, in animal nutrition, biochar has been included in the Catalogue of Feed Materials as a vegetable carbon, a product obtained by carbonizing organic plant material (Reg. EU 2022/1104). It has also attracted attention due to its bioactivity when administered in feed.
Biochar’s unique properties, such as high porosity and extensive surface area, enable it to adsorb harmful substances, including mycotoxins, plant toxins, pathogens, pesticides, and metabolic toxins [
4]. Adsorption can occur physically, where mycotoxins are trapped in the biochar’s pores through van der Waals forces and hydrogen bonding, or chemically, where functional groups (e.g., carboxyl, hydroxyl, and phenolic) interact with mycotoxins. These qualities have led to biochar being recognized globally for its benefits in animal nutrition and environmental management, with ongoing research exploring its potential applications. Several studies have shown that incorporating biochar into animal feed at 1–3% of dry matter can enhance various aspects of animal health and performance, including weight gain, feed efficiency, and product quality [
5,
6,
7]. Dietary biochar has shown benefits across multiple species, including ruminants, swine, poultry, and fish [
8]. In growing pigs, 2% biochar in the diet improved nutrient digestibility, particularly in terms of crude fiber and crude protein, although no significant differences in performance were observed [
9].
Biochar has been shown to enhance growth performance in poultry, improve blood characteristics, reduce fecal microbial shedding, and enhance digestive health and feed efficiency [
10]. Additionally, biochar appears to reduce greenhouse gas emissions, particularly methane from ruminants [
11].
Although studies have demonstrated biochar’s efficacy as a functional ingredient, the fact that is heterogeneous affects how it is applied and its overall effectiveness.
Since biochar’s properties play a key role in its effects on animals and in vitro fermentation, characterizing biochars is necessary for comparisons across studies. Biochar enhances growth rates and/or feed conversion efficiency in pigs, poultry, and ruminants. Improved feed efficiency in pigs may be associated with the increased villus height induced by biochar. However, not all studies on biochar have reported positive effects on nutrient digestibility or performance outcomes.
The physical characteristics of biochar depend on factors related to the production process and feedstock type [
12]. These factors influence biochar properties, including porosity, surface area, particle size, and density [
13]. Higher temperatures tend to produce biochar with a greater porosity and surface area. Temperatures of 350 to 500 °C yield biochar with a higher volatile matter content and lower surface area, while temperatures above 500 °C produce biochar with reduced volatile matter, increased fixed carbon, and an enhanced surface area. The heating rate and residence time also affect biochar’s internal structure. This variability makes biochar versatile but challenges standardization across fields. Therefore, a preliminary in vitro evaluation of biochar’s characteristics is essential for optimizing its use.
This study focuses on characterizing the functional properties of biochar derived from vine pruning residues to determine its suitability as a livestock feed additive.
4. Discussion
Biochar is a highly variable carbon-rich product derived from the thermal decomposition of organic materials in an oxygen-limited environment. Its popularity has increased due to its ability to reduce the environmental impact of biomass and contribute to carbon sequestration. This process helps to store carbon in the soil for long periods, thereby reducing greenhouse gas emissions.
Carbon sequestration is particularly relevant to the carbon credit market, which is expected to grow as efforts to achieve net-zero emissions intensify. The physicochemical properties of biochar are critical for determining its bioactivity and effectiveness in vivo, especially when used as animal feed. These properties, along with the quality, yield, and potential toxicity of biochar, are influenced by various factors, including the type of feedstock, the production technology, and the specific process conditions. Consequently, biochar products are highly heterogeneous, affecting their performance and bioactivity.
Given the wide-ranging applications of biochar, it is essential to understand its specific functional properties, especially when used as a feed ingredient. Our study focused on biochar produced from vine prunings, offering an opportunity to convert this agricultural by-product into a functional and sustainable feed ingredient. This approach not only reduces agro-residual waste but also contributes to resource reusability, thus promoting sustainability in agricultural practices. Vineyards can produce approximately 1 to 10 tons of pruning waste per hectare per year, with winter pruning wood typically destroyed by burning or crushing on the ground [
21].
The chemical characterization of vine biochar showed a dry matter (DM) content of 95.08 ± 0.06%, indicating a highly concentrated product with minimal moisture due to the high temperatures used during pyrolysis [
22]. This low moisture content makes it suitable for animal feeds, as it appears not to significantly alter the moisture content of the feed, which must remain below 12% [
23]. The low water content also reduces the risk of bacterial contamination and fungal growth, making the biochar easier to store safely.
During pyrolysis, organic materials are heated in the absence of oxygen, thereby decomposing organic volatile compounds and water. Higher temperatures drive off more volatiles, leaving a product rich in carbon and other stable compounds [
24].
The absence of detectable ether extract and the low protein content are consistent with high-temperature pyrolysis, which typically degrades these components [
25]. The chemical composition of the VB, particularly its ash content (15.00 ± 0.23%), reflects the presence of significant amounts of minerals. This likely results from the high pyrolysis temperatures, which degrade organic matter, concentrate minerals, and are influenced by the intrinsic characteristics of the original biomass [
26].
Minerals are essential in animal nutrition and are classified as essential, non-essential, or contaminants, such as heavy metals. An in-depth evaluation of the mineral profile is crucial to ensure that the product meets nutritional requirements, while minimizing potential contaminants (Dir. 2002/32/EC).
In this study, an elemental analysis using ICP-MS revealed a varied profile of both macro- and microminerals, including essential nutrients. Importantly, the concentrations of heavy metals, such as arsenic (As), lead (Pb), and cadmium (Cd), were below the threshold levels for biochar as a feed ingredient. This ensures that VB biochar is safe in animal diets without posing risks of toxicity from heavy metals.
The vine biochar analyzed in this study contained high levels of calcium (Ca), potassium (K), magnesium (Mg), and phosphorus (P), as these minerals are less likely to volatilize at lower pyrolysis temperatures (350–500 °C) and are abundant in the original biomass. In animal nutrition, these minerals are classified as macroelements, with the required levels significantly higher than those of the trace elements. Therefore, it is important to define the macroelement content before introducing the biochar into animal feed to ensure compliance with the mineral limits imposed by regulations.
In biochar derived from vine pruning residues, particular attention should be given to copper (Cu). Our results indicate considerable levels of copper, though these are still below the regulatory limits for commercial raw materials. This may be related to its extensive use in agriculture, particularly in vineyards, where copper-based fungicides are applied, leaving copper residues on the biomass after pruning [
27]. Although copper is an essential nutrient in animal physiology, playing a crucial role in enzyme function, immune response, and iron metabolism, its use has recently raised concerns [
28]. Copper can contribute to eutrophication and it is involved in the co-selection of antibiotic-resistant bacteria [
29]. For these reasons, the European Union sets maximum copper levels in animal feed for certain categories to ensure safety and prevent environmental contamination [
30]. Therefore, if VB biochar is used in the preparation of complete feed for animals, it should be considered a potential copper source to ensure compliance with the established European maximum copper level.
Vine pruning biomass is rich in polyphenols, which are organic compounds characterized by multiple phenol units (i.e., aromatic rings with hydroxyl groups attached). The metabolomic profile in this study revealed that the VB extract is rich in small polyphenols containing 4–8 carbon atoms, likely due to the pyrolysis process. During pyrolysis, polyphenols undergo thermal degradation and transformation. Thermal energy breaks the chemical bonds within polyphenolic structures, forming smaller molecules. Functional groups, such as hydroxyl (-OH), carboxyl (-COOH), and methoxy (-OCH
3), attached to polyphenolic rings, are often lost or transformed during pyrolysis [
30].
Among the polyphenols, the VB extract contains hydroxybenzoic acid, gallic acid, cresotinic acid, and hydroxytyrosol, all of which have antioxidant and antimicrobial properties. These compounds scavenge free radicals and interfere with microbial enzymes and proteins, inhibiting their function and leading to microbial cell death [
31]. The VB extract also contains methylcoumarins, which are coumarin molecules with methyl groups (-CH
3) at various positions on the coumarin ring structure. Coumarin derivatives, including methylcoumarins, have been studied for their antioxidant and antimicrobial properties [
32].
Metabolomic analyses confirm the antioxidant activity of the VB extract. The VB extract has an AOX activity that is perfectly in line with the data on the AOX activity of vine pruning waste of 100–500 μmol TE/gr DW [
33]. This property makes biochar from vine waste suitable for various applications, including as a natural source of antioxidants and in animal feed. Antioxidant ingredients could help in reducing oxidative damage in animals, contributing to overall health and performance. They are among the most studied alternatives to antibiotics in animal nutrition, and are integral to agroecology and sustainable development in terms of the One Health approach [
34].
VB biochar inhibits the growth of Escherichia coli strains characterized by fimbrial virulence factors (specifically F4 and F18) and verocytotoxins. Escherichia coli strains were chosen as model organisms because they are primarily responsible for severe enteric and systemic diseases in pig farming, particularly in weaned pigs. The VB extract used in this study significantly inhibited both E. coli strains, likely due to the presence of functional compounds with known antimicrobial properties identified in the extracts. Additionally, the VB extract showed inhibitory effects against E. coli F4+ and F18+ strains, particularly at higher concentrations. The earlier inhibition observed in E. coli F4+ (starting at 1 h) compared with E. coli F18+ (starting at 2 h) suggests that the extract is more effective against certain E. coli pathotypes. The decline in inhibition over time could indicate adaptive bacterial responses or the depletion of active compounds.
Nevertheless, the extract’s ability to inhibit bacterial growth supports its potential role in improving feed safety by reducing pathogenic bacteria. Recent studies, such as Wang et al. [
35], have shown that phenolic compounds like 4-hydroxybenzoic acid can affect bacterial growth and morphology by inhibiting those enzymes involved in quorum sensing (QS) signal synthesis or degradation. QS is a communication mechanism that bacteria use to coordinate behavior, including virulence factor production.
Targeting QS offers an effective strategy for inhibiting virulence without killing the bacteria, thus reducing the risk of resistance development. To better understand the antibacterial mechanism of VB biochar, we investigated the expression of the genes involved in QS signaling and E. coli pathogenicity, focusing on its potential to reduce virulence. Biofilm formation in E. coli is regulated by a complex network of genes and pathways that coordinate bacterial activity based on cell density. In E. coli, the FliA gene encodes sigma factors (σ70 or σ28) that regulate flagellar biosynthesis, including flagellin (FliC). Additionally, FliA controls the transcription of most motility-related genes, including MotA, which is essential for the bacterial flagellar motor. In contrast, the FtsE gene plays a role in cell division and septation, encoding a membrane protein that, together with FtsX, forms the FtsEX complex that regulates the early stages of cell division.
We found that the expression of QS-related genes (FliA, MotA, and FtsE) was downregulated in the presence of the VB extract. This indicates that bioactive components in the biochar may disrupt bacterial communication and biofilm formation. This effect could potentially reduce the virulence and persistence of pathogenic bacteria in the gut, supporting the use of biochar as a functional feed additive.
Previous studies have reported similar downregulation patterns in QS genes in response to various treatments. For example, certain plant extracts or antimicrobial agents downregulate QS genes, interfering with bacterial motility and biofilm formation [
36]. The downregulation of FliA and MotA, which are involved in bacterial motility and chemotaxis, leads to the reduced expression of flagellar components and impaired flagellar motor function, ultimately decreasing motility. Since flagellar motility in pathogenic bacteria is often linked to virulence, the downregulation of FliA and MotA could weaken the bacteria’s ability to invade host tissues or evade immune responses.
Reduced motility also impairs biofilm formation, which is essential for bacterial persistence and infection in various environments. Furthermore, the downregulation of the FtsE gene, which is involved in the divisome complex responsible for cell division, impacts bacterial growth. Reduced FtsE expression can slow growth rates due to inefficient cell division. Phenolic compounds in VB biochar may influence the expression of genes such as FtsE by affecting transcriptional regulators or altering bacterial stress responses. Many phenolic compounds are known for their antibacterial activity, which may involve mechanisms that disrupt cell division.
In this study, we also explored how water-soluble compounds in VB biochar influence the growth of
L. plantarum and
L. reuteri, strains isolated from weaned piglets and previously characterized in vitro and in vivo [
20,
37]. We found no inhibitory effects on beneficial bacteria, such as
L. plantarum and
L. reuteri, suggesting that VB biochar may selectively inhibit pathogenic bacteria while supporting the growth of beneficial microbes. We also observed how
L. plantarum growth was stimulated in the presence of the VB extract, which highlights the prebiotic effect of biochar, promoting a healthy balance in the gut microbiota.
Our findings indicate that VB biochar could exert selective prebiotic activity, as only
L. plantarum showed enhanced growth among the two strains tested. This result aligns with previous studies showing that biochar can either stimulate or inhibit bacterial growth, even within the same species. For instance, Fan Yang et al. [
38] investigated the effects of biochar on various bacterial strains and reported that washed biochar could either increase or decrease colony-forming units (CFUs), emphasizing the role of water-soluble compounds in fresh biochar in modulating bacterial growth. Their results confirmed that the effect is highly strain-dependent. Other studies suggest that biochar can positively influence the gut microbiota and bacterial metabolism. Han et al. [
39] demonstrated that administering rice straw biochar to rats significantly altered cecal bacterial communities by increasing beneficial
Firmicutes and reducing opportunistic pathogens.
However, exactly how biochar affects the growth of different bacterial strains remains unclear, and requires further investigation. Our in vitro study needs to be confirmed by in vivo studies, but translating the effects that we observed to real-world conditions is challenging.
A key factor is to understand the most effective percentage of biochar in the feed in order to optimize the positive effects and minimize any negative effects. High inclusion percentages may negatively impact digestibility due to the material’s porous and absorbent nature. This study also revealed that biochar can selectively modulate microorganism development. This aspect is still poorly understood, and again, further investigation is necessary, both to understand the mechanism of action (strain-dependent effects) and to explore its broader influence, such as its potential to modulate the intestinal microbiota.
