Continuous polyhydroxybutyrate production from biogas in an innovative two-stage bioreactor configuration

Biogas biorefineries have opened up new horizons beyond heat and electricity production in the anaerobic digestion sector. Added-value products such as polyhydroxyalkanoates (PHAs), which are environmentally benign and potential candidates to replace conventional plastics, can be generated from biogas. This work investigated the potential of an innovative two-stage growth-accumulation system for the continuous production of biogas-based polyhydroxybutyrate (PHB) using Methylocystis hirsuta CSC1 as cell factory. The system comprised two turbulent bioreactors in series to enhance methane and oxygen mass transfer: a continuous stirred tank reactor (CSTR) and a bubble column bioreactor (BCB) with internal gas recirculation. The CSTR was devoted to methanotrophic growth under nitrogen balanced growth conditions and the BCB targeted PHB production under nitrogen limiting conditions. Two different operational approaches under different nitrogen loading rates and dilution rates were investigated. A balanced nitrogen loading rate along with a dilution rate (D) of 0.3 day⁻¹ resulted in the most stable operating conditions and a PHB productivity of ~53 g PHB m⁻³ day⁻¹. However, higher PHB productivities (~127 g PHB m⁻³ day⁻¹) were achieved using nitrogen excess at a D = 0.2 day⁻¹. Overall, the high PHB contents (up to 48% w/w) obtained in the CSTR under theoretically nutrient balanced conditions and the poor process stability challenged the hypothetical advantages conferred by multistage vs single-stage process configurations for long-term PHB production.     

Process intensification strategies toward cell culture-based high-yield production of a fusogenic oncolytic virus

We present a proof-of-concept study for production of a recombinant vesicular stomatitis virus (rVSV)-based fusogenic oncolytic virus (OV), rVSV-Newcastle disease virus (NDV), at high cell densities (HCD). Based on comprehensive experiments in 1 L stirred tank reactors (STRs) in batch mode, first optimization studies at HCD were carried out in semi-perfusion in small-scale cultivations using shake flasks. Further, a perfusion process was established using an acoustic settler for cell retention. Growth, production yields, and process-related impurities were evaluated for three candidate cell lines (AGE1.CR, BHK-21, HEK293SF)infected at densities ranging from 15 to 30 × 10⁶ cells/mL. The acoustic settler allowed continuous harvesting of rVSV-NDV with high cell retention efficiencies (above 97%) and infectious virus titers (up to 2.4 × 10⁹ TCID₅₀/mL), more than 4–100 times higher than for optimized batch processes. No decrease in cell-specific virus yield (CSVY) was observed at HCD, regardless of the cell substrate. Taking into account the accumulated number of virions both from the harvest and bioreactor, a 15–30 fold increased volumetric virus productivity for AGE1.CR and HEK293SF was obtained compared to batch processes performed at the same scale. In contrast to all previous findings, formation of syncytia was observed at HCD for the suspension cells BHK 21 and HEK293SF. Oncolytic potency was not affected compared to production in batch mode. Overall, our study describes promising options for the establishment of perfusion processes for efficient large-scale manufacturing of fusogenic rVSV-NDV at HCD for all three candidate cell lines.     

The challenge of estimating kelp production in a turbid marine environment

Coastal kelp forests produce substantial marine carbon due to high annual net primary production (NPP) rates, but upscaling of NPP estimates over time and space remains difficult. We investigated the impact of variable underwater photosynthetically active radiation (PAR) and photosynthetic parameters on photosynthetic oxygen production of Laminaria hyperborea, the dominant NE-Atlantic kelp species, throughout summer 2014. Collection depth of kelp had no effect on chlorophyll a content, pointing to a high photoacclimation potential of L. hyperborea towards incident light. However, chlorophyll a and photosynthesis versus irradiance parameters differed significantly along the blade gradient when normalized to fresh mass, potentially introducing large uncertainties in NPP upscaling to whole thalli. Therefore, we recommend a normalization to kelp tissue area, which is stable over the blade gradient. Continuous PAR measurements revealed a highly variable underwater light climate at our study site (Helgoland, North Sea) in summer 2014, reflected by PAR attenuation coefficients (K_d) between 0.28 and 0.87 m⁻¹. Our data highlight the importance of continuous underwater light measurements or representative average values using a weighted K_d to account for large PAR variability in NPP calculations. Strong winds in August increased turbidity, resulting in a negative carbon balance at depths >3–4 m over several weeks, considerably impacting kelp productivity. Estimated daily summer NPP over all four depths was 1.48 ± 0.97 g C · m⁻² seafloor · d⁻¹ for the Helgolandic kelp forest, which is in the range of other kelp forests along European coastlines.     

Canopy photosynthetic production in a Japanese larch forest. II. Estimation of the canopy photosynthetic production

Seasonal changes and yearly gross canopy photosynthetic production were estimated for an 18 year old Japanese larch (Larix leptolepis) forest between 1982 and 1984. A canopy photosynthesis model was applied for the estimation, which took into account the effect of light interception by the non-photosynthetic organs. Seasonal changes in photosynthetic ability, amount of canopy leaf area and light environment within the canopy were also taken into account. Amount of leaf area was estimated by the leaf area growth of a single leaf. The change of light environment within the canopy during the growing season was estimated with a light penetration model and the leaf increment within the canopy. Canopy respiration and surplus production were calculated as seasonal and yearly values for the three years studied. Mean yearly estimates of canopy photosynthesis, canopy respiration and surplus production were 37, 13 and 23 tCO₂ ha⁻¹ year⁻¹, respectively. Vertical trend, seasonal changes and yearly values of the estimates were analyzed in relation to environmental and stand factors.     

The effects of grazing on the pollen production of grasses

Samples from moss polsters taken over three consecutive years during a controlled grazing experiment in a nature reserve in the Netherlands were analysed for their pollen content, particularly grass pollen. Samples were also taken in the year following the experiment, with grazing pressure strongly diminished. This yielded data regarding the effect of grazing on the pollen production of grasses in open habitats, plantations, and unproductive and productive woods under different grazing pressures.     

Environmental conditions during glycerol bioconversion affect 3-hydroxypropionic acid bioproduction by Limosilactobacillus reuteri DSM 17938

3-Hydroxypropionic acid (3-HP) is a platform molecule whose biological production was carried out by the bacterium Limosilactobacillus reuteri according to a two-step process: first, a growth phase in batch mode on glucose, then a glycerol bioconversion into 3-HP in fed-batch mode. With the objective of improving 3-HP bioproduction, this study aimed at defining the operating conditions during the bioconversion phase that increases the bioproduction performance. A central composite rotatable design allowed testing various pH levels and specific glycerol feeding rates. By establishing response surfaces, optimal conditions have been identified that were different depending on the considered output variable (final 3-HP quantity, 3-HP production yield and production rate). Of them, 3-HP final quantity and 3-HP production yield were maximized at pH 6.0 and at specific glycerol feeding rates of 60 and 55 mg_gly g_CDW⁻¹ h⁻¹, respectively. The specific 3-HP production rate was the highest at the upper limit of the specific substrate feeding rate (80 mg_gly g_CDW⁻¹ h⁻¹) but was not affected by the pH. An additional experiment was carried out at pH 6.0 and a specific glycerol feeding rate of 80 mg_gly g_CDW⁻¹ h⁻¹ to validate the previous observations. In conclusion, the results showed a significant improvement of 3-HP concentration by 13%, of specific production rate by 34% and of 3-HP volumetric productivity by 39%, as compared to the initial values.     

The effect of charcoal production on carbon cycling in African biomes

Using biomass for charcoal production in sub-Saharan Africa (SSA) may change carbon stock dynamics and lead to irreversible changes in the carbon balance, yet we have little understanding of whether these dynamics vary by biome in this region. Currently, charcoal production contributes up to 7% of yearly deforestation in tropical regions, with carbon emissions corresponding to 71.2 million tonnes of CO₂ and 1.3 million tonnes of CH₄. With a projected increased demand for charcoal in the coming decades, even low harvest rates may throw the carbon budget off-balance due to legacy effects. Here, we parameterized the dynamic global vegetation model LPJ-GUESS for six SSA biomes and examined the effect of charcoal production on net ecosystem exchange (NEE), carbon stock sizes and recovery time for tropical rain forest, montane forest, moist savanna, dry savanna, temperate grassland and semi-desert. Under historical charcoal regimes, tropical rain forests and montane forests transitioned from net carbon sinks to net sources, that is, mean cumulative NEE from −3.56 ± 2.59 kg C/m² to 2.46 ± 3.43 kg C/m² and −2.73 ± 2.80 kg C/m² to 1.87 ± 4.94 kg C/m² respectively. Varying charcoal production intensities resulted in tropical rain forests showing at least two times higher carbon losses than the other biomes. Biome recovery time varied by carbon stock, with tropical and montane forests taking about 10 times longer than the fast recovery observed for semi-desert and temperate grasslands. Our findings show that high biomass biomes are disproportionately affected by biomass harvesting for charcoal, and even low harvesting rates strongly affect vegetation and litter carbon and their contribution to the carbon budget. Therefore, the prolonged biome recoveries imply that current charcoal production practices in SSA are not sustainable, especially in tropical rain forests and montane forests, where we observe longer recovery for vegetation and litter carbon stocks.     

Nitrogen and energy losses and methane emissions from beef cattle fed diets with gradual replacement of maize silage and concentrate with grass silage and corn-cob mix

Diets based on large proportions of grassland-based feed are uncommon in forage-based intensive beef production, thus contradicting governmental or commercial strategies to promote the use of grassland-based feed in ruminant production systems. Compared with typical maize silage/concentrate diets, grassland-based diets are associated with impaired nitrogen (N) and energy utilisation because of the comparably lower energy and higher CP content of these feeds. However, quantitative studies concerning the effects of increased dietary proportions of grassland-derived feeds on N and energy losses and utilisation and on methane emissions are missing and the compensation potential of using a limited proportion of an energy-rich forage is unknown. Therefore, we tested five diets with varying types and proportions of forage and concentrate. Three diets consisted of grass silage, maize silage, and concentrate in ratios of, g/kg DM, 100:600:300 (G100; control), 300:500:200 (G300), and 500:300:200 (G500), respectively. Two diets were composed of grass silage, corn-cob mix (CCM), and concentrate in ratios of, g/kg DM, 500:300:200 (G500_CCM), and 750:150:100 (G750_CCM), respectively. A high-protein concentrate (270 g CP/kg DM) was fed to G100, whereas a low-protein concentrate (140 g CP/kg DM) was used in the remaining diets. Diets were fed throughout the entire fattening period to groups of six Limousin-crossbred bulls each. When weighing 246 ± 18 kg, each animal underwent a 7-day total daily faeces and urine collection, which was followed by measuring methane emissions in respiration chambers for 48 h. Total DM intake was similar across all diets, whereas the N intake varied (P < 0.05). Urinary N loss (g/day) was the highest for G750_CCM (28.2) and G100 (26.6) and lowest for G500_CCM (15.2) and G300 (16.9) (P < 0.001). Energy utilisation was comparable among all groups. Metabolisable energy intake decreased numerically only with increasing proportions of grass silage in the diet. Substituting maize silage with CCM counteracted the loss in metabolisable energy intake. Absolute methane emissions were not different across the groups, but methane emission intensity (mg/g body protein retention) varied (P < 0.05), being numerically lower for G100 (349) and G500_CCM (401) compared with the other groups (488 on average). In conclusion, the results show that the grass silage proportion in beef cattle diets can be substantially increased when strategically combined with energy-dense forages, such as CCM. This also limits the need for concentrate and additional protein sources; in addition, the associated urinary N emissions, which are potentially noxious to the environment, are avoided.