Ecological diversity, evolution and metabolism of microbial communities in the wet fermentation of Australian coffee beans

Highlights

• We studied the microbial diversity during wet fermentation of Australian coffee beans

• Hanseniaspora uvarum and Pichia kudriavzevii were the dominant yeasts

• Leuconostoc mesenteroides and Lactococcus lactis were the dominant lactic acid bacteria

• The microorganisms grew significantly during fermentation

• They consumed sugars in the bean and produced metabolites linked with coffee quality

Abstract

The microbial ecology in the fermentation of Australian coffee beans was investigated in this study. Pulped coffee beans were kept underwater for 36 h before air dried. Samples were collected periodically, and the microbial communities were analyzed by culture-dependent and independent methods. Changes in sugars, organic acids and microbial metabolites in the mucilage and endosperm of the coffee beans during fermentation were monitored by HPLC. Culture-dependent methods identified 6 yeast and 17 bacterial species, while the culture-independent methods, multiple-step total direct DNA extraction and high throughput sequencing, identified 212 fungal and 40 bacterial species. Most of the microbial species in the community have been reported for wet fermentation of coffee beans in other parts of the world, but the yeast Pichia kudriavzevii was isolated for the first time in wet coffee bean fermentation. The bacterial community was dominated by aerobic mesophilic bacteria (AMB) with Citrobacter being the predominant genus. Hanseniaspora uvarum and Pichia kudriavzevii were the predominant yeasts while Leuconostoc mesenteroides and Lactococcus lactis were the predominant LAB. The yeasts and bacteria grew significantly during fermentation, utilizing sugars in the mucilage and produced mannitol, glycerol, and lactic acid, leading to a significant decrease in pH. The results of this study provided a preliminary understanding of the microbial ecology of wet coffee fermentation under Australian conditions. Further studies are needed to explore the impact of microbial growth and metabolism on coffee quality, especially flavour.

Introduction

Coffee is one of the most popular and widely consumed beverages worldwide due to its rich and complex flavour and other sensory characteristics (Chin et al., 2015). Postharvest processing of coffee cherry is believed to have a significant impact on the flavour and other quality attributes of coffee, although the exact effects are not well understood. The main objective of primary coffee processing, which can be done either by the dry or wet method, is to remove the flesh of the coffee cherries and preserve the beans by reducing the moisture content of the beans to a stable level of about 12% (Borém et al., 2008; Sfredo et al., 2005). In the dry process, whole cherries are dried for up to 30 days to reach the moisture level and then the beans are mechanically separated from the surrounding layers (Silva et al., 2000; Silva et al., 2008). In the wet process, the pulp is removed by machine, but part of the sticky mucilage remains attached to the bean parchment. This sticky mucilage is removed either by wet fermentation or mechanically by a de-mucilager (Brando, 1999; Silva, 2014).

In the wet process, the pulped beans are kept underwater for 6–72 h before being dried (Knopp et al., 2006; Silva, 2014). A wide range of microorganisms have been isolated from wet coffee fermentation in different regions, including yeasts, lactic acid bacteria (LAB), acetic acid bacteria (AAB), Bacillus spp., Enterobacteriaceae and, occasionally, filamentous fungi (Avallone et al., 2001; Pereira et al., 2015; Pereira et al., 2016). Many species of microorganisms are common to fermentation in different regions, but region-specific species have also been found. For example, in Brazil, a rich diversity of yeasts was found in both wet and dry coffee fermentation throughout the process, where Debaryomyces hansenii, Pichia ofunaensis, P. anomala, and P. fermentans were the predominate species (Pereira et al., 2014; Silva et al., 2000; Silva et al., 2008; Vilela et al., 2010). In India, Saccharomyces marxianus, S. cerevisiae, and Schizosaccharomyces were the most reported isolates (Agate and Bhat, 1966; Velmourougane, 2013). Cryptococcus and Kloeckera species were detected during wet coffee fermentation in Mexico (Avallone et al., 2001), while Hanseniaspora uvarum and Candida species were reported in East Africa (Masoud et al., 2004). In West Africa (Cameroon), Hanseniaspora uvarum, Cladosporium sphaerospermum, Candida quercitrusa, Pichia, Aspergillus, Penicillium and Saccharomycetes were detected using denaturing gradient gel electrophoresis (DGGE) technique of the total direct DNA extracted from dry and wet-processed coffee (Hamdouche et al., 2016).

Similarly, lactic acid bacteria also displayed high diversity for wet coffee fermentation in different regions. Lactobacillus plantarum and Lactobacillus sp. were the predominant isolates detected in both Brazil and India (Silva et al., 2000; Vilela et al., 2010; Velmourougane, 2013). In Mexico, Ethiopia, Taiwan, and Hawaii, Leuconostoc species were the most reported isolates (Leong et al., 2014; Schillinger et al., 2008). Weissella, Leuconostoc mesenteroides, Lactococcus lactis subsp. lactis and Lactobacillus fermentum were reported during both dry and wet fermentation processes in west Africa (Hamdouche et al., 2016). Many bacterial genera other than LAB have also been detected in coffee fermentation such as Klebsiella, Acinetobacter, Bacillus, Escherichia, Pseudomonas and Serratia (Agate and Bhat, 1966; Avallone et al., 2001; Frank et al., 1965; Silva et al., 2000). Therefore, information on the microbial ecology of coffee bean fermentation is crucial not only for understanding the impact of microbial growth and metabolism on coffee quality in general, but also the different characteristics of coffee produced in different regions.

The world market of coffee production is enormous with an estimated 170 million bags of coffee produced in 2018. Brazil is considered the leading producer of coffee beans with 58 million bags of coffee, followed by Vietnam and Columbia (International Coffee Organization, 2019). Australia has a small but growing coffee plantation and processing industry. Coffee beans are grown in tropical areas of Queensland, subtropical areas in southeast Queensland and northeast New South Wales. Coffea arabica is the species mostly grown in Australia, where Catuai Rojo and K7 are the predominant varieties used (Agrifutures Australia, 2017). Several factors potentially facilitate the continuous improvement of the coffee industry in Australia; for instance, the country is free from two serious coffee diseases, namely coffee berry disease and coffee rust that can cause severe crop losses. In addition, the coffee beans are characterized by low caffeine concentrations (10–15% less) and being grown in a cold environment with a long ripening time that produces high-quality beans (Australian Subtropical Coffee Association, 2020). Harvesting is conducted between June to October and performed mostly with a machine. The wet method is considered the most common way to process coffee cherries in Australia and performed in the same way as discussed above. Currently, 50% of the coffee produced in Australia is consumed locally, and the remaining for exporting internationally (Agrifutures Australia, 2017). Virtually no information is available on the microbial ecology of Australian coffee fermentation as no such studies have been reported. The objective of this study is to investigate the microbial ecology and metabolism of wet coffee fermentation under Australian conditions (harvested mechanically, fermented for 36 h, dried mechanically to reach 12% moisture content, the average daily temperature was 20 °C).