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.

Microalgae as Contributors to Produce Biopolymers.

Biopolymers are very favorable materials produced by living organisms, with interesting properties such as biodegradability, renewability, and biocompatibility. Biopolymers have been recently considered to compete with fossil-based polymeric materials, which rase several environmental concerns. Biobased plastics are receiving growing interest for many applications including electronics, medical devices, food packaging, and energy. Biopolymers can be produced from biological sources such as plants, animals, agricultural wastes, and microbes. Studies suggest that microalgae and cyanobacteria are two of the promising sources of polyhydroxyalkanoates (PHAs), cellulose, carbohydrates (particularly starch), and proteins, as the major components of microalgae (and of certain cyanobacteria) for producing bioplastics. This review aims to summarize the potential of microalgal PHAs, polysaccharides, and proteins for bioplastic production. The findings of this review give insight into current knowledge and future direction in microalgal-based bioplastic production considering a circular economy approach. The current review is divided into three main topics, namely (i) the analysis of the main types and properties of bioplastic monomers, blends, and composites; (ii) the cultivation process to optimize the microalgae growth and accumulation of important biobased compounds to produce bioplastics; and (iii) a critical analysis of the future perspectives on the field.

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.

Bacterially assembled biopolyester nanobeads for removing cadmium from water.

Cadmium (Cd)-contaminated waterbodies are a worldwide concern for the environment, impacting human health. To address the need for efficient, sustainable and cost-effective remediation measures, we developed innovative Cd bioremediation agents by engineering Escherichia coli to assemble poly(3-hydroxybutyric acid) (PHB) beads densely coated with Cd-binding peptides. This was accomplished by translational fusion of Cd-binding peptides to the N- or C-terminus of a PHB synthase that catalyzes PHB synthesis and mediates assembly of Cd2 or Cd1 coated PHB beads, respectively. Cd1 beads showed greater Cd adsorption with 441 nmol Cd mg-1 bead mass when compared to Cd2 beads (334 nmol Cd mg-1 bead-mass) and plain beads (238 nmol Cd mg-1 bead-mass). The Cd beads were not ecotoxic and did attenuate Cd-spiked solutions toxicity. Overall, the bioengineered beads provide a means to remediate Cd-contaminated sites, can be cost-effectively produced at large scale, and offer a biodegradable and safe alternative to synthetic ecotoxic treatments.

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.

Proteomic Response of Pseudomonas putida KT2440 to Dual Carbon-Phosphorus Limitation during mcl-PHAs Synthesis.

Pseudomonas putida KT2440, one of the best characterized pseudomonads, is a metabolically versatile producer of medium-chain-length polyhydroxyalkanoates (mcl-PHAs) that serves as a model bacterium for molecular studies. The synthesis of mcl-PHAs is of great interest due to their commercial potential. Carbon and phosphorus are the essential nutrients for growth and their limitation can trigger mcl-PHAs' production in microorganisms. However, the specific molecular mechanisms that drive this synthesis in Pseudomonas species under unfavorable growth conditions remain poorly understood. Therefore, the proteomic responses of Pseudomonas putida KT2440 to the limited carbon and phosphorus levels in the different growth phases during mcl-PHAs synthesis were investigated. The data indicated that biopolymers' production was associated with the cell growth of P. putida KT2440 under carbon- and phosphorus-limiting conditions. The protein expression pattern changed during mcl-PHAs synthesis and accumulation, and during the different physiological states of the microorganism. The data suggested that the majority of metabolic activities ceased under carbon and phosphorus limitation. The abundance of polyhydroxyalkanoate granule-associated protein (PhaF) involved in PHA synthesis increased significantly at 24 and 48 h of the cultivations. The activation of proteins belonging to the phosphate regulon was also detected. Moreover, these results indicated changes in the protein profiles related to amino acids metabolism, replication, transcription, translation, stress response mechanisms, transport or signal transduction. The presented data allowed the investigation of time-course proteome alterations in response to carbon and phosphorus limitation, and PHAs synthesis. This study provided information about proteins that can be potential targets in improving the efficiency of mcl-PHAs synthesis.

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.

Conversion of cheese whey into poly(3-hydroxybutyrate-co-3-hydroxyvalerate) by Haloferax mediterranei.

Haloferax mediterranei was cultivated in highly saline medium using cheese whey as the substrate for the production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate), P(3HB-co-3HV). Acid hydrolysis provided a simple inexpensive method to obtain a cheese whey hydrolysate that was used for cultivation of H. mediterranei. Batch bioreactor cultivation of H. mediterranei resulted in the production of an active biomass concentration of 7.54 g L(-1) with a polymer content of 53%, and a volumetric productivity of 4.04 g L(-1) day(-1). Supplementation of the cultivation medium with micronutrients favored galactose consumption that was used for polymer synthesis after exhaustion of the available glucose. P(3HB-co-3HV) with a 3-hydroxyvalerate content of 1.5 mol% was extracted from the biomass by hypo-osmotic shock. The polymer presented a molecular mass of 4.4×10(5), with a polydispersity index of 1.5. This work demonstrated the feasibility of using cheese whey for the production of a value-added biopolymer with high volumetric productivity, by using a glucose- and galactose-rich substrate obtained by acid hydrolysis of cheese whey. The use of H. mediterranei as the producing strain avoids the need for strict sterility due to the culture's high salinity requirements and, also, allows for polymer extraction by simply contacting the biomass with water.

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.

Adaptation of Scheffersomyces stipitis to hardwood spent sulfite liquor by evolutionary engineering.

BACKGROUND: Hardwood spent sulfite liquor (HSSL) is a by-product of acid sulfite pulping process that is rich in xylose, a monosaccharide that can be fermented to ethanol by Scheffersomyces stipitis. However, HSSL also contains acetic acid and lignosulfonates that are inhibitory compounds of yeast growth. The main objective of this study was the use of an evolutionary engineering strategy to obtain variants of S. stipitis with increased tolerance to HSSL inhibitors while maintaining the ability to ferment xylose to ethanol. RESULTS: A continuous reactor with gradually increasing HSSL concentrations, from 20% to 60% (v/v), was operated for 382 generations. From the final obtained population (POP), a stable clone (C4) was isolated and characterized in 60% undetoxified HSSL. C4 isolate was then compared with both the parental strain (PAR) and POP. Both POP and C4 were able to grow in 60% undetoxified HSSL, with a higher capability to withstand HSSL inhibitors than PAR. Higher substrate uptake rates, 7% higher ethanol efficiency and improved ethanol yield were obtained using C4. CONCLUSION: S. stipitis was successfully adapted to 60% (v/v) undetoxified eucalyptus HSSL. A stable isolate, C4, with an improved performance in undetoxified HSSL compared to PAR was successfully obtained from POP. Owing to its improved tolerance to inhibitors, C4 may represent a major advantage for the production of bioethanol using HSSL as substrate.

PHA production by mixed cultures: a way to valorize wastes from pulp industry.

In this work, hardwood spent sulfite liquor (HSSL), a complex feedstock originating from the pulp industry, was tested for the first time as a substrate for polyhydroxyalkanoate (PHA) production by a mixed microbial culture (MMC) under aerobic dynamic feeding (ADF) conditions. A sequencing batch reactor (SBR) fed with HSSL was operated for 67days and the selected MMC reached a maximum PHA content of 67.6%. The MMC demonstrated a differential utilization of the carbon sources present in HSSL. Acetic acid was fully consumed, while xylose and lignosulphonates were partially consumed (30% and 14%, respectively). The selected culture was characterized by Fluorescence in Situ Hybridization (FISH). Bacteria belonging to the three main classes were identified: Alpha- (72.7±4.0%), Beta- (11.1±0.37%) and Gammaproteobacteria (10.3±0.3%). Within Alphaproteobacteria, a small amount of Paracoccus (4.2±0.51%) and Defluvicoccus related to Tetrad Forming Organisms (9.0±0.28%) were detected.

Improvement on the yield of polyhydroxyalkanotes production from cheese whey by a recombinant Escherichia coli strain using the proton suicide methodology.

In this work Escherichia coli strain CML3-1 was engineered through the insertion of Cupriavidus necator P(3HB)-synthesis genes, fused to a lactose-inducible promoter, into the chromosome, via transposition-mediated mechanism. It was shown that polyhydroxyalkanotes (PHAs) production by this strain, using cheese whey, was low due to a significant organic acids (OA) synthesis. The proton suicide method was used as a strategy to obtain an E. coli mutant strain with a reduced OA-producing capacity, aiming at driving bacterial metabolism toward PHAs synthesis. Thirteen E. coli mutant strains were obtained and tested in shake flask assays, using either rich or defined media supplemented with lactose. P8-X8 was selected as the best candidate strain for bioreactor fed-batch tests using cheese whey as the sole carbon source. Although cell growth was considerably slower for this mutant strain, a lower yield of OA on substrate (0.04 Cmol(OA)/Cmol(lac)) and a higher P(3HB) production (18.88 g(P(3HB))/L) were achieved, comparing to the original recombinant strain (0.11 Cmol(OA)/Cmol(lac) and 7.8 g(P(3HB))/L, respectively). This methodology showed to be effective on the reduction of OA yield by consequently improving the P(3HB) yield on lactose (0.28 Cmol (P(3HB))/Cmol(lac) vs 0.10 Cmol(P(3HB))/Cmol(lac) of the original strain).

Biological treatment of eucalypt spent sulphite liquors: a way to boost the production of second generation bioethanol.

The fermentation of reducing sugars from hardwood (eucalypt) spent sulphite liquor (HSSL) into ethanol by Pichia (Scheffersomyces) stipitis is hindered by concomitant inhibitors of microbial metabolism. The conditions for the HSSL biological treatment step by Paecilomyces variotii were evaluated and optimised. Two different strategies of reactor operation were compared using single batch (B) and sequential batch reactor (SBR). Biological treatment of HSSL in the SBR revealed the best results with respect to the removal of microbial inhibitors. Also, most of inhibitory compounds, acetic acid, gallic acid, pyrogallol, amongst others, were removed from HSSL by P. variotii before the ethanol fermentation. The bio-detoxified HSSL was subjected to a successful fermentation by P. stipitis, attaining a maximum ethanol concentration of 2.4 g L(-1) with a yield of 0.24 g ethanol g sugars(-1).

Design of ionic liquids for lipase purification.

Aqueous two-phase systems (ATPS) are considered as efficient downstream processing techniques in the production and purification of enzymes, since they can be considered harmless to biomolecules due to their high water content and due to the possibility of maintaining a neutral pH value in the medium. A recent type of alternative ATPS is based on hydrophilic ionic liquids (ILs) and salting-out inducing salts. The aim of this work was to study the lipase (Candida antarctica lipase B - CaLB) partitioning in several ATPS composed of ionic liquids (ILs) and inorganic salts, and to identify the best IL for the enzyme purification. For that purpose a wide range of IL cations and anions, and some of their combinations were studied. For each system the enzyme partitioning between the two phases was measured and the purification factors and enzyme recoveries were determined. The results indicate that the lipase maximum purification and recovery were obtained for cations with a C(8) side alkyl chain, the [N(CN)(2)] anion and ILs belonging to the pyridinium family. However, the highest purification parameters were observed for 1-methyl-3-octylimidazolium chloride [C(8)mim]Cl, suggesting that the IL extraction capability does not result from a cumulative character of the individual characteristics of ILs. The results indicate that the IL based ATPS have an improved performance in the lipase purification and recovery.

Utilization of residues from agro-forest industries in the production of high value bacterial cellulose.

Bacterial cellulose (BC), a very peculiar form of cellulose, is gaining considerable importance due to its unique properties. In this study, several residues, from agro-forestry industries, namely grape skins aqueous extract, cheese whey, crude glycerol and sulfite pulping liquor were evaluated as economic carbon and nutrient sources for the production of BC. The most relevant BC amounts attained with the residues from the wine and pulp industries were 0.6 and 0.3 g/L, respectively, followed by biodiesel crude residue and cheese whey with productions of about, 0.1 g/L after 96 h of incubation. Preliminary results on the addition of other nutrient sources (yeast extract, nitrogen and phosphate) to the residues-based culture media indicated that, in general, these BC productions could be increased by ~200% and ~100% for the crude glycerol and grape skins, respectively, after the addition organic or inorganic nitrogen.

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.