## 1 Introduction  
The biofilm mode of living, i.e., bacterial cells encapsulated by a self-produced matrix of extracellular polymeric substances (EPS), has been proven to result in (bacterial) cells becoming highly resistant towards traditional inactivation methods such as the use of antimicrobial agents [1; 2; 3; 4; 5; 6]. Especially at the mature biofilm stage, i.e., a three-dimensional structure of microcolonies interconnected via water channels and protected by a matrix of different polymers, biofilm-associated cells are maximally protected against several stress factors [2; 3; 6]. For this reason, biofilm inactivation studies recently shifted towards novel (non-thermal) inactivation methods such as cold atmospheric plasma (CAP). Plasma can be defined as the fourth state of matter and can be created by the addition of energy to a gas. For the generation of CAP, a specific type of plasma, this energy can be added by means of an electrical discharge at atmospheric pressure and at room temperature. As a result, the gas becomes (partially) ionized and contains a variety of reactive species such as ions, photons, free electrons, radicals, and excited (neutral) species [7; 8; 9; 10; 11]. Although many studies have proven the ability of CAP to significantly reduce the number of viable biofilm-associated cells [12; 13; 14; 15; 16; 17], further optimization is still required to guarantee the complete inactivation of all kinds of (mature) biofilms. Even though the inactivation mechanism of CAP is not yet fully known, especially for biofilms, previous studies indicated that four main factors contributed to CAP's killing potential. These factors are (i) UV radiation, (ii) charged particles, (iii) reactive oxygen species (ROS), and (iv) reactive nitrogen species (RNS) [18; 19]. These species can interact with the cells and result in (i) a damaged (outer) cell membrane, (ii) an altered structure and/or altered functional properties of the intracellular macromolecules, and (iii) a damaged DNA [8; 10; 20]. The type of generated CAP species and their corresponding concentrations proved to be highly influenced by many (processing) conditions, which in turn affects the ability of CAP to cause damage to the cells. Examples of these influencing (processing) conditions are (i) the Gram type of the (biofilm-associated) cells, (ii) the gas flow composition, and (iii) the electrode configuration [21; 22; 23]. A more profound investigation of the influence of these conditions on the generation of certain CAP species, their cell targets, and their effect on the biofilm matrix will therefore enable a further optimization of the CAP technology for biofilm inactivation. Especially the latter is of great importance since the biofilm matrix can (i) prevent some of the CAP species reaching the biofilm-associated cells by providing a physical barrier and/or (ii) interact with some of the CAP species to possibly form new (and less reactive) substances [20; 23; 24]. Moreover, the determination of the specific cell targets can aid to design a highly efficient disinfection schedule by combining CAP treatment with another (mild) inactivation method with a complementary or additional inactivation strategy. Within this study, a next step is taken towards unravelling the CAP inactivation mechanism for mature biofilms while assessing the influence of four (processing) conditions on this inactivation mechanism. More specifically, it will be investigated which specific (types of) CAP species are generated and how these species interact with the biofilm-associated cells and the biofilm matrix. Three of these conditions involve specific plasma characteristics, i.e., (i) the electrode configuration, (ii) the oxygen level of the gas flow, and (iii) the power of the discharge. The influence of these characteristics on the inactivation efficacy of CAP (in terms of log-reductions) has been examined before in the study of [PERSON] et al. [17], which will aid to comment on the lethal effect of the generated CAP species.