2nd Workshop
Resource recovery from waste and wastewater and downstream procedures for PPB biomass

Climate change, water scarcity and soil degradation threaten global food security and may deplete naturalresourcesby 2050. Pig farming generates a significant amount of manure, around 1.3–1.8 billion tons annually in theEuropean Union, containing high nitrogen concentrations that contribute to pollution and greenhouse gasemissions.Currentanimal–based protein production for human consumption is unsustainable and inefficient,requiring a shift towards more sustainable protein production methods[1].This study proposes photo–microbialelectrosynthesis as a versatile solution for bothmanuretreatment and sustainable protein production.Purple phototrophic bacteria (PPB) are highly versatile microorganisms that have recently been proposed as apromising alternative source of protein. Theyperformanoxygenic photosynthesis using a wide range ofsolubleelectron donors, and some can even accept electrons from solid phases such as minerals and graphite electrodes.The mechanism used for electron uptake from a cathode electrode is direct extracellular electron transfer (DEET),which is compatible with carbon fixation via the Calvin–Benson cycle. Inelectro–autotrophic conditions, PPB canachieve up to 60% protein yield, making them a potential alternative source of protein with an amino acid profilewell suited for humanconsumption.[2,3,4].TheVALPIG4FOOD project proposes a novel photo–electro–biorefineryto treat pig manure and convert it intomicrobialprotein and biomethane (Fig. 1). This project aims to increase the sustainability of pig manure treatmentby avoiding the dissipation of nutrients into the soil and atmosphere and reducing carbon emissions to zero. Theproposedbiorefineryrecovers the ammonium fromhydrolyzedpig manure and the biogas generated in theanaerobic digestion to feed a photo–microbial electrosynthesis (photo–MES) system to upgrade biogas tobiomethane and produce PPB protein for human food consumption

Hydrogen has been widely recognized as a promising tool to addressglobal warming problems, due to its highenergy density and the sustainability of its utilization[1].Although being currently not economicallycompetitive[2],hydrogen production through bioprocesses is regarded as a promising approach in terms ofsustainability. This is attributed tothe mild operative conditions[3–4], minimal net greenhouse gas emissions[4],and the potential to exploit waste materials and wastewaters as carbon and energy source[5].In this perspective,the Purple–B project aims to develop atwo–stage sequentialDark andPhoto–fermentationsystem for treating wastegenerated by astronaut crews.The implementation of a two–stage process using food waste and black water orsewage sludge, which are the primary waste streams in a space station, presents an opportunity to maximizehydrogen (H2) recovery from these substrates. This approach creates a direct waste–to–energynexus,simultaneously purifying the effluent and generating a supply of H2.Moreover, the immobilization ofPurpleNon–SulfurBacteria (PNSB) on a hydrophilic matrix enables the system to function in microgravity conditions,such as on planets(e.g.,Mars)or within a gravity–free environment like a space station. By employing animmobilized biomass photobioreactor, the volume of water circulating through the system is minimized, allowingfor better control of biomass growth. Thus, the Purple–B reactor serves as a crucial component within the closedsystem of a space station, effectively transforming waste into energy

Issues regarding the environmental impacts of petroleum–based polymers coupled with petroleum resourcedepletion and thecrude oil prices uncertainty have raised the focus on biodegradable plastics (BPs), obtained fromrenewable resources (the so–called bio–based polymers). Among bio–based polymers, microbial poly–β–hydroxybutyrate (PHB) represents a valid substitute for conventional plastics thanks to its thermoplastics, physicaland mechanical properties [1]. Purple non–sulfur bacteria (PNSB) are capable of accumulating PHB in intracellularinclusions, in response to unbalanced growth conditions characterized by an excessof carbon and a simultaneouslack of other nutrients (e.g., nitrogen, phosphorus, sulfur, or magnesium) [2–3]. Also, PNSB’s versatile metabolismallows them to grow on waste–derived substrates, thus reducing costs for materials [4]. In particular, bread wastesare a valuable substrate for PNSB’s PHB production, due to their high carbon content (up to 70% of carbohydrates,mostly starch) [5]. This work aimed to convert bread wastes into PHB using a two–stage microbial fermentation.The first stage was represented by the lactic fermentation of bread wastes, using theLactobacillus amylovorusDSM 20532. Then, the fermented bread broth was used for the second stage represented by photo–fermentationusing eight different PNSB strainsin pure cultures. The highest PHB production was of 44.5 g·gdry biomass–1,obtained withRhodobacter sphaeroidesPisa7(Fig.1). This study demonstrates the possibility of using carbon–rich food wastes for microbial PHB production, towards a circular economy.

This study investigates the potential of purple non–sulfur bacteria (PNSB) biofilm technology for wastewatertreatment and resource recovery using fuel synthesis wastewater (FSW) as a low–cost substrate. Two biofilmphotobioreactors (BPBRs) were used, with one receiving FSW atfullstrength(100%)and the other at a dilutedstrength(25%). The BPBRs were monitored daily for chemical oxygen demand (COD), optical density, and pH.At the end of the experiment,various resources were recovered,including carotenoids (Crts), bacteriochlorophylls(BChls), total cellular protein (TCP), and polyhydroxyalkanoates (PHAs).Biomass production was higher in 100% wastewater for both suspended and biofilm growth (Figure 1a), withhigherattachment and coverage of the biofilm on the support material(Figure 1 b–c). COD removal was 3225 ±49 mg/L for 100% wastewater and 1302 ± 32 mg/L for 25% wastewater,correspondingto64% and 94% removal,respectively(Figure 1d).This suggests that the diluted wastewater was easier to treat, potentially due to a lowerorganic load.The Crts and BChls production were found within the range of 1.7–4.6μg/gand 1.1–2.7μg/ginboth growths and wastewater concentrations, respectively,indicating notsuitable resources to recoverfrom FSWat both strengths(Figure 1e).TCP content varied from 36–37%and 38–41%in suspended and biofilm growth of100% and 25% wastewater,respectively (Figure 1f). PHB contentincreased with decreasing wastewaterconcentration in both growth modes andwas3–20% (Figure 1g).It indicates that low–strength wastewater hashighTCP and PHB content potential.In conclusion, 100% wastewater concentration had higher COD removal,PNSB growth, and BChls production,but 25% wastewater concentration led to higher PHB, TCP, and carotenoidsproduction.While recovery ofbioresources from low–strength FSW is feasible, it may not be economically viable due to the need for additionalfreshwater sources and reactors for dilution. Therefore, treating full–strength FSW is a moreviable option forresource recovery, but optimizing reactor design is necessary to enhance bioresource production and improvebiofilm growth for more efficient harvesting

The production of polyhydroxyalkanoates (PHA) using phototrophicmixed cultures (PMC)is a promisingtechnology for wastewater treatment andresource recovery[1].However,wastes streams commonly used inPHAproduction have tremendous variations on nutrientsconcentrations, andtheavailabilityof ammonia, coupled oruncoupled with carbon feeding, can impact the selection of PHA producing microorganisms in mixed cultures[2].Likewise,the promising use ofsunlightas free energy sourceforphototrophicPHA production,presentsseasonalfluctuations in outdooroperation,whichmayalsohave animpacton thePMCperformance[1].This researchinvestigated the impact ofdifferentselectionmethodsofPMCenrichedin PHA–accumulating purplephototrophic bacteria (PPB).APMCwas selectedunder light–feast/dark–aerated–famine and winter simulated–outdoor conditions, using fermented domestic wastewater as feedstock, and selectedthree ammonia settings: 1)ammonia available only in the light phase, 2) ammonia always present, and 3) ammonia available only during thedark–aerated–famine phase.The study concludedthat ammonia presence was essential to promote growth of PHA–accumulating bacteria indark–aerated–famine phase,and thatammonia absence in the light–period favored cyanobacteria growth, resultingin decreased phototrophic PHA accumulation capacity.The best performance outcome was obtained underconstant presence of ammonia,attaining a PHA content of 21.6 %gPHA/gVSS (0.6 gPHA/L.day). Moreover,light–feast/dark–aerated–famine operationwas found to guaranteetotal carbon depletion from wastewaterandmaintainingthePHA accumulation performanceofthe systemunder winter conditions,demonstrating its potentialto overcome the constraints of seasonal fluctuations in future outdoor operations

Single cell protein (SCP)can besustainableprotein sourcewhenbased on the recovery of carbon and nutrientsfrom waste–derived resources.The growth of purple phototrophic bacteria (PPB) onpathogen–free sources, suchassterilizedgaseous streams, is a promising option for a safe SCP production.PPBgrowusing awiderange ofelectron/carbon donors, resulting inhigh biomass yields and high protein contents [1].H2fromthefermentationof organic waste, syngas, or generated via water electrolysis using surplus of electricity from renewable sources,and CO2from off–gases, represent promising electron and carbon sources for the sustainable and pathogen–freeproduction of SCP[2].In this study, the potential ofan enrichedPPB consortia forSCP production usingH2andCO2has been evaluated.A series ofsequentialbatch enrichments (over 14)using H2as electron donorwereperformed to obtain a PPB consortium able to achieve an efficient photoautotrophic growth.Subsequently, theinfluence of pH (6.0–8.5), temperature (15–50 ºC) and light intensity (0–50 W·m–2) on the growth kinetics andbiomass yields was investigated using batch tests(Figure 1).Theenvironmental conditionsconsiderablyaffectedtheoverallH2uptake rates,with values up to 61±5 mg COD·d–1under optimal growth conditions(initialpHvaluesof 7,atemperature of 25 ºC and light intensities over 30 W·m–2).Optimal specific uptake rates of2.00±0.14mgCOD·mg COD·d–1were achieved.Lower or higher pH values and temperatures resulted in decreasedrates whereasno photoinhibition was observed atlight intensities up to50 W·m–2.High biomass and protein yields were achieved(~ 1 g CODbiomass⸱g CODH2consumed–1and 3.9–4.4 g protein⸱g–1H2) regardless of the environmental conditions. Thebiomass exhibited high protein contents (>50% w/w),in agreement with PPB grown photoheterotrophically[3].PPB were the dominant bacteria during the experiments (relative abundance over 80% in most tests), with a stablepopulation dominated byRhodobactersp. andRhodopseudomonassp. This study demonstrates the potential ofenriched PPB cultures for H2bioconversion into SCP

Microbial community engineeringfosters thetreatmentandvalorisation oforganic/nutrient residuals from thedairy industry [1].Microbiomesareecologically engineered in non–axenic mixed cultures [2],e.g.,for the selectivemixed–culture fermentation ofcarbohydrates[3,4].Mixed cultures oftheanoxygenicandversatilepurple(PPB)and green (GPB)photoorganoheterotrophic bacteriacanupgradeagri–foodresidualsinto nutrient–rich biomassesand bioproductsat high yield[5,6].Combining them with fermentative bacteria (FB)in anaerobic mixed culturesisanimportantendpoint[1,7].Little is known about metabolic interactionsofFB andphotoorganoheterotrophsincommunities.We engineeredmixed cultures ofPPB and GPBand studied their interactions withFB.Weaddressedeffectsof reactor regimes(batch or chemostat), illumination modes (continuous light, dark, or light/dark cycles; infrared or white light), andsubstrates (acetate, glucose, lactose, cheese whey) on process ecology,conversions, andmetabolicregulation.PPB andGPBwere best selected with fermented organics like acetate, independent of(dis)continuous culturing[7–9].Acetate andinfrared(IR)lightenriched forPPBat93%[8,10], which remained>70% across an irradiancerangefrom 350 (Earth surface)down to3 W m–2[11].Irradiance impacted thegrowth rate, biomass production,and photopigment content.At low light, PPB expressed more proteins of light–harvesting complexes[11].A densePPB biomass (3.8 g VSS L–1)wasobtainedin sequencing batch [10].White light eventually selected for oxygenicgreenmixotrophs, impactingthe anaerobicreactorenvironment[9].Fermentable sugarsbroughtPPB and GPBin competition with FB[7,9]. Reactor regimesdroveselection.Fast–growingFB(>80%)outcompetedthemin batch,under bothdarkandlight.Maintaining a low dilution rate of 0.04h–1in chemostatunder continuous/alternating irradianceestablishedPPB (30%)inmetabolicsymbiosis withFB(>70%)in asingle sludge.Microbial associations of FB and photoorganoheterotrophs can be engineered in anaerobic mixed cultures to treatand valorise carbohydrateresiduals, using microbial ecology.Microbial guildscan be combinedin a single sludge(cheap but low selectivity),or in a two–sludge systemto firstsolubilise/acidify organicsby mixed–culturefermentation before supplying fermentation products to a secondmixed–culture photobioreactorproducing a high–grade PPB or GPB biomass, dependingon treatment/valorisation objectives

Every year, Europe consumes >50 million tonnes of non–biodegradable plastic,10%comingfromPETdemand.In 2020fromthe totalinend–of–lifePETinEU,only52%iscollected for recycling,besides, PET recyclingmostly involves mechanicaldegradationleading to therelease ofmicroplasticsandconsequently,leaching ofchemicalcompounds.[1,2]Theseplastic derived compounds (PDCs),also foundon enzymatic depolymerizationprocesses of PET, are mainly composed ofethylene glycol (EG) and aromatics such as terephthalic acid (TPA),thatcan be furtherusedascarbon sourcesto producenew biodegradable materials.[3]With the wider use ofPurple Phototrophic Bacteria (PPB)as apromisingwaste management technology, itis of utmost importance tounderstand the influencethatcomplexcompoundsfrom PET packaging found in day–to–day products, eventuallybeingfound in waste streams,have in PPB.With this work weaimto understandwhatcouldbetheimpactof PDCs in a mixed culture enriched in PPB notpreviously adaptedto the presence of such compounds. The culture was inoculated in 100 mL serum flasksandilluminatedwith an average of 164 W/m2with halogen lamps after a cut–off UV/VIS filter.Fivedifferent conditions were testedwith increasingconcentrations ofPDCs, starting in0gCOD/L, up to18gCOD/L. The PDCs were a combinationof EGand TPA in molar proportion of 1:1as usually results fromenzymatic PET degradation.To control the normal activity ofthe bacteria,all flasksreceived2 gCOD/Lofbutyricacid(the carbon source used for the selection of the inoculum).The resultsshowedthatthe presence of PDCs upto5gCOD/Ldoes notinhibitthe growth of PPBmixedcultures,withthegrowth profilebeing similarto the control where only butyric wasgiven(Figure 1). Once the PDCconcentration in the medium increased,thelag phaseincreasedup to4timesat the18 gCOD/L condition whencompared tothecontrol. Theresults also showed an absence of consumption ofTPA during the duration of thetest, buton the other hand,afavorableconsumption of EGbythe PPB culturewas observed

In Europe,138 milliontonsof municipal bio–wasteis generated annually, 75% of which is destined forincineration or landfill. This disposal of bio–waste generates a huge negative environmental cost and excessiveeconomic expenditure. These methods have a high carbon footprint, andmost of the nutrientsand resourcespresent are not recovered[1].A high percentage of waste has great potential as a raw material for high value–added products. For example, wastewater and solid organic waste[2–3]contain valuable nutrients that can be usedas raw material for many applications:water regeneration, fertilizers, bio–plastics, cosmetics or proteins[4].It is necessary to find an alternative to the treatment of this urban waste that, in turn,favorsthecirculareconomy. In this sense, the first step begins by changing the concept of WWTP (Wastewater Treatment Plant) toresource recovery plants. In order to achieve this, it is necessary to make use of biotechnology usingmicro–organisms capable of promoting change. One proposal isPPB (purple phototrophic bacteria)due to theirversatile metabolism, capable of assimilating nutrients from the environment and accumulating them into productsof interest such aspolyphosphate, polyhydroxyalkanoate, carotenoids, bacteriochlorophyll, glycogen, proteinorfertilizers[4–5].However, this type ofwastewater does not meet the optimal COD:Nbalance for the development of PPBs.However, this problem can be solved by adding an extrasupply of biodegradable organicmatter. This projectproposes the use of theorganic fraction of municipal solid waste (OFMSW)as a source for obtaining thisorganic matter. This requires priorsolubilizationof the organic matter through the process of thermal hydrolysiswith steam explosion. Thecombination of the liquidfraction (LF)derived from this process with the domesticwastewater makes it possible to obtain a mixture that has a balanced COD:N ratiofor its complete andsimultaneous assimilation

For the purple bacterium Rhodospirillum rubrum, a growth medium was developed,which surprisingly results inmaximal expression of photosynthetic membranes under microaerobic dark conditions. The cultivation process overcomes the necessity for light and opens a new route to obtain photosynthetic products in common darkbioreactors at industrial scales.The dark photosynthesis“ metabolic regime was analyzed in an interdisciplinary approach of stoichiometric andkinetic computational modeling and lab experiments, The experimental determination of redox states of electrontransfer components in combination with simulation studiesindicatesbiochemical mimicking oflight–signalinginthe dark.For further development towards an economically viable process, biogenic wastes ofthemilk/whey industry andviticulture, respectively, are now exploited in fed–batchhigh–celldensity cultivations with a focus on biohydrogenand carotenoid production.

The global market for fruit and vegetable juices is growing due to changes in consumerpreferences, a shift towardshealthier diets, and the popularity of cold–pressed juices. However, as production increases, so does the amount ofwaste and by–products generated. Purple phototrophic bacteria (PPB) have a unique metabolism that allows themto thrive in complex waste streams[1]. Recent studies have shown that PPB can be used to produce high–qualitysingle–cell protein with a high protein concentration[2]. In this study, PPB (Rhodobacter sphaeroides) wasculturedphotoheterotrophically using by–products from cold–pressed soybean, rice, and oat juices as a substrate.The growth was sustained in batch mode, and PPB was able to produce a biomass with remarkable high contentsof high–quality proteins. Positive control (commercial growing medium, [3]) achieved a biomass withca.50%(DW) of proteins, while the addition of soybean by–productsextracts at 50%diminished the growth rateconsiderably (0.72and 0.37g DWL–1d–1, respectively,seeFigure 1)butenhanced the protein content reachingup toca.67% (DW) by uptaking ca. 30% of medium N.Interestingly,the replacement ofthe soymilk basewithmix–cereal by–product extract resulted toabiomassproductivityand proteincontentcomparableto the optimum.Results show thatR. sphaeroideswas not able to grow solely over these by–products, but growth was enabledwhen adding an external source of simple sugars (e.g.,1 g L–1acetate),exhibitinga growth yield of0.33g DWL–1d–1in case of mix–cerealfully basedmedium.Although further analysisisneeded toconfirm the suitability of the obtained PPB biomassas raw material inaquacultureand livestock feeding,the use ofR. sphaeroidesgrowing over vegetable drink ́s by–products seem tobe promising for protein–rich biomass production.These results provide initial evidence on the feasibility ofresource recovery whileproducing high–value biomass from a relevantfoodby–productsourcewhich deemssuitable for its potential downstream valorization as animal feed ingredients.

Purple bacteria contain large amounts of various carotenoids. These natural pigments have three main functions:they harvest and transfer light energy, they have structural roleand they have also protective role as antioxidants.In biotechnology carotenoids are used as natural food colorants and antioxidants. Currently, most of thecommercially utilized carotenoids come from plants or algae: beta–carotene, lutein, or astaxanthin. Purple bacteriacontain hundreds of novel carotenoids, representing a potential source of valuable compounds for food or chemicalindustry.While several methods exist for extracting carotenoids from algal biomass, they are complex, costly, and havelimited applicability on a pilot or industrial scale. Countercurrent chromatography (CCC) is an efficient,automatable, environmentally friendly, and cost–effective technology to recover pigments from microalgae.Considering the great versatility and selectivity of this technology facilitated by a liquid stationary phase, it willenable the recovery of a wide variety of high–value carotenoids, ensuring product diversification for commercial applications.Standard purification protocols include extraction to organic solvents followed by liquid chromatography. Furtherthe structure of novel carotenoids can be analyzed using nuclear magnetic resonance (1H NMR, COSY, 1H–13CHSQC, 1H–13C HMBC, J–resolved, and ROESY) and high–resolution mass spectroscopy to deter to determinetheir exact chemical structur

Industrial activities, pesticides, and improper waste disposal represent the main anthropogenic activities
responsible for soil pollution. The Italian government in 1998 listed fourteen nationally relevant polluted sites in
urgent need an environmental remediation [1]. Among these sites, the city of Taranto – located in southern Italy –
and its nearby industrial area is included as highly polluted district.
The presence of a very large steel industry, an oil refinery, a power plant, and a set of dockyards contributed
altogether to the release of multiple and toxic pollutants in the environment. Although several studies were and
are published focused on air and water pollution, few reports evaluated soil contamination of this area.
Investigation on chemical and physical parameters (pH, electrical conductivity, available P, Organic C) and
contaminant analyses of soil samples collected from a multi-contaminated area located close to Taranto were
performed, [2] showing the presence of hazardous toxic pollutants, such as heavy metals (HMs) and
polychlorinated biphenyls (PCBs). These pollutants are persisting and tend to bioaccumulate along the food-chain.
Effective and sustainable decontamination methods are hence highly needed. Based on the synergistic action
established between plant root system and soil rhizosphere microorganisms, Plant Assisted BioRemediation has
been proved to be efficient in restoring quality of contaminated soils [2, 3].
In this work, the purple non-sulfur bacterium Rhodobacter sphaeroides, a prokaryote able to convert sunlight into
other forms of energy by photosynthesis, was used as plant growth-promoting rhizobacteria. Due to its metabolic
versatility and ability to grow in presence of heavy metals [4, 5], R.sphaeroides can be exploited for environmental
applications, such as bioremediation of polluted sites.
Here, the effect on the growth of Arabidopsis thaliana in PCBs and HMs-contaminated soil from Taranto area,
inoculated with bacterial cells of the wild type 2.4.1 of R. sphaeroides was assessed. These preliminary results
obtained in growth chamber in controlled conditions pose the foundation for the development of a more sustainable
management system for soil bioremediation.

As the global population and living standards continue to rise, it is increasingly important to minimize theenvironmental impact of protein production. Microbial protein answers this challenge, in its simplest approach byusing the route of single–cell protein. An appealingpathis to produce such proteinaceous biomass without usingarable land or fossils, based mainly on carbon dioxideand renewably producedhydrogen gas[1]. While aerobicproduction on these resources(using hydrogen oxidizing bacteria)has been well explored with commercializationon the way, this is far less the case for the phototrophic route using purple bacteria, which enables a superiorhydrogen–to–protein conversion yield[2].This conference presentation will discuss the photoautohydrogenotrophic production of protein using purplebacteria, not only in flasks but also for the first time in a bioreactor(Figure 1). Nutritional quality parameters weredetermined for three species of purple bacteria, all of which demonstrated high protein content (up to 60% of dryweight)withexcellent dietary compatibility,anda fatty acid content dominated by vaccenic acid (82–86%).ARhodobacter capsulatusstrainexhibited the best nutritional and kinetic performance and was selected for growthin a bubble column photobioreactor, achieving a dry weight productivity of 0.8 gDW/L/d, which is higher thanpreviously reported in the literature for this growth mode.Semi–continuous photohydrogenotrophic production was achieved under non–axenicconditions for 32 days.Alternation between visible and infra–redlight was used as an operational tool to suppress microalgaecontamination whilst remaining agoodproductivityin the reactor.These findings suggest that purple bacteria are promising candidatesto sustainably produceprotein without theneed for arable land or fossil fuels. Further research will be necessary to fully explore the functional and structuralproperties of this biomass in food systems and realize its potentialto the fullest