Bacterial dynamics during the anaerobic digestion of toxic citrus fruit waste and semi-continues volatile fatty acids production in membrane bioreactors

Highlights

• Bacterial diversity reduced (49158–65239) after anaerobic digestion of citrus waste.

• The key bacterial phylum affiliated to Firmicutes (37.35–84.73%).

• Treatment POLR8 has greatest abundance of acid forming bacterial (84.37%).

• Treatment UOLR4 has greater acid utilizing bacteria (70.09%).

• Organic loading rate 4 g VS/L⋅d is feasible way to produce volatile fatty acids.

Abstract

Citrus wastes (CW) are normally toxic to anaerobic digestion (AD) because of flavors such as D-limonene. In this study, bacterial community was evaluated during volatile fatty acids (VFAs) production from CW inoculated by sludge in a membrane bioreactor (MBR) using semi-continuous AD with different organic loading rates (OLR). Four treatments including untreated CW filled with 4 and 8 g·VS·L⁻¹d⁻¹ OLR (UOLR4 and UOLR8), pretreated D-limonene-free CW filled with 4 and 8 g·VS·L⁻¹d⁻¹ OLR (POLR4 and POLR8). The initial inoculum and the CW mixture (DAY0) was used as control for comparison. There was an obviously higher bacterial diversity in raw material (66848 sequences in DAY0), while decreased after AD and higher in POLR4 and POLR8 (65239 and 63916) than UOLR4 and UOLR8 (49158 and 51936). The key bacterial associated with VFAs production mainly affiliated to Firmicutes (37.35–84.73%), Bacteroidetes (0.48–36.87%), and Actinobacteria (0.35–29.38%), and the key genus composed of Lactobacillus, Prevotella, Bacillus, Bacteroides and Olsenella which contributed in VFA generation by degradable complex organic compounds. Noticeably, methanogen completely suppressed after MBR-AD and UOLR4 has greater acid utilizing bacteria (70.09%).

Introduction

Until 2016, more than 132 million metric tons of citrus fruits (CF) are produced worldwide production to full fill the requirement [1]. However, improper recycling practices of citrus waste (CW) residues (e.g., citrus peel, etc.) and open dumping were led to a numerous environmental problems like soil and water bodies pollution, malodorous and greenhouse gases emission [2], [3], [4]. Present scenario, more than 80% of cultivated CF is used for production of juice, essential oil, jellies, orange blossom honey and etc. However, after processing, about 60 to 70% CW volume converted and become as homogenous organic waste which can be recover various bio-products. In addition, due to presence of higher concentration of carbohydrates in these kinds of organic residues, most of scientist preferably used as a suitable substrate for the bioethanol or biogas production [5], [6], [7].

In the latest decade, anaerobic digestion (AD) process is presented as one of the best environmentally sustainable technology to recycle CW and convert various valuable products such as hydrogen, volatile fatty acids (VFAs) and biogas [8], [9]. In AD process, a group of indigenous or transformed microbial consortium utilize organic wastes such as CW and converting secondary metabolites through serious of biochemical reaction. However, CW is a challenging organic substrate for AD processes and the methanogens, as the peel oil dominating by D-limonene inhibit methanogenic microorganism existence and their activities [4]. However, CP can be converted in an AD process into the intermediate biochemical VFAs, which has potential to be transformed into bio-plastics, bio-butanol, and pharmaceutical products, and then further use for cosmetics or animal’s foods [10], [11], [12], [13].

Although, CW is vital organic materials for AD, but scientific evidence still point to several challenges with this biological process and the need to improve fermentation process and understand the associated mechanism such as organic loading rates (OLR), CW characteristics, operational parameters, microbial dynamics and metabolic pathways [5], [14], [15]. Some studies have reported that bacterial dynamics associated with AD is more important to clarify the overall performance of an AD within distinctive operation condition [3], [16], [17]. In addition, a particular group of bacteria influence the metabolic pathways and further conversion to other metabolites still need to be articulated, while most of the earlier published literatures are mainly based on modification of physical-chemical parameters or reactors [18], [19]. Beside this, in a previous research works, the optimization of OLR to provide adequate concentration of bio-available organic molecules for microbial cell utilization was reported [3], [20], [21]. Subsequently, overloading the AD bioreactors can reduce the pH and prohibited methanogenic bacterial community, which is mainly connected for biogas production via acidogenesis [22], [23]. Thus, a greater OLR could be beneficial to suppressed methanogenic bacteria growth and further lead to inhibit CH₄ generation during AD, but enhanced the VFAs production and its accumulation.

On the other hand, Ferguson et al. [24] reported that OLR play a vital action during microorganism taxonomy variation during the AD process in bioreactors. Hence, some previous researchers reported that OLR can be used as essential tools that not only change the microbial population during the AD process but also significantly control organic matter degradation, biogas production and then VFAs accumulation [3], [25]. However, bacterial dynamics also change the conversion of the end products during the AD process and further susceptibility of indigenous microorganism for produced metabolites [9], [15], [26], [27]. Therefore, it is necessary to identify those microorganisms’ diversity that inhibit synthesis of VFAs and H₂ in production systems employing various organic fruit residues such as citrus wastes. Camargo et al. [28] obtained greater yield of acetic acid with higher abundance of Clostridium and Ruminiclostridium genera during AD of untreated CW in batch reactors with pH adjusted to 7.0 and mesophilic conditions (37 °C). Subsequently, Torquato et al. [29] recorded similarly hydrogen values (13.4 mmol L⁻¹) by batch reactor-based AD of citrus vinasse. Braga et al. [30] reported predominance of Clostridium and Enterococcus when sugarcane bagasse was the main feeding substrate, families Methanosaetaceae methanogenic archaea and Methanoregulaceae were relatively greater abundance noticed in phase II, while higher abundance in stage I was cellulose-producing genera when fed acidified effluent generated in stage I employing.

It is essential to interpret physical, biological and biochemical data to better understand the mechanism of AD, in order to enhance microbial efficiency during optimal operational condition [31], [32]. Within the literature, several aspects of microbial production of VFAs from food waste were investigated such as the total solid content of the feedstock but none of studies reported microbial dynamics changes during the CW anaerobic digestion and its correlation with physicochemical parameters [4], [33]. Therefore, more investigation are needed to conduct to identify the bacterial community succession in AD of CW and semi-continues VFAs production in membrane bioreactors with increasing organic loading rates.

The main objective of this study was to identify the bacteria dynamics with the influence of different OLR concentration during VFAs production through AD of CW in a side-stream MBR. To the best of the author’s knowledge, this is the first time that the influence of the OLR on bacterial taxonomy was evaluated. Hence, the main innovation of this experiment work is to identify the highest bacterial abundance and VFAs yield with optimal OLR dosage semi-continues VFAs production using membrane bioreactors. As consequence, an experimental test was conducted by AD of CW in membrane reactor based semi-continuous VFAs production and comparing the dominant bacterial community with different OLR under remove or non-removed D-limonene conditions as well as their correlation with physicochemical parameters.