## 6 Factors Affecting the Application of Biofilm Characteristics to Enhance Biofilm Formation  
The type and characteristics of the carrier used affect the attachment of the biofilm. Carriers generally have a negatively charged surface, which makes it difficult for negatively charged bacteria to attach. Additionally, bacteria prefer to attach on rougher, hydrophobic surfaces coated by films. The adhesion of bacteria to the surface can be increased by increasing the interface fluid velocity. The pH has also been shown to affect the surface charge of the bacteria. Higher liquid-phase pH values have been said to cause electronegativity of the bacterial surface as a result of amino acid ozonation. Therefore, pH variations change  
\begin{table}
\begin{tabular}{c c c c c c} \hline \hline \multirow{2}{*}{**Ref**} & **Method of** & **Mature** & **Biofilm** & **Full Carrier** & \multirow{2}{*}{**Biofilm Thickness**} \\  & **Biofilm Enhancement** & **Biofilm** & **Growth Rate** & **Coverage** & \\ \hline
**[PERSON] et al. [9]** & silica-functionalized carriers & day 106 & 1.0 \(\upmu\)m/d & days 32 and 57 & 56.2 \(\pm\) 8.3 \(\upmu\)m on day 32 \\  & & carriers & & & 82.6 \(\pm\) 10.2 \(\upmu\)m on day 57 \\ \hline
**[PERSON] et al. [9]** & dextran-functionalized carriers & - & 0.7 \(\upmu\)m/d & days 32 and 57 & 58.1 \(\pm\) 8.9 \(\upmu\)m on day 32 \\  & & carriers & & & 71.5 \(\pm\) 8.4 \(\upmu\)m on day 57 \\ \hline
**[PERSON] et al. [9]** & Biofilm pre-seeding & - & - & days 32 and 57 & 56.2 \(\pm\) 8.3 \(\upmu\)m on day 32 \\  & & & & & 78.5 \(\pm\) 9.3 \(\upmu\)m on day 57 \\ \hline
**[PERSON] et al. [10]** & AHLs dose & 24 h & - & & 33.56 \(\pm\) 0.29 \(\upmu\)m \\ \hline
**[PERSON] et al. [10]** & Acylase dose & - & - & & 5.71 \(\pm\) 0.11 \(\upmu\)m \\ \hline
**[PERSON] et al. [54]** & Non-modified biocarriers & day 100 & - & day 35 & 182.5 \(\pm\) 44.1 \(\upmu\)m \\ \hline \hline \end{tabular}
\end{table}
Table 3: Comparison of different methods used to enhance biofilm formation.  
the electrical behavior of the bacterial surface, thereby affecting the adhesion of the bacteria to the carriers [59].  
Quorum sensing plays a crucial role in biofilm formation. AHLs are some of the autoinducers produced for quorum sensing [78]. Although these autoinducers are important in biofilm formation, their presence in wastewater induces the synthesis of acylase, which is an AHL-degrading enzyme [72]. The presence of this enzyme affects the quorum sensing, thereby affecting the EPS production and subsequently affecting the biofilm formation. [PERSON] et al. [10] reported that dosing wastewater with acylase reduced the biofilm thickness by 47.95% from 10.97 \(\pm\) 0.34 to 5.71 \(\pm\) 0.11 \(\upmu\)m in municipal wastewater, while a decrease of 63.38% from 13.57 \(\pm\) 0.21 to 4.97 \(\pm\) 0.10 \(\upmu\)m was observed in industrial wastewater. Additionally, c-di-GMP plays a crucial role in the biofilm's formation and its three-dimensional structure. Moreover, c-di-GMP controls the swimming, swarming, and twitching processes and regulates the production of EPS and extracellular DNA, which are essential for the initial attachment of the bacteria to the surface [79]. It has been shown that high levels of EPS and c-di-GMP can coexist [80], although c-di-GMP plays an important role in biofilm formation. High levels of c-di-GMP are linked with reversible attachment and detachment of the biofilm. Increases in c-di-GMP have been associated with activation of the BalA protein, which has been shown to be responsible for biofilm dispersal in _Pseudomonas aeruginosa_. Although c-di-GMP promotes the production of EPS, it can be detrimental to quorum-sensing-dependent biofilm formation [78; 81]. While using the biofilm's properties to enhance biofilm performance seems to be a promising strategy, there is still a lot of work that needs to be done to ensure that these techniques are efficient and will not affect the bacteria that are important in biological wastewater treatment.