Purple non-sulfur bacteria as cell factories to produce a copolymer as PHBV under light/dark cycle in a 4-L photobioreactor.

The present study reports a strategy to produce polyhydroxyalkanoates (PHAs) by culturing the marine bacterium Rhodovulum sulfidophilum DSM-1374. The study was carried out by growing the bacterium anaerobically for 720 h under 16/8 light/dark cycle. Two analytical techniques such as proton magnetic nuclear magnetic resonance (1H NMR) and Fourier transform infrared spectroscopy (FT-IR) were used to determine that the polyester produced was poly-3-hydroxybutirate-co-3-hydroxyvalerate (PHBV). This study showed that the excess of lactate and the limitation of N-P nutrients under a light-dark cycle enhanced PHBV synthesis and achieved a PHBV concentration of 330 mg/L in the R. sulfidophilum culture. During the 30 days of bacterial cultivation, the percentage of polymer in the six harvested dry biomasses gradually increased from 13.7% to 23.4%. In addition, the study showed that PHBV synthesis stopped during the 8-h dark phase and restarted in the light. The light-dark cycle study also showed that R. sulfidophilum DSM-1374 can be grown outdoors because the cells are exposed to the natural light-dark cycle.

Extraction of Polyhydroxyalkanoates from Purple Non-Sulfur Bacteria by Non-Chlorinated Solvents.

In this study, non-chlorinated solvents such as cyclohexanone (CYC) and three ionic liquids, (ILs) (1-ethyl-3-methylimidazolium dimethylphosphate, [EMIM][DMP], 1-ethyl-3-methylimidazolium diethylphosphate, [EMIM][DEP] and 1-ethyl-3-methylimidazolium methylphosphite, [EMIM][MP]) were tested to extract polyhydroxyalkanoates (PHAs) from the purple non-sulfur photosynthetic bacterium (PNSB) Rhodovulumsulfidophilum DSM-1374. The photosynthetic bacterium was cultured in a new generation photobioreactor with 4 L of working volume using a lactate-rich medium. The extracted PHAs were characterized using a thermogravimetric analysis, differential scanning calorimetry, infrared spectroscopy, proton nuclear magnetic resonance and gel permeation chromatography. The most promising results were obtained with CYC at 125 °C with an extraction time of above 10 min, obtaining extraction yields higher than 95% and a highly pure poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHB-HV) with around 2.7 mol% of hydroxylvalerate (HV). A similar yield and purity were obtained with chloroform (CHL) at 10 °C for 24 h, which was used as the referent solvent Although the three investigated ILs at 60 °C for 4 and 24 h with biomass/IL up to 1/30 (w/w) obtained PHAs strongly contaminated by cellular membrane residues, they were not completely solubilized by the investigated ILs.

Bioplastic production by feeding the marine Rhodovulum sulfidophilum DSM-1374 with four different carbon sources under batch, fed-batch and semi-continuous growth regimes.

In the present study, the ability of the marine bacterium Rhodovulum sulfidophilum DSM-1374 to convert, via photo-fermentative process, certain organic acids such as single carbon source (acetate, lactate, malate and succinate) into polyhydroxyalkanoate accumulations within bacterial cells is evaluated. The main goal of the investigation was poly-3-hydroxybutyrate (P3HB) synthesis by a photo-fermentative process. Of the four carbon sources, only succinate simultaneously produced P3HB and H2 (268 mg/L and 1085 mL/L respectively). Malate was the least productive source for P3HB; the other carbon sources (acetate and lactate) produced a significant amount of polymer (596 mg P3HB/L for acetate and 716 mg P3HB/L for lactate) when R. sulfidophilum was cultured in batch growth conditions. Cumulative P3HB increased significantly when the bacterium was grown under two steps: nutrient sufficient conditions (step 1) followed by macronutrient deficient conditions (step 2). The highest cumulative P3HB was observed at the end of step 2 (1000 mg/L) when R. sulfidophilum was fed with lactate under phosphorus starvation. When grown over 1200 h, under a semi-continuous regimen, the harvested dry-biomass reached a constant content of P3HB (39.1 ± 1.6 % of cell dry-weight), in the semi-steady state condition. Since lactate is an abundant byproduct of world industries, it can be used to mitigate the environmental impact in a modern circular bio-economy.

Production of Biohydrogen and/or Poly-ß-hydroxybutyrate by Rhodopseudomonas sp. Using Various Carbon Sources as Substrate.

The polyhydroxyalkanoates (PHA) are family of biopolyesters synthesized by numerous bacteria which are attracting a great attention due to their thermoplastic properties. Polyhydroxybutyrate (PHB) is the most common type of PHA which presents thermoplastic and biodegradable properties. It is synthesized under stressful conditions by heterotrophic bacteria and many photosynthetic microorganisms such as purple non-sulfur bacteria and cyanobacteria. Biological hydrogen (H2) production is being evaluated for use as a fuel since it is a promising substitute for carbonaceous fuels owing to its high conversion efficiency and high specific content. In the present work, the purple non-sulfur photosynthetic bacterium Rhodopseudomonas sp. for the simultaneous H2 photo-evolution and poly-ß-hydroxybutyrate (PHB) production has been investigated. Three different types of carbon sources were tested in the presence of glutamate as a nitrogen source in a batch cultivation system, under continuous irradiance. The results indicated the fact that the type of carbon source in the culture broth affects in various ways the metabolic activity of the bacterial biomass, as evidenced by the production of PHB and/or H2 and biomass. The best carbon source for PHB accumulation and H2 production by Rhodopseudomonas sp. turned out to be the acetate, having the highest H2 production (2286 mL/L) and PHB accumulation (68.99 mg/L, 18.28% of cell dry weight).

Effects of pH, temperature and salinity on P3HB synthesis culturing the marine Rhodovulum sulfidophilum DSM-1374.

Rhodovulum sulfidophilum DSM-1374 is a potential producer of polyester when growing in phototrophic conditions. The present study investigated on a polyester product (P3HB) by culturing Rhodovulum sulfidophilum DSM-1374 in two different photobioreactors (PBR-1 and PBR-2) both with 4-L working volumes. PBR-1 is equipped with an internal rotor having 4 paddles to mix the bacterial culture while PBR-2 has an internal coil-shaped rotor. After selecting PBR-1, which best performed in the preliminary experiment, the effect of different stressing growth conditions as pH (7.0, 8.0, and 9.0), temperature (25, 30, and 35 °C), and medium salinity (1.5, 2.5, 3.5, and 4.5%) were tested. When the pH of the culture was set to 8.0, the capability of the bacterium to synthetize the polyester increased significantly reaching a concentration of 412 mg (P3HB)/L; the increase of the pH at 9.0 caused a reduction of the P3HB concentration in the culture. The medium salinity of 4.5% was the best stress-growth condition to reach the highest concentration of polyester in the culture (820 ± 50 mg (P3HB)/L) with a P3HB mass fraction in the dry biomass of 33 ± 1.5%. Stresses caused by culture temperature are another potential parameter that could increase the synthesis of P3HB.

Whey and molasses as inexpensive raw materials for parallel production of biohydrogen and polyesters via a two-stage bioprocess: New routes towards a circular bioeconomy.

Consecutive dark-fermentation and photo-fermentation stages were investigated for a profitable circular bio-economy. H2 photo-production versus poly(3-hydroxybutyrate) (P3HB) accumulation is a modern biotechnological approach to use agro-food industrial byproducts as no-cost rich-nutrient medium in eco-sustainable biological processes. Whey and molasses are very important byproducts rich in nutrients that lactic acid bacteria can convert, by dark-fermentation, in dark fermented effluents of whey (DFEW) and molasses (DFEM). These effluents are proper media for culturing purple non-sulfur bacteria, which are profitable producers of P3HB and H2. The results of the present study show that Lactobacillus sp. and Rhodopseudomonas sp. S16-VOGS3 are two representative genera for mitigation of environmental impact. The highest productivity of P3HB (4.445 mg/(L·h)) was achieved culturing Rhodopseudomonas sp. S16-VOGS3, when feeding the bacterium with 20% of DFEM; the highest H2 production rate of 4.46 mL/(L·h) was achieved when feeding the bacterium with 30% of DFEM.

Poly-3-hydroxybutyrate and H2 production by Rhodopseudomonas sp. S16-VOGS3 grown in a new generation photobioreactor under single or combined nutrient deficiency.

The main goal of this investigation was setting up a growth strategy to separate H2 evolution from P3HB synthesis in order to increase cumulative P3HB in Rhodopseudomonas cells. The accumulation of poly-3-hydroxybutyrate (P3HB) was investigated culturing Rhodopseudomonas sp. S16-VOGS3 with three carbon substrates either as acetate, butyrate or lactate and with two nitrogen sources either as ammonium or glutamate. The investigation was carried out under several stress conditions caused by single or double nutrient deficiency. The content of P3HB in cell dry weight (CDW) was 21.8% with lactate; 24.6% with acetate and 27.6% with butyrate under sulfur deficient conditions. The P3HB content increased significantly culturing Rhodopseudomonas sp. S16-VOGS3 with butyrate following three phases of growth: phase-1, nutrient sufficient conditions; phase-2, nitrogen-deficiency and phase-3, sulfur-deficient conditions. Under this last phase, the highest P3HB content was achieved (34.4% of CDW). A combined production of P3HB and molecular H2 was obtained when Rhodopseudomonas sp. S16-VOGS3 was cultured with either acetate or butyrate under nitrogen sufficiency (glutamate) or nitrogen deficiency.

Hydroxytyrosol rich-mixture from olive mill wastewater and production of green products by feeding Rhodopseudomonas sp. S16-FVPT5 with the residual effluent.

This study disserts on the exploitation of olive mill wastewater (OMW) for the production of both bio-based poly-ß-hydroxybutyrate (PHB) and hydrogen (H2) by using the residual effluent as feedstock for growing purple bacteria after the recovery of hydroxytyrosol-rich mixtures. In particular, Rhodopseudomonas sp. S16-FVPT5 was fed with either the virgin OMW or dephenolized-OMW (d-OMW). For polyphenols removal, the OMW was treated with activated carbon; subsequently, acidified ethanol (pH = 3.1) at 50 °C was used as extractor solvent for obtaining hydroxytyrosol-rich mixtures. The maximum hydroxytyrosol content in the resultant polyphenolic mixture was 2.02 g/L. The highest co-production of PHB (315 mg PHB/L) and H2 (2236 mL H2/L) were achieved feeding Rhodopseudomonas sp. S16-FVPT5 with pure d-OMW. The highest hydrogen yield (4.55 L(H2)/Ld-OMW) was obtained feeding the bacterium with d-OMW, diluted at 25%; by increasing the content of d-OMW into the culture broth the hydrogen yield progressively decreased. Lower results were obtained by feeding the bacterium with a synthetic medium, the cumulative hydrogen was 1855 mL H2/L); the PHB was 101 mg PHB/L. The highest theoretical light conversion efficiency was 2.36% with the synthetic medium and 1.99% when feeding Rhodopseudomonas sp. S16-FVPT5 with d-OMW diluted with water 50%, v/v.

The aquatic fern Azolla as a natural plant-factory for ammonia removal from fish-breeding fresh wastewater.

This study has investigated the potential of an Azolla-Anabaena symbiosis, a marriage between the cyanobacterium Anabaena azollae and the aquatic fern (Azolla), to remove ammonia from freshwater fish breeding areas. Experiments were carried out under artificial light of 20, 70, and 140 µmol m(-2) s(-1). We investigated three different water temperatures for the growing Azolla, ranging from sub-optimal to optimal temperatures (15, 22, and 28 °C). The capability of Azolla to remove ammonia from wastewater was demonstrated, and the highest ammonia concentration tolerated by the symbiosis between Azolla-anabaena without any toxic effect on the aquatic ferns was ascertained. The shortest time taken to remove ammonia from wastes, 2.5 cm deep and at 28 °C, was 40 min. The ammonia removal rate (A RR) was both light and temperature dependent and the highest rate (6.394 h(-1)) was attained at light intensity of 140 µmol m(-2) s(-1) and at a temperature of 28 °C; the lowest (0.947 h(-1)) was achieved at 20 µmol m(-2) s(-1) and 15 °C. The depth of the fish-wastewater pool also affected the A RR with the relation between A RR and the depth being a hyperbolic function.

Dephenolization of stored olive-mill wastewater, using four different adsorbing matrices to attain a low-cost feedstock for hydrogen photo-production.

This investigation deals with the conversion of olive-mill wastewater (OMW) into several feedstocks suitable for hydrogen photo-production. The goal was reached by means of two sequential steps: (i) a pre-treatment process of stored-OMW for the removal of polyphenols, which made it possible to obtain several effluents, and (ii) a photo-fermentative process for hydrogen production by means of Rhodopseudomonas palustris sp. Four different adsorbent matrices (Azolla, granular active carbon, resin, and zeolite) were used to dephenolize stored-OMW. The four liquid fractions attained by using the above process created the same number of effluents, and these were diluted with water and then used for hydrogen photo-production. The maximum hydrogen production rate (14.31 mL/L/h) was attained with the photo-fermenter containing 25% of the effluent, which came from the pre-treatment of stored-OMW using granular active carbon. Using the carbon effluent as feedstock, the greatest light conversion efficiency of 2.29% was achieved.

Hydrogen photoproduction by Rhodopseudomonas palustris 42OL cultured at high irradiance under a semicontinuous regime.

The main goal of this study was to increase the hydrogen production rate improving the culture technique and the photobioreactor performances. Experiments were carried out at a constant culture temperature of 30°C and at an average irradiance of 480 W m(-2) using a cylindrical photobioreactor (4.0 cm, internal diameter). The culture technique, namely, the semicontinuous regime for growing Rhodopseudomonas palustris 42OL made it possible to achieve a very high daily hydrogen production rate of 594 ± 61 mL (H(2)) L(-1) d(-1). This value, never reported for this strain, corresponds to about 25 mL (H(2)) L(-1) h(-1), and it was obtained when the hydraulic retention time (HRT) was of 225 hours. Under the same growth conditions, a very high biomass production rate (496 ± 45 mg (dw) L(-1) d(-1)) was also achieved. Higher or lower HRTs caused a reduction in both the hydrogen and the biomass production rates. The malic-acid removal efficiency (MA(re)) was always higher than 90%. The maximal hydrogen yield was 3.03 mol H(2) mol MA(-1) at the HRT of 360 hours. The highest total energy conversion efficiency was achieved at the HRT of 225 hours.

The recovery of polyphenols from olive mill waste using two adsorbing vegetable matrices.

Olive mill wastewater (OMW) is considered one of the most pollutive waste materials in the Mediterranean basin. However, its phenolic fraction should be recovered, since it has been shown to have incredible benefits for health. In the present study, the adsorbent and desorbent capacities of Azolla and granular activated carbon (GAC) were investigated. The GAC was found to be more efficient than Azolla in both the adsorption and the desorption of phenols. The total characterization of two powder products obtained from Azolla and GAC desorption is reported, together with their antioxidant and antiradical activities. In the Azolla powder product, total polyphenols were more than twice as numerous as those found in the GAC powder product. The GAC powder contained hydroxytyrosol in concentrations that were 3.5 times higher than those of Azolla. On the other hand, both powder products showed great antiradical activities: the IC50 was found to be 102 mg ml⁻¹ for the Azolla and 199 mg ml⁻¹ for the GAC powders respectively. The oxygen radical absorbance capacity was very high: 4097 µmol TE g⁻¹ Azolla powder product and 1277 µmol TE g⁻¹ of GAC powder products.

Production of bio-fuels (hydrogen and lipids) through a photofermentation process.

The purple non-sulfur photosynthetic bacteria Rhodopseudomonas palustris (strain 42OL) was investigated for a co-production of both bio-H(2) and biodiesel (lipids). The investigation was carried out using malic and glutamic acids in a fed-batch cultivation system under continuous irradiances of 36, 56, 75, 151, 320, 500, and 803 W m(-2). Boltzmann's sigmoidal regression model was used to determine growth kinetic parameters during hydrogen photoevolution. The upper limit of volumetric hydrogen photoevolution was 15.5 + or - 0.9 ml l(-1) h(-1). During the entire cultivation period (408 h), the highest average hydrogen production rate (HPR(av)) of 11.1 + or - 3.1 ml l(-1) h(-1) was achieved at an irradiance of 320 W m(-2). Biomasses stored at the end of each experimental set were analyzed in order to determine lipid content, which ranged from a minimum of 22 + or - 1% to a maximum of 39 + or - 2% of biomass dry weight.

Green energy from Rhodopseudomonas palustris grown at low to high irradiance values, under fed-batch operational conditions.

Rhodopseudomonas palustris was grown under continuous irradiances of 36, 56, 75, 151, 320, 500, and 803 W m(-2), for a co-production of both bio-H(2) and biodiesel (lipids) using fed-batch conditions. The highest overall bio-H(2) produced [4.2 l(H(2)) l(culture) (-1)] was achieved at 320 W m(-2), while the highest dry biomass (3.18 g l(-1)) was attained at 500 W m(-2). Dry biomass contained between 22 and 39% lipid. The total energy conversion efficiency was at its highest (6.9%) at 36 W m(-2).

Growth characteristics of Rhodopseudomonas palustris cultured outdoors, in an underwater tubular photobioreactor, and investigation on photosynthetic efficiency.

The underwater tubular photobioreactor is a fully controlled outdoor system to study photosynthetic bacteria. Before growing bacteria cells outdoors, two modified van Niel medium (vN-A, vN-B) were tested under artificial light. During exponential growth, the specific growth rates were 0.0416 and 0.0434 h(-1), respectively; vN-B was chosen for outdoor experiments. The growth behavior of Rhodopseudomonas palustris was investigated under a natural light-dark cycle (sunrise-sunset, 15L/9D) and a forced light-dark cycle (9:00-19:00, 10L/14D). Relationships between solar radiations, daily growth rates, and biomass output rates were also investigated. After determining the elemental biomass molar composition and its combustion heat, some trends of photosynthetic efficiency (PE) were obtained over daylight. The PE trends were always of the oscillatory type, with the exception of that achieved at low biomass concentration. Under a natural light/dark cycle, the maximum PE (11.2%) was attained at sunset, while under a forced light/dark cycle, the highest PE (8.5%) was achieved in the morning. Three initial biomass concentrations were investigated (0.65, 1.01, and 1.54 g l(-1)). The stoichiometric equation for bacteria cells indicated that 87.7% of the carbon of acetic acid was converted to biomass and only 12.3% was lost as CO(2).

Hydrodynamic alterations during cyanobacteria (Arthrospira platensis) growth from low to high biomass concentration inside tubular photobioreactors.

The rheological behavior of an Arthrospira culture was studied from low to high biomass concentration. Two tubular undulating row photobioreactors (TURP-5r and TURP-10r), with a very short light path of 1.0 cm, were used during batch growth. In TURP-5r, the biomass concentration increased to 14.5 g(dw) L(-1), and alterations of the physical properties and hydrodynamic behavior occurred as a result. In the past, the rheological characteristics of photosynthetic-microbe cultures were rarely investigated because of the low biomass concentration attained in the systems. Developing closed photobioreactor technologies, the optimum biomass concentration rises and the viscosity, the generalized Reynolds number (N'(Re)), and the power required for culture recycling are also subject to alteration. Starting from a biomass concentration of 4.1 g(dw) L(-1), the Arthrospira culture already exhibits the characteristics of a non-Newtonian fluid. As a result of culture recycling from 2.0 to 20.5 g(dw) L(-1) and an available power of 1.67 W row(-1), we demonstrated that N'(Re) is reduced from 6265 to 1148. Our experimental results showed that N'(Re) of 2345 can be reached only at a cell concentration below 11.1 g(dw) L(-1), while at a cell concentration below 4.1 g(dw) L(-1) N'(Re) = 4080 was reached. The power consumption (P(c)) for culture recycling increased noticeably when the cell concentration rose; the highest P(c) increase attained was from 2.0 to 4.1 g(dw) L(-1). This is the range within which the Arthrospira culture changes from a Newtonian to a non-Newtonian fluid.

Growth characteristics of Arthrospira platensis cultured inside a new close-coil photobioreactor incorporating a mandrel to control culture temperature.

The aim of this study was to investigate Arthrospira growth inside a new CCP incorporating a mandrel for culture temperature control. Some hydrodynamic aspects and photobioreactor performances were investigated as well. The bioreactor incorporated A. platensis grown under batch and semicontinuous conditions. Two systems were used to recycle Arthrospira cultures: a peristaltic pump and an airlift system. When the pump recycled the culture, we achieved a very high Dean number (De=3,950), which decreased a great deal when the pump was replaced with the airlift system. During outdoor Arthrospira batch growth, a cell concentration of 16.4 g (DW)l-1 was reached after 9 days. However, the maximum chlorophyll content of the biomass (2.0% of DW) was achieved on the fifth and sixth days. The highest daily biomass output rate was obtained using the airlift system, when the CCP was operated under a semicontinuous regime: the gross output rate was 2.85+/-0.37 g (DW) l-1 d-1 and the net was 2.32+/-0.11 g (DW) l-1 d-1. The advantages of the airlift system may be due to the low concentration of oxygen built up inside Arthrospira culture and the lack of cell damage due to the pump system. Thus, oxygen and pump stress may have been avoided.

Dilution of solar radiation through "culture" lamination in photobioreactor rows facing south-north: a way to improve the efficiency of light utilization by cyanobacteria (Arthrospira platensis).

Efficient utilization of solar radiation for the photoautotrophic production of cyanobacterium biomass was achieved, using small pipes (ID = 0.01 m) arranged in rows in two photobioreactors facing south-north. A high Arthrospira yield of 47.7 g m(-2) (installation area) d(-1) was attained under outdoor conditions in a tubular undulating row photobioreactor (TURP-10r). During the summer, under a semicontinuous culture regime, the optimal biomass concentration (OBC) in TURP-5r was 6.0 g L(-1): it was 5.0 g L(-1) in TURP-10r. These OBCs made it possible to produce a biomass output rate of 2.7 +/- 0.2 g L(-1) d(-1) in the former and 2.1 +/- 0.1 g L(-1) d(-1) in the latter. When Arthrospira was grown at a preset dilution rate (0.3 d(-1)), sunrise cell density (SrCD) variations were not proportional to the drop of solar radiation. The SrCD was comparatively high at high solar radiation and decreased abruptly with decreasing solar radiation. There was a tendency to stabilize at low solar radiation. In both photobioreactors, the chlorophyll content of the Arthrospira biomass (% of the dry weight) was higher at sunrise than at sunset. A comparison of the chlorophyll biomass content in the TURPs showed no significant differences. Night biomass losses were very high (> 30% of the daylight productivity) when the culture temperature was kept constant at 31 +/- 1.0 degrees C: these losses fell to < 20% of the daylight productivity, when the night temperature of the cultures decreased according to the environmental temperature. Dilution of solar radiation was carried out using two quasi-laminated bioreactors. The rows of S-N facing bioreactors showed a very high growth yield in TURP-10r [about 2.1g (d.w.) MJ(-1)]. In TURP-10r, the high photic ratio (R(f) = 6), the high surface-to-volume ratio (S(ill)/V = 400 m(-1)) and the S-N facing of the rows (better than an E-W orientation) allowed for good results.