Acidogenic fermentation of organic wastes represents a strategic platform for the co-production of H2, CO2, and volatile fatty acids (VFAs), which are potential key intermediates for cost-effective polyhydroxyalkanoate (PHAs) biosynthesis. This typically relies on carbon sources that are too expensive and hinder the commercialization of PHAs. This study provides metagenomic insights into the microbial dynamics underpinning the acidogenic conversion of waste melon under increasing organic loading rates (OLRs). Metabarcoding revealed that Megasphaera dominated the community, with its abundance rising markedly from 5 to 20 gCOD/L, accompanied by relevant contributions from Solobacterium, Prevotella, and Clostridium. These taxa were associated with the formation of acetic, propionic, and butyric acids and with enhanced hydrogenogenesis. Higher OLRs, up to 20 gCOD/L, promoted hydrogen-producing species while suppressing lactic acid bacteria, thereby improving H2 and VFAs yields up to 26.7% v/v and 13 gCOD/L, respectively. By linking microbial shifts to metabolic outputs, this work advances the understanding of acidogenic pathways essential for integrating dark fermentation-derived H2, CO2, and VFAs into sustainable PHAs production systems.

Wine production generates significant quantities of by-products each year, among which surplus grape must is notable for its high content of fermentable sugars, organic acids, and polyphenols. As wine consumption declines and grape must surpluses grow, identifying sustainable valorization strategies becomes increasingly critical. This study investigates for the first time the potential use of red and white grape musts as substrates for polyhydroxyalkanoates (PHAs) production by two well-characterized bacterial strains, Cupriavidus necator DSM 545 and Hydrogenophaga pseudoflava DSM 1034, under both batch and fed-batch fermentation regimes. Both musts supported microbial growth and PHAs accumulation. In batch cultures, C. necator DSM 545 achieved a PHB content of up to 61.5% of cell dry weight (CDW), while H. pseudoflava DSM 1034 reached 67.9% PHB on grape must, with yields and biomass comparable to or exceeding those obtained with synthetic sugar-based media. Under fed-batch conditions with red must, C. necator DSM 545 sustained growth and PHB production across multiple feeding feeding periods, outperforming the control medium. Conversely, H. pseudoflava DSM 1034 displayed initial growth but failed to increase biomass over time, suggesting that this strain may be poorly suited for use in fed-bacth-based applications, likely due to nutrient depletion or the accumulation of inhibitory compounds. Overall, these results proved that grape musts are promising feedstocks for sustainable PHAs production. Their integration into circular economy frameworks offers a valuable opportunity for waste recovery and the development of bioplastics within the agri-food industry, especially in light of the increasing grape must surpluses recently experienced worldwide.

Nowadays, in the agricultural and agro-industrial sectors, there is increasing attention on the wine industry. The environmental impact of wine production, particularly in terms of residues generation, is a growing concern. Among generated residues, wine lees, rich in organic matter, phenols compounds, and with low pH, pose environmental challenges due to their disposal requirements. Despite their usage for biogas production and extraction of compounds, such as antioxidants, tartaric acid and ethanol, their potential in bioplastic production, specifically polyhydroxyalkanoates (PHAs), remains largely untapped. This study aims to evaluate the potential use of wine lees as a medium sustaining bacterial growth and PHAs accumulation. Specifically, Cupriavidus necator DSM 545 and Hydrogenophaga pseudoflava DSM 1034, were cultivated in the liquid phase of wine lees obtained from the Prosecco winemaking. On pure distilled wine lees, after mild feedstock pre-treatments, C. necator DSM 545 reached a CDW (cell dry weight) of 2.97 g/L and accumulated PHAs was 1.27 g/L, pair to 42.90 % of CDW. On the same substrate, CDW for H. pseudoflava DSM 1034 was 3.96 g/L and PHAs reached values of 1.60 g/L and 40.42 % CDW. These results obtained on wine lees are similar or even better than those achieved in the control growths of the two strains on pure glucose. This is the first approach for the utilization of wine lees for PHAs production, highlighting their potential use in the PHAs industry, and offering a sustainable alternative for both residues management and bioplastic production.

Polyhydroxyalkanoates (PHAs) are biodegradable biopolymers with the potential to replace fossil-based plastics. However, their widespread adoption is currently limited by the high cost of carbon substrates. Whey permeate is rich in lactose and minerals and thus represents a potential sustainable carbon source. Cupriavidus necator is commonly used for PHAs production; however, its inability to utilize lactose significantly limits PHAs yields from dairy residues. In this study, lactose from permeate was enzymatically hydrolyzed into glucose and galactose using Maxilact® LGI 5000, an industrial β-galactosidase formulation. After determining the optimal conditions for the use of Maxilact® LGI 5000, the growth of C. necator DSM 545 was evaluated in minimal medium supplemented with enzymatically treated whey permeate. In the hydrolyzed permeate, the biomass and accumulated PHAs reached values above 5 g/L and around 60 % of cell dry weight (CDW), respectively, comparable or even higher than the yields obtained in the benchmark experiment with pure sugars. A Simultaneous Saccharification and Fermentation (SSF) setup was also adopted and provided a more streamlined approach to bioconversion. In conclusion, although further optimization of the process conditions is necessary, these results indicate that the use of whey permeate combined with β-galactosidase can greatly enhance PHAs production.

This study utilized a circular economy approach to convert unripe rice, a low-cost by-product of the rice milling industry, into biofuels using a biorefinery process. The recombinant yeast Saccharomyces cerevisiae ER T12.7 strain was tested for its ability to produce ethanol from unripe rice. In hydrolysis trials with 20 % (dw/v) unripe rice, ER T12.7 showed superior saccharification yields comparable to the commercial enzyme, STARGENTM 002. In 1-L bioreactor tests, ER T12.7 produced ethanol as efficiently as the parental ER V1 strain under simultaneous saccharification and fermentation conditions. The spent fermentation broth from both amylolytic strains was evaluated for biomethane production, achieving high yields of up to 373.61 mL CH4/g volatile solids. This research is the first to demonstrate process integration to produce ethanol and methane from rice waste sequentially, highlighting the potential of unripe rice in biorefining for a circular economy.

The utilization of agri-food wastes holds significant importance from both environmental and economic standpoints. Whey permeate, a by-product of cheese manufacturing with high lactose content, could be considered a promising substrate for microbial growth to yield value-added products. Whey permeate was therefore investigated as a potential feedstock for the production of polyhydroxyalkanoates (PHAs) by Hydrogenophaga pseudoflava DSM1034. Although the ability of this microorganism to grow and accumulate PHAs on whey permeate has been previously demonstrated, the PHAs yields were notably low. Therefore, a meticulous inoculum optimization was performed, taking into consideration adaptation to lactose-containing substrates, initial bacterial concentration, and growth kinetics. In media containing lactose as a carbon source, the lag phase, exceeding 40 hours in non-optimized conditions, decreased to only 4 hours. In whey permeate, a cell dry weight (CDW) of 5.49 ± 0.08 g/L was obtained. PHAs fraction in CDW and PHAs yield were 52.48 ± 3.14 % of CDW and 0.144 g/g of consumed sugar, respectively. Furthermore, these results were achieved in flask without hydrolysis of lactose contained in the permeate. Moreover, no galactose remained unutilized in the spent broth demonstrating that H. pseudoflava DSM1034 metabolized both sugars contained in lactose. Additionally, in culture media containing mixtures of glucose and galactose, microbial growth was lower compared to growth on comparable amounts of lactose. This study underscores the potential of whey permeate as a valuable resource for the production of PHAs, offering a sustainable solution for the utilization of dairy industry by-products, mitigating its environmental impact and promoting sustainable resource utilization.

The exploitation of agri-food wastes is of great importance for environmental and economic reasons. Date wastes are attractive biomasses that could be used as a carbon source for the growth of microorganisms to obtain added-value products. In this work, spoilage date syrup, containing 102.01 and 101.00 g/L of glucose and fructose, respectively, was assessed as a feedstock for the production of bioethanol and polyhydroxyalkanoates (PHAs) by Saccharomyces cerevisiae MEL2 and Cupriavidus necator DSM 545, respectively. The waste date syrup was first evaluated as a carbon source for microbial growth and resulted to sustain the growth of both strains. 47.95 g/l of ethanol, corresponding to the 93.52% of the theoretical yield, were obtained from the fermentation of date syrup by S. cerevisiae MEL2, here adopted as a proficient bioethanol yeast strain. Furthermore, C. necator DSM 545, a well-known PHAs-producer, was able to accumulate up to 79.20 % (w/w on dry mass) of PHAs. This study demonstrates that bioethanol and PHAs can be obtained from date wastes, contributing to developing cost-effective exploitation of these residues with economic and environmental advantages.

In the challenge of mitigating greenhouse gases produced mainly by fossil fuels, the recovery of agricultural and food waste to produce biohydrogen by dark fermentation is considered a promising source for the future. However, industrial-scale production remains challenging and several parameters significantly influencing the application of the technology need to be finely tuned. In the present study, an evaluation of the production of biohydrogen from cassava and pineapple waste, widely available in many African countries, was carried out using different microbial consortia originating from industrial biogas plants in Italy. The production of biohydrogen was defined both by screening the most appropriate inoculum and by using a "centered composite design" to optimize the inoculum pretreatment time and the pH. From cassava waste, which is abundant of starch (43%), the highest hydrogen levels (62.32 mL H2/gVS) were obtained at pH 7.72 adopting a 4-h heat treated inoculum originating from an anaerobic digester fed with corn and barley silage. Higher hydrogen yields (75.50 mL H2/gVS) were achieved at pH 6 once pineapple, which has a high content of hemicellulose, was converted by another selected inoculum, isolated from an olive pomace treating plant, where hemicellulose is one of the most common polysaccharide. The correlation coefficients R2 and adjusted R2 are close to unity, proving the model to be appropriate to express the concentration of hydrogen produced.This combined approach performed successfully and represents a strong strategy to optimize the production of biohydrogen from agricultural residues even in African contexts.(c) 2023 The Author(s). Published by Elsevier Ltd on behalf of Hydrogen Energy Publications LLC. This is an open access article under the CC BY-NC-ND license (http:// creativecommons.org/licenses/by-nc-nd/4.0/).

Anaerobic co-digestion of protein-rich substrates is a prominent strategy for converting valuable feedstocks into methane, but it releases ammonia, which can inhibit the overall process. This study developed a cutting-edge combined culturomic and metagenomic approach to investigate the microbial composition of an ammonia-tolerant biogas plant. Newly-isolated microorganisms were used for bioaugmentation of stressed batch reactors fed with casein, maize silage and their combination. A co-culture enriched with proteolytic bacteria was isolated, selected and compared with the proteolytic collection strain Pseudomonas lundensis DSM6252. The co-culture and P. lundensis were combined with the ammonia-resistant archaeon Methanoculleus bourgensis MS2 to boost process stability. A microbial population pre-adapted to casein was also tested for evaluating the digestion of protein-rich feedstock. The promising results suggest combining proteolytic bacteria and M. bourgensis could exploit microbial co-cultures to improve anaerobic digestion stability and ensure stable productivity even under the harshest of ammonia conditions.

Organic fraction of municipal solid waste (OFMSW) has the potential to sustain large-scale biofuel production. So far, OFMSW is mainly converted into biogas by anaerobic digestion (AD), and limited research is available on its use to produce bioethanol. This paper reports, for the first time, the conversion of starch-rich OFMSW to bioethanol by using a novel yeast co-secreting both glucoamylase and alpha-amylase enzymes. As such, OFMSW can be converted to bioethanol without adding costly enzymes following a consolidated bioprocessing (CBP) approach. The OFMSW, sampled at an industrial AD plant, was processed to bioethanol with an outstanding yield, approaching 100 % of the theoretical maximum. Moreover, the co-conversion of OFSMW with starch-rice waste, namely discolored rice (DR) available in large quantities close to the AD plant, was performed to test the feasibility of valorizing different waste substrates simultaneously. The ethanol levels reached 60 g/L, indicating that both the developed process and yeast strain have important features towards ethanol production from organic waste streams.

Biohydrogen by dark fermentation using organic wastes is a promising technology towards energy transition and green economy. Unfortunately, H2 production is still limited as several parameters must be finely tuned. This study aims at the development of an efficient process to convert African lignocellulosic residues into H2. An alkaline H2O2 pre-treatment of coffee and pineapple skins, which are hugely produced as waste in many African countries, has been developed. The goal was to get simple sugars, under mild conditions, to foster H2 production. Once pre-treated, the feedstocks were converted by a heat-treated inoculum into H2 at two pH values (5.5 and 6.0). The results indicate that coffee and pineapple skins were efficiently transformed into H2 (up to 47.99 and 91.80 mL/gVS, respectively) at an optimal pH of 6 with a concentration of 1.25 % H2O2. These results proved that H2O2 pre-treatment and the fine tuning of pH can be the key drivers to boost H2 production of lignocellulosic matrices. This combined approach performed successfully and stands for a promising way towards the optimization of the H2-producing processing from waste streams also in African countries. This will contribute to the United Nation (UN)'s 'Agenda 2030' for green energy and sustainable economic growth.

Traditional plastics reshaped the society thanks to their brilliant properties and cut-price manufacturing costs. However, their protracted durability and limited recycling threaten the environment. Worthy alternatives seem to be polyhydroxyalkanoates, compostable biopolymers produced by several microbes. The most common 3-hydroxybutyrate homopolymer has limited applications calling for copolymers biosynthesis to enhance material properties. As a growing number of researches assess the discovery of novel comonomers, great endeavors are dedicated as well to copolymers production scale-up, where the choice of the microbial carbon source significantly affects the overall economic feasibility. Diving into novel metabolic pathways, engineered strains, and cutting-edge bioprocess strategies, this review aims to survey up-to-date publications about copolymers production, focusing primarily on precursors origins. Specifically, in the core of the review, copolymers precursors have been divided into three categories based on their economic value: the costliest structurally related ones, the structurally unrelated ones, and finally various low-cost waste streams. The combination of cheap biomasses, efficient pretreatment strategies, and robust microorganisms paths the way towards the development of versatile and circular polymers. Conceived to researchers and industries interested in tackling polyhydroxyalkanoates production, this review explores an angle often underestimated yet of prime importance: if PHAs copolymers offer advanced properties and sustainable end-of-life, the feedstock choice for their upstream becomes a major factor in the development of plastic substitutes.

Cocoa pod husks (CPH) and cocoa bean shells (CBS) are the main by-products of the cocoa industry and a source of bioactive compounds. These residues are not completely used and thrown in the fields without any treatment, causing environmental problems. Looking for a holistic valorization, the aim of this work was first to deeply characterize CPH and CBS in their chemical composition, amino acid, and fatty acid profiles, as well as their application as antioxidants. CBS had a high level of protein (17.98% DM) and lipids (16.24% DM) compared with CPH (4.79 and 0.35% DM respectively). Glutamic acid and aspartic acid were the predominant amino acids. The total phenolic compounds (TPC) detected in the ethanolic extracts of CPH and CBS were similar to pyrogallol as the main detected polyphenol (72.57 mg/L). CBS ethanolic extract showed a higher antioxidant activity than CPH. Both extracts increased the oxidation stability of soybean oil by 48% (CPH) and 32% (CBS). In addition, alkaline pretreatment of CPH was found suitable for the release of 15.52 ± 0.78 g glucose/L after subsequent saccharification with the commercial enzyme Cellic®. CTec2. Alkaline hydrolyzed and saccharified CPH (Ahs-CPH) was assessed for the first time to obtain polyhydroxy alkanoate (PHAs) and bioethanol. Ahs-CPH allowed the growth of both Cupriavidus necator DSM 545 and Saccharomyces cerevisiae Fm17, well-known as PHA- and bioethanol-producing microbes, respectively. The obtained results suggest that such agricultural wastes have interesting characteristics with new potential industrial uses that could be a better alternative for the utilization of biomass generated as million tons of waste annually.

Due to their long domestication time course, many industrial Saccharomyces cerevisiae strains are adopted in numerous processes mostly for historical reasons instead of scientific and technological needs. As such, there is still significant room for improvement for industrial yeast strains relying on yeast biodiversity. This paper strives to regenerate biodiversity with the innovative application of classic genetic methods to already available yeast strains. Extensive sporulation was indeed applied to three different yeast strains, specifically selected for their different origins as well as backgrounds, with the aim of clarifying how new variability was generated. A novel and easy method to obtain mono-spore colonies was specifically developed, and, to reveal the extent of the generated variability, no selection after sporulation was introduced. The obtained progenies were then tested for their growth in defined mediums with high stressor levels. A considerable and strain-specific increase in both phenotypic and metabolomic variability was assessed, and a few mono-spore colonies were found to be of great interest for their future exploitation in selected industrial processes.

The environmental concerns regarding fossil plastics call for alternative biopolymers such as polyhydroxyalkanoates (PHAs) whose manufacturing costs are however still too elevated. Autotrophic microbes like Cupriavidus necator, able to convert CO2 and H2 into PHAs, offer an additional strategy. Typically, the preferred source for CO2 and H2 are expensive pure gases or syngas, which has toxic compounds for most PHAs-accumulating strains. In this work, for the first time, H2 and CO2 originating from an acidogenic reactor were converted autotrophically into poly(3-hydroxybutyrate) P(3HB). During the first stage, a mixed microbial community continuously catabolized melon waste into H2 (26.7 %) and CO2 (49.2 %) that were then used in a second bioreactor by C. necator DSM 545 to accumulate 1.7 g/L P(3HB). Additionally, the VFAs (13 gCOD/L) produced during acidogenesis were processed into 2.7 g/L of P(3HB-co-3HV). This is the first proof-of-concept of using acidogenic-derived H2 and CO2 from fruit waste to produce PHAs.

Natural yeast with superior fermentative traits can serve as a platform for the development of recombinant strains that can be used to improve the sustainability of bioethanol production from starch. This process will benefit from a consolidated bioprocessing (CBP) approach where an engineered strain producing amylases directly converts starch into ethanol. The yeast Saccharomyces cerevisiae L20, previously selected as outperforming the benchmark yeast Ethanol Red, was here subjected to a comparative genomic investigation using a dataset of industrial S. cerevisiae strains. Along with Ethanol Red, strain L20 was then engineered for the expression of α-amylase amyA and glucoamylase glaA genes from Aspergillus tubingensis by employing two different approaches (delta integration and CRISPR/Cas9). A correlation between the number of integrated copies and the hydrolytic abilities of the recombinants was investigated. L20 demonstrated important traits for the construction of a proficient CBP yeast. Despite showing a close relatedness to commercial wine yeast and the benchmark Ethanol Red, a unique profile of gene copy number variations (CNVs) was found in L20, mainly encoding membrane transporters and secretion pathway proteins but also the fermentative metabolism. Moreover, the genome annotation disclosed seven open reading frames (ORFs) in L20 that are absent in the reference S288C genome. Genome engineering was successfully implemented for amylase production. However, with equal amylase gene copies, L20 proved its proficiency as a good enzyme secretor by exhibiting a markedly higher amylolytic activity than Ethanol Red, in compliance to the findings of the genomic exploration. The recombinant L20 dT8 exhibited the highest amylolytic activity and produced more than 4 g/L of ethanol from 2% starch in a CBP setting without the addition of supplementary enzymes. Based on the performance of this strain, an amylase/glucoamylase ratio of 1:2.5 was suggested as baseline for further improvement of the CBP ability. Overall, L20 showed important traits for the future construction of a proficient CBP yeast. As such, this work shows that natural S. cerevisiae strains can be used for the expression of foreign secreted enzymes, paving the way to strain improvement for the starch-to-bioethanol route.

This paper is a microbiological voyage throughout the main discoveries and research activities of scholars who studied or taught in this field at Padova University during its 800-year history. The ancient practice of variolization, the so-called miracle of Legnaro and the pellagra debate, whose observations and investigations were driven at Padova even before microbiology was established as a scientific discipline, along with the modern era research activities in the field of agricultural, environmental and food microbiology, undoubtedly suggest the hallmark and everyday role played by microbes in overwhelmingly global, but profoundly human, adventures.

Traditional plastics represent a tremendous threat to the environment because of increases in polluting manufacturing as well as their very extended degradation time. Polyhydroxyalkanoates (PHAs) are polymers with similar performance to plastic but are compostable and synthesizable from renewable sources and therefore could be a replacement for fossil-based plastics. However, their production costs are still too high, thus demanding the investigation of new and cheap substrates. In this sense, agricultural wastes are attractive because they are inexpensive and largely available. Specifically, fruit and vegetables are rich in sugars that could be fermented into PHAs. In this work two strains, Cupriavidus necator DSM 545 and Hydrogenophaga pseudoflava DSM 1034, well-known PHA-producing microbes, were screened for their ability to grow and accumulate PHAs. Ten different fruit and vegetable processing waste streams, never before reported in combination with these strains, were tested. Residues from red apple and melon were found to be the most suitable feedstocks for PHA production. Under specific selected conditions, C. necator DSM 545 accumulated up to 7.4 and 4.3 g/L of 3-hydroxybutyrate (3HB) from red apple and melon, respectively. Copolymer production was also obtained from melon. These results confirm the attractiveness of food processing waste as a promising candidate for PHA production. Ultimately, these novel substrates draw attention for future studies on process optimization and upscaling with C. necator.

Broken rice, a low-cost starchy residue of the rice industry, can be an interesting substrate to reduce the polyhydroxyalkanoates (PHAs) production cost. However, since the most common PHAs-producing strains lack amylases, this waste must be firstly hydrolysed by additional commercial enzymes. In this work, the acidogenesis phase of the anaerobic digestion was exploited as efficient hydrolysis step to convert broken rice into volatile fatty acids (VFAs) to be used as PHAs carbon source by Cupriavidus necator DSM 545, one of the most promising PHAs-producing microbes. Broken rice, both non-hydrolysed and enzymatically hydrolysed, was processed in two continuous stirred tank reactors, at hydraulic retention times (HRT) of 5, 4 and, 3 days, to produce VFAs. The highest VFAs levels were obtained from non-hydrolysed broken rice which was efficiently exploited for PHAs accumulation by C. necator DSM 545. PHAs contents were higher after 96 h of incubation and, noteworthy, reached the highest value of 0.95 g/L in the case of 4 days HRT without any chemicals supplementation, except vitamins. Moreover, in view of a biorefinery approach, the residual solid fraction was used for methane production resulting in promising CH4 levels. Methane yields were very promising again for 4 days HRT. As such, this HRT resulted to be the most suitable to obtain effluents with high promise in terms of both PHAs accumulation and CH4 production. In addition, these results demonstrate that broken rice could be efficiently processed into two valuable products without any costly enzymatic pre-treatment and pave the way for future biorefining approaches where this by-product can be converted in a cluster of added-value compounds. Techno-economical estimations are in progress to assess the feasibility of the entire process, in view of supporting the low-cost conversion of organic waste into valuable products.

Starch-rich by-products could be efficiently exploited for polyhydroxyalkanoates (PHAs) production. Unfortunately, Cupriavidus necator DSM 545, one of the most efficient PHAs producers, is not able to grow on starch. In this study, a recombinant amylolytic strain of C. necator DSM 545 was developed for the one-step PHAs production from starchy residues, such as broken rice and purple sweet potato waste. The glucodextranase G1d from Arthrobacter globiformis I42 and the alpha-amylase amyZ from Zunongwangia profunda SM-A87 were co-expressed into C. necator DSM 545. The recombinant C. necator DSM 545 #11, selected for its promising hydrolytic activity, produced high biomass levels with noteworthy PHAs titers: 5.78 and 3.65 g/L from broken rice and purple sweet potato waste, respectively. This is the first report on the engineering of C. necator DSM 545 for efficient amylase production and paves the way to the one-step conversion of starchy waste into PHAs.