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.

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.

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.

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.

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.

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.

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.

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.

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.

Molecular weight and thermal properties of polyhydroxyalkanoates produced from fermented sugar molasses by open mixed cultures.

Polyhydroxyalkanoates (PHAs) produced from fermented molasses and synthetic feeds containing single volatile fatty acids (VFAs) by an open mixed culture enriched in glycogen accumulating organisms (GAOs) were characterized with regards to molecular weight and thermal properties. The polymer contained five types of monomers, namely 3-hydroxybutyrate, 3-hydroxy-2-methylbutyrate, 3-hydroxyvalerate, 3-hydroxy-2-methylvalerate and 3-hydroxyhexanoate in different ratios depending on the VFA composition of the substrate. Polymers produced from fermented molasses had weight average molecular weights (M(w)) in the range (3.5-4.3)x10(5)g/mol and polydispersity indexes (PDI) of 1.8-2.1 while polymers produced from synthetic VFAs had M(w) of (4.5-9.0)x10(5)g/mol and PDI of 1.7-3.9. Thermal properties such as glass transition temperature (-14 degrees C to 4.8 degrees C), melting temperature (89-174 degrees C) and melting enthalpy (0-82.1J/g) were controlled in broad ranges by the monomer composition. The decomposition temperatures of the polymers produced were between 277.2 degrees C and 294.9 degrees C, and independent of monomer composition and molecular weight.

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.

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.

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.

Metabolic modelling of polyhydroxyalkanoate copolymers production by mixed microbial cultures.

BACKGROUND: This paper presents a metabolic model describing the production of polyhydroxyalkanoate (PHA) copolymers in mixed microbial cultures, using mixtures of acetic and propionic acid as carbon source material. Material and energetic balances were established on the basis of previously elucidated metabolic pathways. Equations were derived for the theoretical yields for cell growth and PHA production on mixtures of acetic and propionic acid as functions of the oxidative phosphorylation efficiency, P/O ratio. The oxidative phosphorylation efficiency was estimated from rate measurements, which in turn allowed the estimation of the theoretical yield coefficients. RESULTS: The model was validated with experimental data collected in a sequencing batch reactor (SBR) operated under varying feeding conditions: feeding of acetic and propionic acid separately (control experiments), and the feeding of acetic and propionic acid simultaneously. Two different feast and famine culture enrichment strategies were studied: (i) either with acetate or (ii) with propionate as carbon source material. Metabolic flux analysis (MFA) was performed for the different feeding conditions and culture enrichment strategies. Flux balance analysis (FBA) was used to calculate optimal feeding scenarios for high quality PHA polymers production, where it was found that a suitable polymer would be obtained when acetate is fed in excess and the feeding rate of propionate is limited to approximately 0.17 C-mol/(C-mol.h). The results were compared with published pure culture metabolic studies. CONCLUSION: Acetate was more conducive toward the enrichment of a microbial culture with higher PHA storage fluxes and yields as compared to propionate. The P/O ratio was not only influenced by the selected microbial culture, but also by the carbon substrate fed to each culture, where higher P/O ratio values were consistently observed for acetate than propionate. MFA studies suggest that when mixtures of acetate and propionate are fed to the cultures, the catabolic activity is primarily guaranteed through acetate uptake, and the characteristic P/O ratio of acetate prevails over that of propionate. This study suggests that the PHA production process by mixed microbial cultures has the potential to be comparable or even more favourable than pure cultures.

Microbial characterisation of polyhydroxyalkanoates storing populations selected under different operating conditions using a cell-sorting RT-PCR approach.

The identity of polyhydroxyalkanoates (PHA) storing bacteria selected under aerobic dynamic feeding conditions, using propionate as carbon source (reactor P), was determined by applying reverse transcriptase-polymerase chain reaction (RT-PCR) on micromanipulated cells and confirmed by fluorescence in situ hybridisation (FISH). Four genera, Amaricoccus, Azoarcus, Thauera and Paraccoccus were detected, the latter only rarely present. All the biomass was involved in PHA storage as shown by Nile Blue staining. By quantitative FISH, their specific amount was determined in this and two other systems using acetate as the carbon substrate (sequencing batch reactor [SBR] A and A1). SBR A and reactor P had the same sludge retention time (SRT, 10 days), while reactor A1 was operated with the SRT of 1 day and the double organic loading rate (OLR). Systems fed with acetate (41.1 +/- 2.2 and 49.4 +/- 1.4% total Bacteria, for A and A1, respectively) became enriched in Thauera independently on the SRT and OLR, while it was only present in a minor amount when propionate was used as a substrate (1.9 +/- 0.2% total Bacteria). Amaricoccus was present in both reactors operated at 10 days SRT, favoured in the one fed with propionate (61.4 +/- 1.9% total bacteria), and almost completely removed at the SRT of 1 day. Azoarcus cells were found in all the analysed systems (3.9 +/- 0.3, 23.3 +/- 1.5 and 45.9 +/- 1.5 for P, A and A1, respectively), while Paracoccus was scarcely present.

The influence of process parameters on the characteristics of polyhydroxyalkanoates produced by mixed cultures.

The characterization of polyhydroxyalkanoates (PHA) produced by mixed cultures is fundamental for foreseeing the possible final applications of the polymer. In this study PHA produced under aerobic dynamic feeding (ADF) conditions are characterized. The PHA produced shows a stable average molecular weight ([symbol: see text]) in the range (1.0-3.0) x 10(6), along three years of reactor operation. Attempts to improve the amount of PHA produced did not introduce significant variations on the values [symbol: see text]. Along this period, the polydispersity indices (PDI) were between 1.3 and 2.2. The use of different carbon sources allowed the tailoring of polymer composition: homopolymers of poly(3-hydroxybutyrate), P(3HB), were obtained with acetate and butyrate, whereas a mixture of acetate and propionate, and propionate and valerate, gave terpolymers of 3-hydroxybutyrate (3HB), 3-hydroxyvalerate (3HV), and 2-methyl-3-hydroxyvalerate (2M3HV). All of these PHA had [symbol: see text] between 2.0 x 10(6) and 3.0 x 10(6). Thermal characterization of the produced polymers showed values of glass transition temperature, melting temperature, melting enthalpy, and crystallinity slightly lower than those obtained for PHA from pure cultures. The introduction of a purification step during the polymer extraction process allowed the elimination of possible contaminants but did not significantly improve the polymer quality.

Elucidation of metabolic pathways in glycogen-accumulating organisms with in vivo 13C nuclear magnetic resonance.

Glycogen-accumulating organisms (GAOs) are found in enhanced biological phosphorus removal systems where they compete with polyphosphate-accumulating organisms for external carbon substrates. (13)C nuclear magnetic resonance ((13)C-NMR) was used to elucidate the metabolic pathways operating in an enriched GAO culture dominated by two known GAOs (81.2%). The experiments consisted of adding (13)C-acetate (labelled on position 1 or 2) to the culture under anaerobic conditions, and operating the culture through a cycle consisting of an anaerobic, an aerobic and a further anaerobic phase. The carbon transformations over the cycle were monitored using in vivo(13)C-NMR. The two-carbon moieties in hydroxybutyrate and hydroxyvalerate were derived from acetate, while the propionyl precursor of hydroxyvalerate was primarily derived from glycogen, with only a small fraction originating from acetate. Comparison of the labelling patterns in hydroxyvalerate at the end of the first and the second anaerobic periods in pulse experiments with 2-(13)C-acetate showed that the Entner-Doudoroff (ED) pathway was used for the breakdown of glycogen. This conclusion was further supported by the labelling pattern on glycogen observed in the pulse experiments with 1-(13)C-acetate, which can only be explained by the operation of ED with recycling of pyruvate and glyceraldehyde 3-phosphate via gluconeogenesis. The activity of the ED pathway for glycogen degradation by GAOs is demonstrated here for the first time. In addition, the decarboxylating part of the tricarboxylic acid cycle was confirmed to operate also under anaerobic conditions.