Highly complex substrates lead to dynamic bacterial community for polyhydroxyalkanoates production.

Mixed microbial cultures (MMC) and waste/surplus substrates, as hardwood spent sulfite liquor, are being used to decrease polyhydroxyalkanoates' (PHA) production costs. The process involves two or three steps, being the selection step a crucial one. For the industrial implementation of this strategy, reactor stability in terms of both performance and microbial community presence has to be considered. A long-term operation of a sequencing batch reactor under feast/famine conditions was performed along with microbial community identification/quantification using FISH and DGGE. The community was found to be extremely dynamic, dominated by Alphaproteobacteria, with Paracoccus and Rhodobacter present, both PHA-storing microorganisms. 16S rRNA gene clone library further revealed that side populations' non-PHA accumulators were able to strive (Agrobacterium, Flavobacteria, and Brachymonas). Nevertheless, reactor performance in terms of PHA storage was stable during operation time. The monitoring of the MMC population evolution provided information on the relation between community structure and process operation.

Unveiling PHA-storing populations using molecular methods.

Enrichment of mixed microbial cultures (MMCs) in polyhydroxyalkanoate (PHA)-storing microorganisms must take place to develop a successful PHA production process. Moreover, throughout the operational period of a MMC system, the population needs to be checked in order to understand the changes in the performance that eventually occurred. For these reasons, it is necessary to monitor the population evolution, in order to identify the different groups of microorganisms and relate them with the storage capacity and kinetics of the MMC. Regarding this particular process, several culture-independent molecular techniques were already applied, with the use of hybridization techniques such fluorescence in situ hybridization and also PCR-based methods like denaturing gradient gel electrophoresis, terminal restriction fragment length polymorphism, pyrosequencing, and quantitative PCR standing out. This review intends, thus, to look at the molecular methods currently applied in monitoring the PHA-storing population evolution and how they can be combined with the evolutionary engineering step in order to optimize the overall process.

Impact of a Pretreatment Step on the Acidogenic Fermentation of Spent Coffee Grounds.

Acidogenic fermentation (AF) is often applied to wastes to produce short-chain organic acids (SCOAs)-molecules with applications in many industries. Spent coffee grounds (SCGs) are a residue from the coffee industry that is rich in carbohydrates, having the potential to be valorized by this process. However, given the recalcitrant nature of this waste, the addition of a pretreatment step can significantly improve AF. In this work, several pretreatment strategies were applied to SCGs (acidic hydrolysis, basic hydrolysis, hydrothermal, microwave, ultrasounds, and supercritical CO2 extraction), evaluated in terms of sugar and inhibitors release, and used in AF. Despite the low yields of sugar extracted, almost all pretreatments increased SCOAs production. Milder extraction conditions also resulted in lower concentrations of inhibitory compounds and, consequently, in a higher concentration of SCOAs. The best results were obtained with acidic hydrolysis of 5%, leading to a production of 1.33 gSCOAs/L, an increase of 185% compared with untreated SCGs.

Enzymatic Potential of Filamentous Fungi as a Biological Pretreatment for Acidogenic Fermentation of Coffee Waste.

Spent coffee grounds (SCGs) are a promising substrate that can be valorized by biotechnological processes, such as for short-chain organic acid (SCOA) production, but their complex structure implies the application of a pretreatment step to increase their biodegradability. Physicochemical pretreatments are widely studied but have multiple drawbacks. An alternative is the application of biological pretreatments that include using fungi Trametes versicolor and Paecilomyces variotii that naturally can degrade complex substrates such as SCGs. This study intended to compare acidic and basic hydrolysis and supercritical CO2 extraction with the application of these fungi. The highest concentration of SCOAs, 2.52 gCOD/L, was achieved after the acidification of SCGs pretreated with acid hydrolysis, but a very similar result, 2.44 gCOD/L, was obtained after submerged fermentation of SCGs by T. versicolor. This pretreatment also resulted in the best acidification degree, 48%, a very promising result compared to the 13% obtained with the control, untreated SCGs, highlighting the potential of biological pretreatments.

Biotreatments Using Microbial Mixed Cultures with Crude Glycerol and Waste Pinewood as Carbon Sources: Influence of Application on the Durability of Recycled Concrete.

Two eco-friendly healing bioproducts generated from microbial mixed cultures (MMC) for the production of polyhydroxyalkanoates (PHA) were used as surface treatments, with two residual materials used as the substrates, namely crude glycerol and pinewood bio-oil. Their ability to improve the durability of concrete samples containing recycled aggregates was assessed. To determine this protective capacity, 180 samples were analyzed using different tests, such as water penetration under pressure, capillary absorption, freeze-thaw and water droplet absorption test. Three types of conditions were used: outdoor-indoor exposure, re-application of biopolymers and application in vertical exposure conditions. The results showed reductions of up to 50% in the water penetration test and a delay in the water droplet absorption test of up to 150 times relative to the reference. The surface application of these bioproducts significantly reduced the degree of water penetration in recycled concrete, increasing its useful lifespan and proving to be a promising treatment for protecting concrete surfaces.

FISH in Suspension or in Adherent Cells.

Fluorescence in situ hybridization (FISH) enables the detection and enumeration of microorganisms in a diversity of samples. Short-length oligonucleotide DNA probes complementary to 16S or 23S rRNA sequences are generally used to target different phylogenetic levels. The protocol for the application of FISH to aggregated or suspended cells in mixed microbial communities is described in this chapter, with a special emphasis on environmental samples.

Enrichment of a mixed microbial culture of PHA-storing microorganisms by using fermented hardwood spent sulfite liquor.

Pulp and paper factories produce several residues that can be explored and valorized through polyhydroxyalkanoate (PHA) production via a three-step process. The objective of this work was focused on the selection step. Acidified hardwood spent sulfite liquor (HSSL), a fermented waste stream from a pulp and paper factory, was used to select a mixed microbial culture (MMC) in a sequencing batch reactor (SBR) operated for 156 days under different operational conditions. The MMC adapted to the imposed conditions, revealing its robustness whenever the operational parameters were changed. Feast-to-Famine ratio was kept below or equal to 0.2, with constant production of a copolymer of P(3HB-co-3 HV), and with storage contents values over 30 %. Changes in the operational conditions, namely cycle length, and organic load rate (OLR), successfully led to the selection of an MMC with a stable accumulation capacity and an increased biomass concentration. Next Generation Sequencing analysis was performed on samples collected during the SBR operational period. The analysis of the microbial composition of the MMC showed a rise in PHA-accumulating bacteria over time. Acidovorax and Comamonas species were found mainly to drive the PHA storage process during the first two periods of operation. After an increase in the OLR, in the last period, a shift towards Comamonas dominance occurred, suggesting a higher tolerance to the inhibitory compounds of the HSSL for this genus.

Community structure evolution and enrichment of glycogen-accumulating organisms producing polyhydroxyalkanoates from fermented molasses.

An open mixed culture was enriched with glycogen-accumulating organisms (GAOs) by using a sequencing batch reactor and treating an agroindustrial waste (sugar cane molasses) under cyclic anaerobic-aerobic conditions. Over a 1-year operating period, the culture exhibited a very stable GAO phenotype with an average polyhydroxyalkanoate (PHA) content of 17% total suspended solids. However, the GAO microbial community evolved over the course of operation to a culture exhibiting unusual characteristics in producing PHAs comprised of short-chain-length monomers, namely, 3-hydroxybutyrate, 3-hydroxy-2-methylbutyrate, 3-hydroxyvalerate, and 3-hydroxy-2-methylvalerate, and also, up to 31 mol% of the medium-chain-length (MCL) monomer 3-hydroxyhexanoate (3HHx). Microbial community analysis by fluorescence in situ hybridization revealed a concurrent long-term drift in the GAO community balance, from mainly "Candidatus Competibacter phosphatis" to mainly Defluviicoccus vanus-related organisms. The production of 3HHx was confirmed by (13)C nuclear magnetic resonance (NMR) and appeared to be related to the increased presence of D. vanus-related GAOs. These results suggest a broadened spectrum of material, chemical, and mechanical properties that can be achieved for biopolymers produced by open mixed cultures from fermented waste. The increased spectrum of polymer properties brings a wider scope of potential applications.

Pyrethroid pesticide metabolite, 3-PBA, in soils: method development and application to real agricultural soils.

3-Phenoxybenzoic acid (3-PBA) is a shared metabolite of several synthetic pyrethroid pesticides (SPs) resulting from environmental degradation of parent compounds and thus occurs frequently as a residue in samples. Hence, the importance of 3-PBA evaluation after pyrethroid application. There is a gap of analytical methods to determine 3-PBA in soil samples. Therefore, an analytical method that combines the solid-phase extraction (SPE) and gas chromatography-mass spectrometry (GC/MS) detection has been developed for the determination of 3-PBA in soil samples. The analytical method was validated in terms of linearity, sensitivity, intra- and inter-day batch precisions, recoveries, and quantification limits. An SPE method using a Strata X cartridge allows obtaining limits of detection and quantification equal to 4.0 and 13.3 ng g-1, respectively. Under optimized conditions, the method average recovery levels ranged from 70.3 to 93.5% with a relative standard deviation below 3.4%. Method intra- and inter-day precision was under 5.0 and 4.8%, respectively. The developed method was applied to 11 agricultural soil samples in the north of Portugal. The developed methodology allowed for the determination of the pyrethroid metabolite, 3-PBA, in agricultural soil samples at levels of few ng g-1. Graphical abstract ᅟ.

Deltamethrin impact in a cabbage planted soil: Degradation and effect on microbial community structure.

Synthetic pyrethroids (SPs) are one of the most common pesticides used worldwide. Their use has greatly increased in the last decades and its' continuous application lead to added pesticides concentration in soil. Consequently, SPs may enter the food chain, affecting the environment and human health. The degradation over time of the pyrethroid pesticide deltamethrin applied to cabbages was monitored. The evolution was followed both on cabbages and the surrounding soils, and the soil microbial community characterized by next-generation sequencing of the 16S rRNA gene. The main shift in the microbial community structure was observed during the first 30 days after pesticides' application. The modification in the microbial community composition, where an increased abundance of Nocardioides sp. and Sphingomonas sp. were observed, was correlated respectively with the conversions of deltamethrin and its metabolite, 3-phenoxybenzoic acid (3-PBA). Although deltamethrin was not found in any of the tested samples (soil and cabbage) after 180 days, it caused an environmental impact much further than the 7 days security interval. These findings suggest that deltamethrin application can disturb soil microbial community and that natural biodegradation can have an important part in pesticides soil decontamination.

Strategies for PHA production by mixed cultures and renewable waste materials.

Production of polyhydroxyalkanoates (PHA) by mixed cultures has been widely studied in the last decade. Storage of PHA by mixed microbial cultures occurs under transient conditions of carbon or oxygen availability, known respectively as aerobic dynamic feeding and anaerobic/aerobic process. In these processes, PHA-accumulating organisms, which are quite diverse in terms of phenotype, are selected by the dynamic operating conditions imposed to the reactor. The stability of these processes during long-time operation and the similarity of the polymer physical/chemical properties to the one produced by pure cultures were demonstrated. This process could be implemented at industrial scale, providing that some technological aspects are solved. This review summarizes the relevant research carried out with mixed cultures for PHA production, with main focus on the use of wastes or industrial surplus as feedstocks. Basic concepts, regarding the metabolism and microbiology, and technological approaches, with emphasis on the kind of feedstock and reactor operating conditions for culture selection and PHA accumulation, are described. Challenges for the process optimization are also discussed.

Phytotoxicity of pyrethroid pesticides and its metabolite towards Cucumis sativus.

Pyrethroid pesticides residues have been frequently detected in soils and have been recognized to contribute to soil toxicity. The phytotoxic impact of pesticides was evaluated in Cucumis sativus (C. sativus) seeds. Percentage of seed germination, root elongation, shoot length and leaf length were considered as endpoints to assess the possible acute phytotoxicity of soil by the exposure to pyrethroid pesticides (cypermethrin, deltamethrin and cyhalothrin) and its metabolite phenoxybenzoic acid (3-PBA), in a concentration range between 50 and 500µgkg-1. For germination percentage, it was only observed a negative impact when seeds were exposed to the metabolite. Cypermethrin showed impact in the three studied endpoints of seed development, while deltamethrin merely affected the root length. Concerning pigments content, it can be said that chlorophylls and total carotenoids median values increased for cypermethrin and deltamethrin. This increase was more pronounced to deltamethrin in joint effect with the organic solvent dimethyl sulphoxide (DMSO). When exposed to cyhalothrin and 3-PBA, no statistically significant differences were observed for C. sativus seeds to all the assessed endpoints of seed development and the investigated pigments content. This research brings new data concerning the relative sensitivity of C. sativus seeds to pyrethroids pesticides commonly found in agricultural facilities, as well as critical understanding and development of using C. sativus for phytotoxicity assessments efforts for pesticide exposures.

Denitrifying phosphorus removal: linking the process performance with the microbial community structure.

This study investigated the link between the process performance of two denitrifying phosphorus (P) removal systems and their microbial community structure. Two sequencing batch reactors (SBRs) were operated with either acetate or propionate as the sole carbon source, and were gradually acclimatised from anaerobic-aerobic to anaerobic-anoxic conditions. It was found that the propionate SBR was able to sustain denitrifying P removal after acclimatisation, while the enhanced biological phosphorus removal (EBPR) activity in the acetate reactor collapsed after the aerobic phase was eliminated. The results suggested that the anoxic glycogen production rate in the acetate SBR was insufficient to support the anaerobic glycogen demand for acetate uptake. The chemical transformations in each SBR suggested that different types of polyphosphate-accumulating organisms (PAOs) were present in each system, possessing different affinities for nitrate. Microbial characterisation with fluorescence in situ hybridisation (FISH) revealed that Accumulibacter was the dominant organism in each reactor, although different cell morphotypes were observed. A coccus morphotype was predominant in the acetate SBR while the propionate SBR was enriched in a rod morphotype. It is hypothesised that the coccus morphotype corresponds to an Accumulibacter strain that is unable to use nitrate as electron acceptor but is able to use oxygen, and possibly nitrite. The rod morphotype is proposed to be a PAO able to use nitrate, nitrite and oxygen. This hypothesis is in agreement with literature studies focussed on the identity of denitrifying PAOs (DPAOs), as well as a recent metagenomic study on Accumulibacter.

Advances in enhanced biological phosphorus removal: from micro to macro scale.

The enhanced biological phosphorus removal (EBPR) process has been implemented in many wastewater treatment plants worldwide. While the EBPR process is indeed capable of efficient phosphorus (P) removal performance, disturbances and prolonged periods of insufficient P removal have been observed at full-scale plants on numerous occasions under conditions that are seemingly favourable for EBPR. Recent studies in this field have utilised a wide range of approaches to address this problem, from studying the microorganisms that are primarily responsible for or detrimental to this process, to determining their biochemical pathways and developing mathematical models that facilitate better prediction of process performance. The overall goal of each of these studies is to obtain a more detailed insight into how the EBPR process works, where the best way of achieving this objective is through linking together the information obtained using these different approaches. This review paper critically assesses the recent advances that have been achieved in this field, particularly relating to the areas of EBPR microbiology, biochemistry, process operation and process modelling. Potential areas for future research are also proposed. Although previous research in this field has undoubtedly improved our level of understanding, it is clear that much remains to be learned about the process, as many unanswered questions still remain. One of the challenges appears to be the integration of the existing and growing scientific knowledge base with the observations and applications in practice, which this paper hopes to partially achieve.

Microbial selection strategies for polyhydroxyalkanoates production from crude glycerol: Effect of OLR and cycle length.

Crude glycerol from biodiesel manufacture can be used as carbon source for microbial fermentations. The production of polyhydroxyalkanoates by manipulating the Sequencing Batch Reactor (SBR) selection stage of microbial mixed cultures (MMC) using high organic loading rates (OLR, 50CmM/day) and different cycles lengths (6, 12 and 24h) were optimized. Batch-production of polyhydroxybutyrate (PHB) presented an accumulation capacity in the high range (0.44g/g) after 3 pulses of 50CmM, with a final content of 59% PHB/wt., for the culture selected with 50CmM/day and a 24h cycle length. These values were in the range to those obtained with pure cultures and higher than the ones for MMC. With this strategy three main advantages in terms of the PHA production can be considered: utilization of a real waste without the resort to pure microbial cultures and a pre-fermentation step, consolidating the role of MMC in the valorisation of complex wastes/by-products.

Synthesis of polyhydroxyalkanoates from different short-chain fatty acids by mixed cultures submitted to aerobic dynamic feeding.

The production of polyhydroxyalkanoates from acetate and propionate by two mixed cultures well adapted to each of these substrates was evaluated. Sludge fed with acetate (A), produced a homopolymer of hydroxybutyrate (HB), whereas sludge fed with propionate (P) produced a copolymer of HB and HV (hydoxyvalerate). Switching the substrate feeds, propionate to sludge A and acetate to culture P, a terpolymer of HB, HV and hydroxymethylvalerate (HMV) was obtained with culture A and a copolymer of P(HB/HV) by sludge P. Regardless of the population used, the polymer yield and productivity were much higher for acetate than for propionate. Feeding a mixture of acetate and propionate, in equal parts, to both cultures resulted in an increase of HV units produced per C mol of propionate consumed, relative to the situation where only propionate was used. The individual use of butyrate and valerate by culture A was also studied. Butyrate produced a homopolymer whereas valerate was stored as a terpolymer of P(HB/HV/HMV). The polymer yields on acetate and butyrate were higher than those on propionate and valerate. The polymer productivity was higher for acetate and propionate than for butyrate and valerate. Results showed that the polymer composition, and consequently the polymer properties, could be manipulated by varying the volatile fatty acid feed composition and/or the population.

Recent advances in polyhydroxyalkanoate production by mixed aerobic cultures: from the substrate to the final product.

Numerous bacteria have been found to exhibit the capacity for intracellular polyhydroxyalkanoates (PHA) accumulation. Current methods for PHA production at the industrial scale are based on their synthesis from microbial isolates in either their wild form or by recombinant strains. High production costs are associated with these methods; thus, attempts have been made to develop more cost-effective processes. Reducing the cost of the carbon substrates (e.g., through feeding renewable wastes) and increasing the efficiency of production technologies (including both fermentation and downstream extraction and recovery) are two such examples of these attempts. PHA production processes based on mixed microbial cultures are being investigated as a possible technology to decrease production costs, since no sterilization is required and bacteria can adapt quite well to the complex substrates that may be present in waste material. PHA accumulation by mixed cultures has been found under various operational conditions and configurations at both bench-scale and full-scale production. The process known as "feast and famine" or as "aerobic dynamic feeding" seems to have a high potential for PHA production by mixed cultures. Enriched cultures submitted to a transient carbon supply can synthesize PHA at levels comparable to those of pure cultures. Indeed, the intracellular PHA content can reach around 70% of the cell dry weight, suggesting that this process could be competitive with pure culture PHA production when fully developed. Basic and applied research of the PHA production process by mixed cultures has been carried out in the past decade, focusing on areas such as microbial characterization, process configuration, reactor operational strategies, process modeling and control, and polymer characterization. This paper presents a review of the PHA production process with mixed cultures, encompassing the findings reported in the literature as well as our own experimental results in relation to each of these areas.

Methods for detection and visualization of intracellular polymers stored by polyphosphate-accumulating microorganisms.

Polyphosphate-accumulating microorganisms (PAOs) are important in enhanced biological phosphorus (P) removal. Considerable effort has been devoted to understanding the biochemical nature of enhanced biological phosphorus removal (EBPR) and it has been shown that intracellular polymer storage plays an important role in PAO's metabolism. The storage capacity of PAOs gives them a competitive advantage over other microorganisms present that are not able to accumulate internal reserves. Intracellular polymers stored by PAOs include polyphosphate (poly-P), polyhydroxyalkanoates (PHAs) and glycogen. Staining procedures for qualitative visualization of polymers by optical microscopy and combinations of these procedures with molecular tools for in situ identification are described here. The strengths and weaknesses of widely used polymer quantification methods that require destruction of samples, are also discussed. Finally, the potential of in vivo nuclear magnetic resonance (NMR) spectroscopy for on-line measurement of intracellular reserves is reported.

Bio-oil upgrading strategies to improve PHA production from selected aerobic mixed cultures.

Recent research on polyhydroxyalkanoates (PHAs) has focused on developing cost-effective production processes using low-value or industrial waste/surplus as substrate. One of such substrates is the liquid fraction resulting from pyrolysis processes, bio-oil. In this study, valorisation of bio-oil through PHA production was investigated. The impact of the complex bio-oil matrix on PHA production by an enriched mixed culture was examined. The performance of the direct utilization of pure bio-oil was compared with the utilization of three defined substrates contained in this bio-oil: acetate, glucose and xylose. When compared with acetate, bio-oil revealed lower capacity for polymer production as a result of a lower polymer yield on substrate and a lower PHA cell content. Two strategies for bio-oil upgrade were performed, anaerobic fermentation and vacuum distillation, and the resulting liquid streams were tested for polymer production. The first one was enriched in volatile fatty acids and the second one mainly on phenolic and long-chain fatty acids. PHA accumulation assays using the upgraded bio-oils attained polymer yields on substrate similar or higher than the one achieved with acetate, although with a lower PHA content. The capacity to use the enriched fractions for polymer production has yet to be optimized. The anaerobic digestion of bio-oil could also open-up the possibility to use the fermented bio-oil directly in the enrichment process of the mixed culture. This would increase the selective pressure toward an optimized PHA accumulating culture selection.

Production of polyhydroxyalkanoates from fermented sugar cane molasses by a mixed culture enriched in glycogen accumulating organisms.

Batch production of polyhydroxyalkanoates (PHAs) under aerobic conditions by an open mixed culture enriched in glycogen accumulating organisms (GAOs) with fermented sugar cane molasses as substrate was studied. The produced polymers contained five types of monomers, namely 3-hydroxybutyrate (3HB), 3-hydroxyvalerate (3HV), 3-hydroxy-2-methylbutyrate (3H2MB), 3-hydroxy-2-methylvalerate (3H2MV) and the medium chain length monomer 3-hydroxyhexanoate (3HHx). With fermented molasses as substrate, PHA was produced under concurrent consumption of stored glycogen with yields of 0.47-0.66 C-mol PHA per C-mol of total carbon substrate and with rates up to 0.65 C-mol/C-molX h. In order to investigate the role of glycogen during aerobic PHA accumulation in GAOs, synthetic single volatile fatty acids (VFAs) were used as substrates and it was found that the fate of glycogen was dependent on the type of VFA being consumed. Aerobic PHA accumulation occurred under concurrent glycogen consumption with acetate as substrate and under minor concurrent glycogen production with propionate as substrate. With butyrate and valerate as substrates, PHA accumulation occurred with the glycogen pool unaffected. The composition of the PHA was dependent on the VFA composition of the fermented molasses and was 56-70 mol-% 3HB, 13-43 mol-% 3HV, 1-23 mol-% 3HHx and 0-2 mol-% 3H2MB and 3H2MV. The high polymer yields and production rates suggest that enrichment of GAOs can be a fruitful strategy for mixed culture production of PHA from waste substrates.