Role of the Electroactive and Non-electroactive Surface Area (EASA and nEASA) for Electroactive Biofilms in Bioelectrochemical Systems

The role of the electroactive surface area (EASA) and of the non-electroactive surface area (nEASA) was studied to better understand electroactive biofilm’s (EAB) growth and performance in four different systems. Those systems consisted in four 1L glass bottles filled with mineral medium and substrates, a stainless-steel cathode and a bioanode. Four different types of bioanode were assembled in order to study the EASA and nEASA role. A potentiostat controlled the anodic potential, which was fixed in every system at + 0.2 V vs SHE (standard hydrogen electrode). To measure the EASA of every system, cyclic voltammetries (CVs) were carried out at different scan rates. Comparing them with the one obtained with a reference system, each EASA is easily calculated. The nEASA, instead, was measured calculating the geometric volume. The obtained results demonstrate the fundamental role of the EASA and, moreover, the necessity to reduce as much as possible the nEASA in order to enhance the performance

Bioelectrochemical hydrogen production with hydrogenophilic dechlorinating bacteria as electrocatalytic agents

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Assessment of long-term fermentability of pha-based materials from pure and mixed microbial cultures for potential environmental applications

The use of polyhydroxyalkanoates (PHA) as slow-release electron donors for environmental remediation represents a novel and appealing application that is attracting considerable attention in the scientific community. In this context, here, the fermentation pattern of different types of PHA-based materials has been investigated in batch and continuous-flow experiments. Along with commercially available materials, produced from axenic microbial cultures, PHA produced at pilot scale by mixed microbial cultures (MMC) using waste feedstock have been also tested. As a main finding, a rapid onset of volatile fatty acids (VFA) production was observed with a low-purity MMC-deriving material, consisting of microbial cells containing 56% (on weight basis) of intracellular PHA. Indeed, with this material a sustained, long-term production of organic acids (i.e., acetic, propionic, and butyric acids) was observed. In addition, the obtained yield of conversion into acids (up to 70% gVFA/gPHA) was higher than that obtained with the other tested materials, made of extracted and purified PHA. These results clearly suggest the possibility to directly use the PHA-rich cells deriving from the MMC production process, with no need of extraction and purification procedures, as a sustainable and effective carbon source bringing remarkable advantages from an economic and environmental point of view

Assessment of Long-Term Fermentability of PHA-Based Materials from Pure and Mixed Microbial Cultures for Potential Environmental Applications

The use of polyhydroxyalkanoates (PHA) as slow-release electron donors for environmental remediation represents a novel and appealing application that is attracting considerable attention in the scientific community. In this context, here, the fermentation pattern of different types of PHA-based materials has been investigated in batch and continuous-flow experiments. Along with commercially available materials, produced from axenic microbial cultures, PHA produced at pilot scale by mixed microbial cultures (MMC) using waste feedstock have been also tested. As a main finding, a rapid onset of volatile fatty acids (VFA) production was observed with a low-purity MMC-deriving material, consisting of microbial cells containing 56% (on weight basis) of intracellular PHA. Indeed, with this material a sustained, long-term production of organic acids (i.e., acetic, propionic, and butyric acids) was observed. In addition, the obtained yield of conversion into acids (up to 70% gVFA/gPHA) was higher than that obtained with the other tested materials, made of extracted and purified PHA. These results clearly suggest the possibility to directly use the PHA-rich cells deriving from the MMC production process, with no need of extraction and purification procedures, as a sustainable and effective carbon source bringing remarkable advantages from an economic and environmental point of view

Simplified Reactor Design for Mixed Culture-Based Electrofermentation toward Butyric Acid Production

Mixed microbial culture (MMC) electrofermentation (EF) represents a promising tool to drive metabolic pathways toward the production of a specific compound. Here, the MMC-EF process has been exploited to obtain butyric acid in simplified membrane-less reactors operated by applying a difference of potential between two low-cost graphite electrodes. Ten values of voltage difference, from -0.60 V to -1.5 V, have been tested and compared with the experiment under open circuit potential (OCP). In all the tested conditions, an enhancement in the production rate of butyric acid (from a synthetic mixture of glucose, acetate, and ethanol) was observed, ranging from 1.3- to 2.7-fold relative to the OCP. Smaller enhancements in the production rate resulted in higher values of the calculated specific energy consumption. However, at all applied voltages, a low flow of current was detected in the one-chamber reactors, accounting for an average value of approximately -100 µA. These results hold a substantial potential with respect to the scalability of the electrofermentation technology, since they pinpoint the possibility to control MMC-based bioprocesses by simply inserting polarized electrodes into traditional fermenters

Efficient utilization of monosaccharides from agri-food byproducts supports Chlorella vulgaris biomass production under mixotrophic conditions

Microalgae are promising resources for the sustainable production of biofuels, feed, and high-value chemicals. Several strains can grow heterotrophically or mixotrophically on multiple organic substrates even if the high cost associated to their use can hinder scalability and economical sustainability of the overall process. The use of agri-food waste biomass hydrolysates might make the cultivation procedure more sustainable, while at the same time valorising underutilized by-products. In this study, Chlorella vulgaris biomass production and sugar utilization was investigated during mixotrophic cultivation on hydrolysates of two inexpensive and widely available recalcitrant agri-food waste biomasses: barley straw (BS) and citrus processing waste (CPW). CPW hydrolysate supported enhanced biomass production, compared to BS digestate, likely because of the presence, beside glucose, of significant amounts of galactose, which is rapidly metabolized by the algae. Notably, when pure monosaccharides were provided as sole organic carbon, growth stopped before complete sugar consumption. Arrested growth in presence of pure monosaccharides correlated with a drastic drop in extracellular pH, which appears to depend on both carbon and nitrogen sources. Our results indicate that mixotrophic cultivation of C. vulgaris on BS or CPW hydrolysates results in more efficient conversion of organic carbon into biomass, compared to growth on pure sugars, indicating that these agri-food by-products can be utilized as valid feedstocks for sustainable algal biomass production

Exploitation of wasted bread as substrate for polyhydroxyalkanoates production through the use of Haloferax mediterranei and seawater

The use of the halophile microorganism Haloferax mediterranei, able to synthesize poly(hydroxybutyrate-hydroxyvalerate) (PHBV), is considered as a promising tool for the industrial production of bioplastic through bioprocessing. A consistent supplementation of the growth substrate in carbohydrates and minerals is overall necessary to allow its PHBV production. In this work, wasted bread was used as substrate for bioplastic production by microbial fermentation. Instead of the consistent and expensive minerals supplement required for Hfx. mediterranei DSM1411 growth, microfiltered seawater was added to the wasted bread-derived substrate. The suitable ratio of wasted bread homogenate and seawater, corresponding to 40:60, was selected. The addition of proteases and amylase to the bread homogenate promoted the microbial growth but it did not correspond to the increase of bioplastic production by the microorganism, that reach, under the experimental conditions, 1.53 g/L. An extraction procedure of the PHBV from cells, based on repeated washing with water, followed or not by a purification through ethanol precipitation, was applied instead of the conventional extraction with chloroform. Yield of PHBV obtained using the different extraction methods were 21.6 ± 3.6 (standard extraction/purification procedure with CHCl3:H2O mixture), 24.8 ± 3.0 (water-based extraction), and 19.8 ± 3.3 mg PHAs/g of wasted bread (water-based extraction followed by ethanol purification). Slightly higher hydroxyvalerate content (12.95 vs 10.78%, w/w) was found in PHBV obtained through the water-based extraction compared to the conventional one, moreover, the former was characterized by purity of 100% (w/w). Results demonstrated the suitability of wasted bread, supplemented with seawater, to be used as substrate for bioplastic production through fermentation. Results moreover demonstrated that a solvent-free extraction, exclusively based on osmotic shock, could be used to recover the bioplastic from cells

Production of Short-chain Fatty Acid from CO2 Through Mixed and Pure Culture in a Microbial Electrosynthesis Cell

The continuous accumulation of atmospheric CO2 requires the development of new technologies for its mitigation. Carbon capture and utilization (CCU) technologies aim to convert CO2 into precious compounds like chemicals and fuels. Biological fixation is an attractive CCU strategy in terms of cost, sustainability and variety of products. Chemoautotrophic microorganisms such as methanogens and acetogens are able to reduce CO2 into acetate and methane, respectively. Acetogens bacteria are able to use CO2 for cell growth through the Wood Liujhundal pathway, moreover, the final precursor (i.e. Acetyl-CoA) of the autotrophic metabolism, is also used in energy metabolism with acetate production as a waste product. Furthermore, it is possible to obtain multicarbon products of autotrophic origin starting from acetyl-CoA and acetate. The biotechnological use of these microorganisms requires the presence of H2 as substrate, which is used as an electron donor in the pathway. This reaction can be sustained by a biocathode in a microbial electrosynthesis cell, in which the reducing power is generated by a polarized electrode. This study proposes the use of a microbial electrosynthesis cell for conversion to acetate in H-cells by either a mixed culture enriched with Acetobacterium woodii or a pure culture of Acetobacterium woodii, to observe the difference in terms of acetate production and reducing power consumption efficiency. The mixed culture was obtained from a mixture of activated sludge and anaerobic digestate, treated by a protocol capable to select acetogenic microorganisms without the use of specific chemical inhibitors (2-Bromoethanesulfonate). Both inoculums were tested at room temperature (25°C) in the cathodic chamber of the H-cell at potentials in the range of -0.7 to -1.1 V vs SHE. The obtained results showed that the enriched mixed culture produced at -0.7 vs SHE a mixture of volatile fatty acids including C4 and C5 molecules with an overall coulombic efficiency of 50%, while at the potential of -0.9 vs SHE methane constituted the main product of the biocathode. The pure culture, on the other hand, showed a specific production of acetate with a coulombic efficiency of 44% at -0.9 vs SHE and 63% at -1.1 vs SHE. Furthermore, a drastic decrease in biocathode biomass was observed in pure culture, suggesting a higher tendency to form biofilms on the electrode unlike the mixed culture, which showed a standard growth profile in the bulk

Chlorine-free Extractions of Mixed-Culture Polyhydroxyalkanoates Produced from Fermented Sewage Sludge at Pilot Scale

In this study, various conventional and innovative methods were investigated for the recovery of polyhydroxyalkanoates (PHA) from a single batch of biomass produced at a pilot scale from mixed microbial cultures (MMCs) and fermented sewage sludge as a feedstock. Sustainable chlorine-free methods using NaOH and/or H2O2, as well as extraction in nontoxic ethyl acetate, were analyzed. Interestingly, the combined treatment of biomass with NaOH and H2O2 solutions demonstrated good recovery (70 wt %) and high purity (92 wt %) of the polymer in small-scale trials. Moreover, when the coupled treatment was performed on a larger biomass quantity (approximately 200 g), it achieved high purity and recovery yield (93 and 88 wt %, respectively), indicating the feasibility of this extraction method on a larger scale

Recirculation factor as a key parameter in continuous-flow biomass selection for polyhydroxyalkanoates production

The effectiveness of polyhydroxyalkanoates (PHA) production with mixed microbial cultures (MMC) largely depends on the selection of PHA-storing microorganisms, conventionally performed in sequencing batch reactors (SBR). These, although easily allow the establishment of the required feast and famine (FF) regime, can represent a factor of cost increase when the process is scaled up. Here, a novel continuous-flow process for MMC selection under FF conditions has been developed by using two sequentially operated reactors. The feast reactor, having a tubular configuration, was continuously fed with a synthetic mixture of acetic and propionic acids (at an organic loading rate of 2.12 gCOD/L d) and the effluent of this reactor was in part sent to the CSTR famine reactor. The recirculation factor (RC), that is the ratio between the recirculation flow rate and the feeding flow rate to the feast reactor, was the main parameter investigated. Four different runs were performed with the RC varying from 1 to 8 and the increase in its value caused a decrease of the biomass residence time in each reactor. The intracellular PHA content in the feast reactor almost linearly increased up to RC 4 (with a value of 34 ± 2 %, wt/wt) and dropped at the RC 8 condition that, however, showed the maximum PHA content (58 ± 5 %, wt/wt) during the accumulation tests. Indeed, the relative abundance of sequences affiliated with putative PHA-storing bacteria increased up to 90.5 % at RC 8 and were dominated by members of the Alphaproteobacteria class mostly represented by the genus Meganema (74 %)