# Dark Fermentation of Fruit Juice Effluents: Effects of Substrate Concentration
## 1 Introduction  
The fruit juice industry involves significant water consumption during production, leading to the generation of large volumes of wastewater [1, 2]. This wastewater is typically discharged into municipal sewage systems and treated at wastewater treatment plants (WWTPs) alongside domestic wastewater. In general, the wastewater produced by the fruit juice industry is characterized by its exceptionally high organic load, including a large proportion of easily biodegradable compounds, low pHs, nutrient imbalances, and seasonal fluctuations in both effluent volume and organic load [3]. These variations are largely due to the seasonal nature of fruit production and processing [4, 5]. Given these variable characteristics, it is essential that wastewater from the fruit juice sector undergo pre-treatment to meet quality standards before being discharged into municipal sewage systems, thereby reducing potential environmental impacts. In recent decades, anaerobic digestion has emerged as an effective method for treating various types of industrial wastewater with a high organic content [3, 4, 6, 7, 8, 9]. The benefits of anaerobic digestion include low sludge production, high conversion efficiency, reduced energy consumption, and the production of valuable byproducts like biogas [3]. A keyphase in anaerobic digestion is acidogenic fermentation, where carbohydrates are converted into volatile fatty acids (VFAs), alcohols, carbon dioxide, and hydrogen [10]. This process not only aids in treating organic waste but also offers the potential for hydrogen production, a clean and renewable energy source. The feasibility of hydrogen production is especially important in today's economy, which is heavily dependent on fossil fuels and faces challenges such as depleting reserves, rising costs, greenhouse gas emissions, and widespread air pollution. Thus, the search for alternative, non-polluting, and sustainable energy sources has become crucial. Hydrogen offers numerous advantages as an alternative to conventional fossil fuels. These include the absence of harmful emissions--since hydrogen combustion produces only water--higher combustion efficiency, and approximately 50% greater efficiency in internal combustion engines compared to gasoline [11]. Additionally, hydrogen has 2.75 times the energy content of any hydrocarbon, and it can be transported more efficiently through existing natural gas pipelines than electricity via power lines. Hydrogen can be produced through various methods, including thermochemical, electrochemical, and biological processes. However, thermochemical processes often require hydrocarbon feedstocks, mainly derived from fossil fuels, while electrochemical processes demand a continuous electricity supply. In contrast, microbial fermentation presents an attractive alternative as it does not depend on external energy inputs, operates under ambient conditions, and when waste substrates are used, it achieves both renewable energy production and efficient waste treatment [12; 13]. Among the biological methods for hydrogen production, acidogenic fermentation is particularly advantageous due to its high hydrogen yield, simple operational control, low production costs, and independence from light [14]. Dark fermentation is usually compared with two-stage anaerobic digestion. On the one hand, dark fermentation converts more efficiently carbohydrate-rich organic substrates into hydrogen and presents the advantage of lower operational costs [15; 16]. On the other hand, two-stage anaerobic digestion involves both hydrogen and methane production phases. However, the hydrogen yield alone is typically lower than that of dark fermentation [15]. In the literature, it has been described that numerous operational factors--such as pH, temperature, reactor design, inoculum type, nutrient concentrations, the presence of metal ions, substrate type, and substrate concentration--can significantly influence the efficiency of hydrogen production through acidogenic fermentation [17; 18; 19].