Removal of Quebracho and Tara tannins in fungal bioreactors: Performance and biofilm stability analysis

Highlights

• Tannins were treated in non-sterile reactors inoculated with A. tubigensis.

• Stable fungal biofilm developed in the reactor treating Quebracho tannins (QT).

• Fungal biofilm turned into a suspended culture during Tara tannin (TT) treatment.

• TT and QT concentrations strongly affected tannin removal due to substrate inhibition.

• Long-term COD removal of TT up to 90% was achieved with a suspended culture.

Abstract

Tannins are polyphenolic compounds produced by plants that are used in the vegetable tanning of leather at industrial scale. Quebracho tannin and Tara tannin are intensively used by the tanning industry and are two of the most recalcitrant compounds that can be found in tannery wastewaters. In this study two reactors fed with Quebracho tannin and Tara tannin, respectively, were inoculated with polyurethane foam cubes colonized with a fungal strain biofilm of Aspergillus tubingensis MUT 990. A stable biofilm was maintained in the reactor fed with Quebracho tannin during 180 days of operation. Instead, biofilm got detached from the foam cubes during the start-up of the reactor fed with Tara tannin and a bacterial-based suspended culture was developed and preserved along the operational period (226 days). Soluble chemical oxygen demand removals up to 53% and 90% and maximum elimination capacities of 9.1 g sCOD m⁻³ h⁻¹ and 37.9 g sCOD m⁻³ h⁻¹ of Quebracho and Tara tannins, respectively, were achieved in the reactors without the addition of co-substrates. Next generation sequencing analysis for bacteria and fungi showed that a fungal consortium was developed in the reactor fed with Quebracho tannin while fungi were outcompeted by bacteria in the reactor fed with Tara tannin. Furthermore, Quebracho and Tara tannins were successfully co-treated in a single reactor where both fungi and bacteria were preserved.

Introduction

Tannins are abundantly applied in leather tanning industry due to their prerogative of precipitating proteins (Lorenz et al., 2014). In fact, tannins blind to the collagen proteins of the animal skin making leather more durable and not putrescible. Tannins are water-soluble polyphenolic compounds produced by plants (Barbehenn and Peter Constabel, 2011) and could be subdivided into hydrolysable (gallotannins and ellagitannins), condensed (or proanthocyanidins) and complex tannins (Khanbabaee and Ree, 2001). Tara tannin (TT) is obtained from the fruit pods of Cæsalpinia spp., which principal components are hydrolysable tannins based on a galloylated quinic acid structure (Sciences et al., 2007). Quebracho tannin (QT) is obtained from the wood of Schinopsis spp. and is composed mainly of condensed tannins that are more difficult to be biodegraded than those found in TT given that hydrolysable tannins are easier to be biodegraded (He et al., 2008). Conventional activated sludge systems (Li et al., 2009) and anaerobic digestion (Mannucci et al., 2010) are not effective to treat tannins due to their low biodegradability and high soluble chemical oxygen demand (sCOD) (Lofrano et al., 2013). In fact, high concentration of tannins can inhibit the biological treatment (Munz et al., 2009). Then, tannins are usually removed by means of chemical processes in tannery wastewater treatment plants (WWTPs).

Although the leather tanning industry is known to be of prime economic importance in many countries, the concern about the environmental impacts related to the production of leather and the release of various recalcitrant pollutants in tannery wastewaters (Lofrano et al., 2013; Romer et al., 2011) has been increasing in the last decades. Current biological wastewater treatments are engineered biological ecosystems based on bacteria, which are ineffective in the removal of several recalcitrant compounds such as tannins (Mannucci et al., 2010). Fungi play a role in the biodegradation of phenols, chlorinated phenolic compounds, chlorinated alkanes and alkenes, polycyclic aromatic hydrocarbons, petroleum hydrocarbons and other emerging contaminants (Harms et al., 2011). Although fungi represent a promising biological resource in environmental biotechnology, they have rarely been applied in wastewater treatment due to a lack of knowledge regarding the optimal process conditions and due to their lack of stability under non-sterile conditions (Espinosa-Ortiz et al., 2016). Most research in wastewater treatment with fungi has been focused on the degradation of pharmaceuticals, dyes and pesticides at lab-scale, with important limitations and, above all, mostly under sterile conditions (Svobodová and Novotný, 2017). In fact, the main operational concerns are related to bacterial contamination and to the robustness of the bioprocess in the long-run since fungi are easily outcompeted by bacteria. Nowadays, operation of a fungal-based bioreactor able to maintain stable fungal growth and performance, under sterile and non-sterile conditions, is still challenging. Furthermore, the fungal degradation of most recalcitrant compounds frequently requires the supply of an external carbon source (co-substrate), since recalcitrant compounds might not be suitable substrates for fungal growth (Palli et al., 2016). Nevertheless, among recalcitrant compounds present in tannery wastewaters, tannins represent a potential carbon source for fungi, despite their antimicrobial properties (Mingshu et al., 2006). Alternative biological treatment processes able to effectively remove this fraction could lead to environmental and economical advantages (Giaccherini et al., 2017). Moreover, Aspergillus spp. and Penicillium spp. have been isolated in tannery wastewaters and reported as microorganisms capable to biodegrade tannins (Murugan et al., 2007). In particular, Aspergillus (section Nigri) can grow on tannic acid (TA) as carbon source and was found in tannery wastewaters with high QT concentration (León-Galván et al., 2010). Based on this findings, it can be hypothesised that fungi would be able to grow in the recalcitrant fraction of wastewater containing a high concentration of tannins, such as tannery wastewater. Most of the research reported in the literature about the biodegradation of natural tannins and polyphenolic compounds by fungi has been performed in petri dishes or flasks using tea by-products (Ni et al., 2015), tannin extracts (Belmares et al., 2009) or on TA (Van Diepeningen et al., 2004). To the authors' knowledge, no previous experiments have been reported with continuously fed bioreactors (under sterile or non-sterile conditions) for the removal of QT, TT or other natural tannins with fungi.

The aims of the present work were i) to demonstrate the proper performance ii) to evaluate the stability and iii) to assess the bacterial and fungal diversity of a novel fungal bioreactor configuration operated under non-sterile conditions to reach an effective removal of Tara and Quebracho tannins. TT was selected as a representative of hydrolysable tannins and QT as a representative of condensed tannins, among the natural tannins applied as tanning agents. The evolution of the system was evaluated by means of combined physical-chemical analyses and molecular techniques for bacteria and fungi. Almost no works in literature have analysed both communities and their relationship during the treatment of tannins in bioreactors.