Anaerobic digestion (AD) is a sustainable technology for converting organic waste into biogas. The solid by-product of AD, the digestate, is generally used as fertiliser or as a soil amendment in current crop management. A large variety of micro-organisms are involved in organic matter conversion into biogas. Among these bacteria, clostridia, which are endospore-forming bacteria, are naturally present and are mainly brought by the digester's feeding (such as slurry manure or sludge). Therefore, they contaminate the biogas plant and, of course, the digestate. Identified pathogenic representatives of this cluster include Clostridium botulinum, C. perfringens and C. tetani [[1], [2], [3]]. Bacterial spores are dormant forms of vegetative cells that can develop and cause intoxication once the environmental conditions are suitable for their development. The presence of pathogens in organic waste, especially spore-forming bacteria, is of growing concern for public health since the relationship between the use of digestate as a soil amendment and human disease remains unclear [4]. Because of their high resistance to conventional hygienisation processes, more innovative sanitation approaches are needed [5]. Different hygienisation treatments have been described, but there is a lack of data concerning the mechanisms involved. Thus, more thorough insight is needed to understand the mechanisms involved (such as membrane cell permeation and inhibition of enzymatic activities) during treatment to inactivate both the vegetative cells and spores forming Clostridium bacteria.

As described by several authors, through sporulation, spore-forming bacteria can have a complex structure, including spore core, cortex region, and coat layers, to protect themselves from adverse conditions [[6], [7], [8]]. The spore's coat is composed of a thick proteinaceous multi-layered structure made up of highly cross-linked polypeptides that confer resistance to some chemical and lytic enzymes but play little or no role in heat resistance [8]. The spore core is the analogue of the protoplast of a growing cell and includes DNA, ribosomes, and tRNA. It also contains large amounts of pyridine-2,6- dicarboxylic acid (dipicolinic acid [DPA]) chelated in a 1:1 ratio with Ca2 (CaDPA) that contribute to the extreme resistance of the spores [9]. Finally, the cortex of the spore is comprised of peptidoglycan and occupies as much as half of the spore volume [6,[8], [9], [10]]. The cortex of the spore is essential and is involved in the heat resistance of the spore.

Several technologies and treatments have been developed and applied for many years to control spoilage and pathogenic micro-organisms in different products. However, the removal of resistant pathogens and bacterial endospores in the specific field of hygienisation of biowaste is of growing interest, mainly due to the use of biowaste as fertilisers. The total removal of pathogenic bacteria in biowaste remains difficult, regardless of the process used, probably because of the complexity of the material structure. Thus, explaining why the impact of a specific treatment (as physical or chemical treatment) is still poorly understood. To date, thermal processing is still commonly used to inactivate spores in food matrices. However, even if f:rom an energetic and economical point of view, thermal pre-hygienization of waste is generally the most cost-effective treatment [4], alternative methods have to be developed to improve the efficiency of spore removal. According to Ross et al. [11], combining two or more nonthermal processes (such as ultrasonication, high pressure, pulsed electric fields and acidification) can also enhance microbial inactivation and allows the use of lower individual treatment intensities. Volatile fatty acids (VFAs) and their various salts are frequently used as food additives and preservatives but can also have interesting applications in biowaste hygienisation [[12], [13], [14]].

VFAs are known to affect vegetative cell activity through two main mechanisms: cytoplasmic acidification with subsequent uncoupling of energy production and regulation, and accumulation of the dissociated acid anion to toxic levels [11,15]. To our knowledge, little information is available on the bactericidal effects of VFAs treatment against C. perfringens. One explanation could be that the complex structure of spores has limited their detection and characterization methods compared to vegetative cells. In this context, flow cytometry (FCM) could be considered a suitable method to characterise micro-organisms at the single cell level. It is a fast method of obtaining multi-parametric data from thousands of individual cells within a sample [16]. Fluorescent dyes can provide much better information about cell properties, such as viability and the physiological state of cells, compared to other methods. Recently, Ganguly et al. [17] developed an efficient method using a combination of FCM and fluorescence-assisted cell sorting (FACS) to quantify the culture heterogeneity of P. thermosuccinogenes.

Most of the time, the determination of process efficiency on the removal of different bacteria genders is evaluated through conventional microbiological methods such as the enumeration of bacterial populations (before and after acidic or thermal treatment). To our knowledge, little information is available on the mechanisms involved in the inactivation of Clostridium perfringens by a combined application of VFAs and moderate heat treatment at the cell level. Thus, only partial or no information is available concerning the structural changes induced in bacterial spores after treatment. In this study, efforts were made to provide more accurate information and quantification of the impact of thermal, acidic and combined VFA-thermal treatments not only to inactivate vegetative cells but also to limit the sporulation ability of Clostridium perfringens.