Structure of fungal communities in sequencing batch reactors operated at different salinities for the selection of triacylglyceride-producers from a fish-canning lipid-rich waste stream.

Oleaginous fungi natively accumulate large amounts of triacylglycerides (TAG), widely used as precursors for sustainable biodiesel production. However, little attention has been paid to the diversity and roles of fungal mixed microbial cultures (MMCs) in sequencing batch reactors (SBR). In this study, a lipid-rich stream produced in the fish-canning industry was used as a substrate in two laboratory-scale SBRs operated under the feast/famine (F/F) regime to enrich microorganisms with high TAG-storage ability, under two different concentrations of NaCl (SBR-N: 0.5 g/L; SBR-S: 10 g/L). The size of the fungal community in the enriched activated sludge (EAS) was analyzed using 18S rRNA-based qPCR, and the fungal community structure was determined by Illumina sequencing. The different selective pressures (feeding strategy and control of pH) implemented in the enrichment SBRs throughout operation increased the abundance of total fungi. In general, there was an enrichment of genera previously identified as TAG-accumulating fungi (Apiotrichum, Candida, Cutaneotrichosporon, Geotrichum, Haglerozyma, Metarhizium, Mortierella, Saccharomycopsis, and Yarrowia) in both SBRs. However, the observed increase of their relative abundances throughout operation was not significantly linked to a higher TAG accumulation.

Simplified engineering design towards a competitive lipid-rich effluents valorization.

Medium- and long-chain fatty acids and glycerol contained in the oily fraction of many food-industry effluents are excellent candidates to produce biobased high-value triacylglycerides (TAGs) and polyhydroxyalkanoates (PHAs). The typical process configuration for TAGs recovery from lipid-rich streams always includes two steps (culture enrichment plus storage compounds accumulation) whereas, for PHAs production, an additional pretreatment of the substrate for the obtainment of soluble volatile fatty acids (VFAs) is required. To simplify the process, substrate hydrolysis, culture enrichment, and accumulation (TAG and PHA storage) were coupled here in a single sequencing batch reactor (SBR) operated under the double growth limitation strategy (DGL) and fed in pulses with industrial waste fish oil during the whole feast phase. When the SBR was operated in 12 h cycles, it was reached up to 51 wt % biopolymers after only 6 h of feast (TAG:PHA ratio of 50:51; 0.423 CmmolBIOP/CmmolS). Daily storage compound production was observed to be over 25% higher than the reached when enrichment and accumulation stages were carried in separate operational units. Increasing the feast phase length from 6 to 12 h (18 h cycle) negatively affected the DGL strategy performance and hence system storage capacity, which was recovered after also extending the famine phase in the same proportion (24 h cycle). Besides, the carbon influx during the feast phase was identified as a key operational parameter controlling storage compounds production and, together with the C/N ratio, culture selection. The different cycle configurations tested clearly modulated the total fungal abundances without no significant differences in the size of the bacterial populations. Several PHA and TAG producers were found in the mixed culture although the PHA and TAG productions were poorly associated with the increased relative abundances (RAs) of specific operational taxonomic units (OTUs).

Dynamics of PHA-Accumulating Bacterial Communities Fed with Lipid-Rich Liquid Effluents from Fish-Canning Industries.

The biosynthesis of polyhydroxyalkanoates (PHAs) from industrial wastes by mixed microbial cultures (MMCs) enriched in PHA-accumulating bacteria is a promising technology to replace petroleum-based plastics. However, the populations' dynamics in the PHA-accumulating MMCs are not well known. Therefore, the main objective of this study was to address the shifts in the size and structure of the bacterial communities in two lab-scale sequencing batch reactors (SBRs) fed with fish-canning effluents and operated under non-saline (SBR-N, 0.5 g NaCl/L) or saline (SBR-S, 10 g NaCl/L) conditions, by using a combination of quantitative PCR and Illumina sequencing of bacterial 16S rRNA genes. A double growth limitation (DGL) strategy, in which nitrogen availability was limited and uncoupled to carbon addition, strongly modulated the relative abundances of the PHA-accumulating bacteria, leading to an increase in the accumulation of PHAs, independently of the saline conditions (average 9.04 wt% and 11.69 wt%, maximum yields 22.03 wt% and 26.33% SBR-N and SBR-S, respectively). On the other hand, no correlations were found among the PHAs accumulation yields and the absolute abundances of total Bacteria, which decreased through time in the SBR-N and did not present statistical differences in the SBR-S. Acinetobacter, Calothrix, Dyella, Flavobacterium, Novosphingobium, Qipengyuania, and Tsukamurella were key PHA-accumulating genera in both SBRs under the DGL strategy, which was revealed as a successful tool to obtain a PHA-enriched MMC using fish-canning effluents.

Valorization of lipid-rich wastewaters: A theoretical analysis to tackle the competition between polyhydroxyalkanoate and triacylglyceride-storing populations.

The lipid fraction of the effluents generated in several food-processing activities can be transformed into polyhydroxyalkanoates (PHAs) and triacylglycerides (TAGs), through open culture biotechnologies. Although competition between storing and non-storing populations in mixed microbial cultures (MMCs) has been widely studied, the right selective environment allowing for the robust enrichment of a community when different types of accumulators coexist is still not clear. In this research, comprehensive metabolic analyses of PHA and TAG synthesis and degradation, and concomitant respiration of external carbon, were used to understand and explain the changes observed in a laboratory-scale bioreactor fed with the lipid-rich fraction (mainly oleic acid) of a wastewater stream produced in the fish-canning industry. It was concluded that the mode of oxygen, carbon, and nitrogen supply determines the enrichment of the culture in specific populations, and hence the type of intracellular compounds preferentially accumulated. Coupled carbon and nitrogen feeding regime mainly selects for TAG producers whereas uncoupled feeding leads to PHA or TAG production function of the rate of carbon supply under specific aeration rates and feast and famine phases lengths.

A framework based on fundamental biochemical principles to engineer microbial community dynamics.

Microbial communities are complex but there are basic principles we can apply to constrain the assumed stochasticity of their activity. By understanding the trade-offs behind the kinetic parameters that define microbial growth, we can explain how local interspecies dependencies arise and shape the emerging properties of a community. If we integrate these theoretical descriptions with experimental 'omics' data and bioenergetics analysis of specific environmental conditions, predictions on activity, assembly and spatial structure can be obtained reducing the a priori unpredictable complexity of microbial communities. This information can be used to define the appropriate selective pressures to engineer bioprocesses and propose new hypotheses which can drive experimental research to accelerate innovation in biotechnology.

A novel strategy for triacylglycerides and polyhydroxyalkanoates production using waste lipids.

Lipids are one of the main components of the organic matter present in the effluents of the food-processing industry. These waste streams can be biotransformed into valuable triacylglycerides (TAGs) and polyhydroxyalkanoates (PHAs), precursors of biofuels and biomaterials alternative to petroleum-based products. These compounds are yielded by mixed microbial cultures, and considering that both TAG and PHA accumulators may coexist within the community, it seems crucial to define those operational strategies that might control the selection of the dominant metabolic pathways (TAG or PHA accumulation). In this work, residual fish-canning oil was used as a carbon source in a two-stage process (culture selection and intracellular compounds accumulation) in which the substrate was simultaneously hydrolyzed in these two stages without the need for a previous fermentation unit. It was pretended to maximize preferential TAG or PHA storage in the accumulation reactor by the imposition of certain selective pressures in the enrichment one. Uncoupling C and N feedings and limiting nitrogen availability in the medium, allowed to maximize PHA production (82.3 wt% of PHAs, 0.80 CmmolPHA/CmmolS). Besides, when low pH in the famine phase was considered as additional selective pressure, it was possible to shift the ratio TAG:PHA from 4:96 obtaining 43.0 wt% of TAGs (0.67 CmmolTAG/CmmolS). Therefore, this novel and simplified process demonstrated versatility and efficiency in the storage of TAGs and PHAs from a unique residual feedstock and using an open culture proving that product selection can be harnessed if choosing the right operational conditions in the enrichment stage.

Optimization of an enriched mixed culture to increase PHA accumulation using industrial saline complex wastewater as a substrate.

Polyhydroxyalkanoates (PHA) appear as good candidates to substitute conventional petroleum-based plastics since they have similar properties but with the advantage of being biodegradable. Wastewater streams with high organic content are feasible substrates for PHA production resulting in an opportunity for waste recovery. One of the main challenges is the optimization of the selection of microorganisms with high PHA storage capacity. This microbial selection is performed in sequencing batch reactors (SBR) operated under an aerobic feast/famine (F/F) regime. In the present study, a settling stage was added at the end of the feast phase of the enrichment cycle of a SBR fed with pre-acidified cooked mussel processing wastewater (containing up to 12 g NaCl/L). Settling and subsequent supernatant discharge favoured the wash-out of non-accumulating microorganisms as well as the removal of substances that enhanced their undesired development (proteins and carbohydrates). Microbial analysis performed by fluorescence in situ hybridization (FISH) technique showed shifts in the microbial community; the presence of genus Paracoccus increased whereas genera Comamonas decreased. Moreover, the process efficiency was improved with the increase of the PHA production yield (YPHA) and the maximum PHA storage capacity (max. PHA) from 0.48 to 0.72 CmmolPHA/CmmolVFA and from 40 to 60 wt%, respectively. The polymer composition also changed, its HB:HV ratio varied from 83:17 to 70:30. Results obtained in the present study showed that settling after the feast phase promoted the removal of carbon sources that did not contribute to PHA production and the washout of non-storing bacteria, which favoured the culture enrichment.