Dark Fermentation Effluent Research Articles

Mixotrophic cultivation of microalgae-bacteria consortia enhances dark fermentation effluent treatment

Dark fermentation (DF) is a waste treatment bioprocess which produces biohydrogen and volatile fatty acids (VFAs) such as acetate or butyrate. DF can be coupled with microalgae cultivation, allowing VFA conversion into valuable biomass. Nevertheless, the process is hindered by slow butyrate consumption. In this study, novel artificial microalgae-bacteria consortia were used as a strategy to accelerate butyrate removal. Three microalgal strains with various trophic metabolisms, Chlorella sorokiniana, Euglena gracilis and Ochromonas danica, were cultivated on DF effluent that was either sterile or contained endogenous bacteria. Bacteria did not impact microalgal biomass production of C. sorokiniana or E. gracilis while accelerating butyrate removal rates 2 to 10-fold. O. danica greatly impacted microbial diversity, probably due to its phagotrophic metabolism. These results show that bacteria in organic rich effluents can greatly aid in substrate removal while allowing microalgal growth, inspiring bioprocesses coupling raw fermentation effluents with microalgae biomass production and valorization.

A dual-chamber Microbial Electrolysis Cell for electromethanosynthesis from the effluent of cheese whey dark fermentation

Dark fermentation of cheese whey (CW) for the production of biohydrogen generates an acidic effluent, containing high concentrations of volatile fatty acids, which needs to be further treated before disposal and possibly further valorised. This study develops a dual-chamber Microbial Electrolysis Cell (MEC) that achieves simultaneously the reduction of the organic content of this effluent to environmentally acceptable levels, along with bio-electrochemical reduction of CO2 to CH4. The MEC was operated for 140 days and the effect of the following conditions on the MEC performance was examined: (a) the feed concentration of the acidic fermentate (in the range 6–81 gCOD/L), (b) the conductivity of the feed modified via KCl addition (range 2–22 mS/cm), (c) the MEC operation mode (with or without catholyte renewal) and (d) the solids content (modified via CW filtration prior to its use). The results showed that high COD removal (>95 %) was achieved in all cases, along with a CH4 production of up to 1.1 mmol/gCODconsumed. The best performance of the cell was obtained for a feed COD concentration of ∼30 gCOD/L and a feed conductivity of ∼15 mS/cm; these conditions resulted in a COD removal exceeding 99 %, a CH4 production of 1.1 mmolCH4/gCODconsumed and a net energy production of 15.8 % compared to the energy demand of the system. The electrochemical study of the system revealed that higher and lower feed COD concentrations were characterized by higher internal resistances. The results indicate that the MEC can be exploited for further treatment and valorization of a high-strength effluent along with the production of CH4 with an energy surplus, as an efficient waste-to-energy technology.

Investigating the efficiency of a two-stage anaerobic-aerobic process for the treatment of confectionery industry wastewaters with simultaneous production of biohydrogen and polyhydroxyalkanoates.

The scope of the current study was to investigate the efficiency of a two-stage anaerobic-aerobic process for the simultaneous treatment and valorization of selective wastewater streams from a confectionary industry. The specific wastewater (confectionary industry wastewater, CIW) was a mixture of the rinsing eluting during washing of the cauldrons in which jellies and syrups were produced, and contained mainly readily fermentable sugars, being thus of high organic load. The first stage of the process was the dark fermentation (DF) of the CIW in continuous, attached-biomass systems, in which the effect on hydrogen yields and distribution of metabolites were studied for different packing materials (ceramic or plastic), hydraulic retention times, HRTs (12 h–30 h) and feed substrate concentration (20 g COD/L- 50 g COD/L). In the second stage, the effectiveness of the aerobic treatment of the DF effluents was evaluated in terms of the reduction of the organic load and the production of polyhydroxyalkanoates (PHAs) through an enriched mixed microbial culture (MMC). The MMC was developed in a continuous draw and fill system, in which the accumulation potential of PHAs was studied. It was shown that the hydrogen production rates decreased for increasing substrate concentration and HRTs, with a maximum of 12.70 ± 0.35 m3 H2/m3 initial CIW achieved for the lowest HRT and feed concentration and using ceramic beads as packing material. Butyrate, acetate and lactate were the main metabolites generated in all cases, in different ratios. The distribution of metabolites during DF was shown to highly affect the efficiency of the second process in terms of both the reduction of organic load and the PHAs yields. The highest removal of organic load achieved after 48 h of aerobic treatment was 84.0 ± 0.9 %, whereas the maximum PHAs yield was 21.46 ± 0.13 kg PHAs/m3 initial CIW.

Combining pretreatments and co-fermentation as successful approach to improve biohydrogen production from dairy cow manure

The present paper is focused on enhancing the production of biohydrogen (bioH2) from dairy cow manure (DCM) through dark fermentation (DF). Two enhancement production strategies have been tested: i) the combination of H2O2 with sonification as pretreatment and ii) the co-fermentation with cheese whey as co-substrate. Concerning the pretreatment, the best combination was investigated according to the response surface methodology (RSM) by varying H2O2 dosage between 0.0015 and 0.06 g/gTS and ultrasonic specific energy input (USEI) between 35.48 and 1419.36 J/gTS. The increase of carbohydrates concentration was used as target parameter. Results showed that the combination of 0.06 g/gTS of H2O2 with 1419.36 J/gTS of USEI maximized the concentration of carbohydrates. The optimized conditions were used to pretreat the substrate prior conducting DF tests. The use of pretreatment resulted in obtaining a cumulative bioH2 volume of 51.25 mL/L and enhanced the bioH2 production by 125% compared to the control test conducted using raw DCM. Moreover, the second strategy, i.e. co-fermentation with cheese whey (20% v/v) as co-substrate ended up to enhancing the DF performance as the bioH2 production reached a value of 334.90 mL/L with an increase of 1372% compared to the control DF test. To further improve the process, dark fermentation effluents (DFEs) were valorized via photo fermentation (PF), obtaining an additional hydrogen production aliquot.

Enrichment of a Mixed Culture of Purple Non-Sulfur Bacteria for Hydrogen Production from Organic Acids

Hydrogen (H2) as a clean fuel holds global potential and can be produced through bio-processes. To enhance bioH2 yields, integrated systems have been proposed, combining dark fermentation (DF) of wastewater with a subsequent photofermentation (PF) stage involving purple non-sulfur (PNS) bacteria. Mixed cultures of PNS bacteria and their microbial ecology have been relatively understudied despite the known benefits of mixed cultures in industrial applications. The aim of this study was to obtain various mixed cultures of PNS bacteria under different environmental conditions during the enrichment stage. Four different mixed cultures were obtained (A, B, C, and D). However, in the H2 production phase, only Consortium A, which had been enriched with malic acid as the carbon source, exposed to 32 W m−2 of irradiance, and subjected to intermittent agitation, produced H2 with a yield of 9.37 mmol H2 g−1 COD. The consortia enriched were a hybrid of PF and DF bacteria. Especially in Consortium A, Rhodopseudomonas palustris was the dominant organism, and various DF bacteria were positively associated with H2 production, with their dominance comparable to that of PNS bacteria. Despite the reported low yields, optimizing environmental conditions for this culture could potentially enhance hydrogen production from DF effluents.

Comparative study of exoelectrogenic utilization preferences and hydrogen conversion among major fermentation products in microbial electrolysis cells

This study comparatively investigated the exoelectrogenic utilization and hydrogen conversion of major dark fermentation products (acetate, propionate, butyrate, lactate, and ethanol) from organic wastes in dual-chamber microbial electrolysis cells (MECs) alongside their mixture as a simulated dark fermentation effluent (DFE). Acetate-fed MECs showed the highest hydrogen yield (1,465 mL/g chemical oxygen demand), near the theoretical maximum yield, with the highest coulombic efficiency (105%) and maximum current density (7.9 A/m2), followed by lactate-fed, propionate-fed, butyrate-fed, mixture-fed, and ethanol-fed MECs. Meanwhile, the highest hydrogen production rate (514 mL/L anolyte∙d) was observed in ethanol-fed MECs despite their lower coulombic efficiency. Butyrate was the least favored substrate, followed by propionate, leading to significantly delayed startup and reaction. The active anodic microbial community structure varied considerably among the MECs utilizing different substrates, particularly between Geobacter and Acetobacterium dominance. The results highlight the substantial effect of the DFE composition on its utilization and current-producing bioanode development.

Enhanced hydrogen and methane production from date fruit wastes using semi continuous two-stage anaerobic digestion process with increasing organic loading rates

Biological conversion of date wastes under continuous conditions is a challenge due to their high content of biodegradable organic matter. In this study, hydrogen (H2) and methane (CH4) production in a two-stage process to increase the energy recovery from date pulp wastes (DPW) was investigated. In the first stage, the effect of increasing organic leading rate (OLR) on dark fermentation (DF) performances was evaluated in a semi continuously stirred tank reactors (SCSTR) without nutrients addition. The highest volumetric H2 production rate (VHPR) and specific H2 yield (SHY) of 0.76 L/L.d and 81 LH2/kg volatile solid (VS), respectively were achieved at high OLR of 11.74 gVS/L.d. In the second stage, the DF effluent was used as substrate to feed the SCSTR of anaerobic digestion (AD). The highest volumetric methane production rate (VMPR) of 2.92 L/L.d was achieved with a high OLR (4.25 gVS/L.d) and short hydraulic retention time (HRT, 8 days), producing specific methane yield (SMY) of 530 LCH4/kgVS. Thus, the two-stage process can be an effective strategy for high rate renewable energy recovery (13 kJ/gVSinlet) and organic waste processing.

Sequential dark fermentation and microbial electrolysis cells for hydrogen production: Volatile fatty acids influence and energy considerations

Hydrogen production from food waste by coupling dark fermentation (DF) and microbial electrolysis cells (MEC) was studied. Metabolic patterns in DF, their effects on MECs efficiency, and the energy output of the coupling were investigated. Mesophilic temperature and acidic pH 5.5 resulted in 72 ± 20 mL H2/g CODin and a butyrate-enriched profile (C2/C4, 0.5–0.6) contrasting with an acetate-enriched profile (C2/C4, 1.8–1.9) and 36 ± 10 mL H2/g CODin at pH 7. Assessment in series of the DF effluents in MECs resulted in a higher hydrogen yield (566–733 mL H2/g CODin) and volatile fatty acids (VFAs) removal (84–95%) obtained from pH 7 effluents compared to pH 5.5 effluents (173–186 mL H2/g CODin and 29–59%). Finally, the output energy was lower in DF at pH 7, however, these effluents retrieved the highest energy in the MEC, showing the importance of process pH and VFAs profile to balance the coupling.

Utilization of dark fermentation effluent for algal cultivation in a modified airlift photobioreactor for biomass and biocrude production

Developing an efficient photobioreactor (PBR) and reducing freshwater dependence are among the significant challenges for generating 3rd generation biomass feedstock. Addressing these, the present study focused on developing a modified airlift (MoAL) PBR. Its performance was further evaluated and compared with the traditional airlift PBR by cultivating microalgae in dark fermentation spent wash. Lower mixing time and higher interfacial mass transfer coefficient was observed in the MoAL PBR having a perforated draft tube. Experimentally, the MoAL exhibited the maximum biomass concentration of 3.18 g L−1, which was 30% higher than that of the conventional airlift PBR. The semi-continuous operation of the MoAL (with water recycling) achieved the maximum biomass productivity of 0.83 g L−1 d−1, two folds superior to that of batch culture. The comprehensive biomass characterization (proximate, ultimate, and thermochemical) further confirmed its potential for bioenergy application. Considering that, hydrothermal liquefaction of the biomass resulted in a maximum biocrude yield of 31% w/w with a higher heating value (HHV) of 36.6 MJ kg−1. In addition, the biocrude comprised 66.6% w/w lighter fraction (<343 °C), including 21.5% w/w of heavy naphtha, 20.5% w/w of kerosene, and 24.6% w/w of diesel. The results can help develop sustainable technology for simultaneous wastewater remediation and biocrude production.

Maximizing performance of microbial electrolysis cell fed with dark fermentation effluent from water hyacinth

The performance of microbial electrolysis cell (MEC) fed with dark fermentation effluent (DEF) from water hyacinth (WH) was enhanced in this study. First, the single effects of the auxiliary processes, including centrifugation, dilution, buffering, and external power input, were investigated. Then, the interaction of these processes was further evaluated using response surface methodology (RSM) and a combination of artificial neural network (ANN) and particle swarm optimization (PSO). Statistical analysis results revealed that ANN-PSO outperformed RSM in predictability. Consequently, the ANN-PSO approach determined that a 2.2-fold dilution of centrifuged-DFE (∼1.64 g of soluble metabolite products per L), buffer concentration of 75 mM, and an applied voltage of 0.7 V were the optimal conditions for simultaneously maximizing H2 production yield and energy efficiency of DFE@WH-fed MEC. Under co-optimized conditions, H2 yield (560.8 ± 10.8 mL/g-VS) and electrical energy recovery (162.2 ± 4.7%) significantly improved compared to unoptimized conditions.

Simultaneous Production of Biohydrogen (bioH2) and Poly-Hydroxy-Alkanoates (PHAs) by a Photoheterotrophic Consortium Bioaugmented with Syntrophomonas wolfei

Mixed cultures represent better alternatives to ferment organic waste and dark fermentation products in anerobic conditions because the microbial associations contribute to electron transfer mechanisms and combine metabolic possibilities. The understanding of the microbial interactions in natural and synthetic consortia and the strategies to improve the performance of the processes by bioaugmentation provide insight into the physiology and ecology of the mixed cultures used for biotechnological purposes. Here, synthetic microbial communities were built from three hydrogen (bioH2) and poly-hydroxy-alkanoates (PHA) producers, Clostridium pasteurianum, Rhodopseudomonas palustris and Syntrophomonas wolfei, and a photoheterotrophic mixed consortium C4, and their performance was evaluated during photofermentation. Higher hydrogen volumetric production rates (H2VPR) were determined with the consortia (28–40 mL/Lh) as compared with individual strains (20–27 mL/Lh). The designed consortia reached the highest bioH2 and PHA productions of 44.3 mmol and 50.46% and produced both metabolites simultaneously using dark fermentation effluents composed of a mixture of lactic, butyric, acetic, and propionic acids. When the mixed culture C4 was bioaugmented with S. wolfei, the bioH2 and PHA production reached 32 mmol and 50%, respectively. Overall, the consumption of organic acids was above 50%, which accounted up to 55% of total chemical oxygen demand (COD) removed. Increased bioH2 was observed in the condition when S. wolfei was added as the bioaugmentation agent, reaching up to 562 mL of H2 produced per gram of COD. The enhanced production of bioH2 and PHA can be explained by the metabolic interaction between the three selected strains, which likely include thermodynamic equilibrium, the assimilation of organic acids via beta-oxidation, and the production of bioH2 using a proton driving force derived from reduced menaquinone or via electron bifurcation.

Integration of Dark Fermentation with Microbial Electrolysis Cells for Biohydrogen and Methane Production from Distillery Wastewater and Glycerol Waste Co-Digestion

This study aimed to enhance dark fermentative hydrogen production from co-digestion of distillery wastewater (DW) and glycerol waste (GW) through integration with microbial electrolysis cells. First, the optimal proportion of DW and GW in hydrogen production was investigated in batch mode. The results show that DW and GW co-digestion at a ratio of 99:1 (% v/v) gave the highest hydrogen yield of 149.5 mL-H2/g − VSadded. Continuous hydrogen production using the optimal proportion was conducted in a continuously stirred tank reactor. As a result, a maximal hydrogen yield of 99.7 mL-H2/g − VSadded was achieved, and the dominant hydrogen-producing bacterium was Clostridium sensu stricto 7. The dark fermentation effluent from the continuously stirred tank reactor was later used to produce methane using batch MECs. The maximum methane yield of 115.1 mL-CH4/g − VSadded was obtained under an applied voltage of 1 V and continuous stirring at 120–140 rpm. Microbial community analysis revealed that Metahnobacterium, Methanomethylovorans, Methanoculleus, and Methanosarcina were the methanogenic archaea in the microbial electrolysis cell reactor.

Combined biohydrogen and polyhydroxyalkanoates production from sheep cheese whey by a mixed microbial culture

The present study investigates the combined production of biohydrogen and polyhydroxyalkanoates (PHA) from sheep cheese whey through a 3-stage bioprocess, i.e. dark fermentation, selection of PHA storing microorganisms, and PHA accumulation. Batch dark fermentation tests (Stage I) were performed on raw cheese whey under different pH operating conditions, avoiding either the addition of inoculum or substrate pre-treatment to support the economic and technical feasibility of the proposed process. The performance of the fermentative stage was assessed in terms of biohydrogen and soluble metabolites production yields. The dark fermentation effluent was used as organic acid-rich feedstock either for selecting and harvesting PHA storing microorganisms from a mixed microbial culture without the addition of external nutrient sources (Stage II) or for the PHA accumulation by the selected biomass (Stage III). The results of the study support the possibility of achieving combined recovery yields of 5.3 L biohydrogen and 7.6 g PHA per litre of fed sheep cheese whey in the case of optimal dark fermentation pH setting (pH = 6). Such outcomes underline the untapped potential of sheep cheese whey for the recovery of high-added value bioproducts.

Optimizing the production of docosahexaenoic fatty acid by Crypthecodinium cohnii and reduction in process cost by using a dark fermentation effluent

Recovery of omega-3 fatty acids (FAs) from microalgae constitutes a valuable alternative to fish oil, and offers the ability to isolate a single ω-3 FA, instead of a mixture. In the framework of the concept of circular economy and sustainability, in the current work, an alternative carbon source was used as feed for oleaginous microalga Crypthecodinium cohnii cultivation and, consequently, production of the omega-3 FA, docosahexaenoic acid (DHA). The conditions that improve DHA production in a fed-batch culture of C. cohnii, using an organic acid-rich liquid fraction of dark fermented biowaste as feed, were studied and optimized. The process was mathematically approached focusing on a proper Design of Experiments (DoE) method coupled with Response Surface Methodology (RSM). The temperature of 23 °C and the use of yeast extract (YE) as initial nitrogen source favored the amount of DHA recovered. In addition, the increase of the volatile FA (VFA) concentration of the feed enhanced the recovered cell biomass. In order to propose a final fermentation protocol with industrial potential and evaluate the annual total cost for the production of DHA, fermentation process was scaled-up and economically evaluated through cost analysis. The increase of bioreactor size from 1 to 20 m3 leads to a significant decrease in the cost of produced DHA while leveling at higher capacities.

Enhanced hydrogen production from water hyacinth by a combination of ultrasonic-assisted alkaline pretreatment, dark fermentation, and microbial electrolysis cell

In this study, hydrogen (H2) production from water hyacinth (WH) was enhanced by the integration of the ultrasonic-assisted alkaline (UAA) pretreatment, dark fermentation (DF), and microbial electrolysis cell (MEC). The results showed that UAA pretreatment improved around 350% in H2 production in the DF stage and nearly 400% in the whole process compared to un-pretreated. The H2 yield in the DF stage reached the maximum value of 110.4 mL/g-VS at a WH concentration of 20 g-TS/L. However, high concentrations of co-produced soluble metabolite products (SMPs) and suspended solid in DF effluent adversely affected the efficiency of the MEC stage. Consequently, a WH concentration of 5 g-TS/L was optimal for the UAA-DF-MEC process that achieved the highest H2 yield of 565.8 mL/g-VS. It suggests that other auxiliary processes (e.g., dilution, centrifugation, effective methanogen inhibition, etc.) need to be developed to further improve the H2 production from WH via the UAA-DF-MEC process.

High current density via direct electron transfer by hyperthermophilic archaeon, Geoglobus acetivorans, in microbial electrolysis cells operated at 80 °C

Utilization of hyperthermophilic electro-active microorganisms in microbial electrolysis cells (MECs) that are used for hydrogen production from organic wastes offers significant advantages, such as increased reaction rate and enhanced degradation of insoluble materials. However, only a limited number of hyperthermophilic bioelectrochemical systems have been investigated so far. This study is the first to illustrate hydrogen production in hyperthermophilic MECs with a maximum rate of 0.57 ± 0.06 m3 H2/m3d, where an iron reducing archaeon, Geoglobus acetivorans, was used as inoculum. In fact, this is the first study to report that G. acetivorans, as the fourth hyperthermophilic electro-active archaeon. In single chamber MECs operated at 80 °C with a set potential of 0.7 V, a peak current density of 1.53 ± 0.24 A/m2 has been attained and this is the highest record of current produced by pure culture hyperthermophilic microorganisms. Turnover cyclic voltammetry curve illustrated a sigmoidal shape (midpoint of −0.40 V vs. Ag/AgCl), and together with linear relation of scan rate and peak anodic current, proves the biofilm attachment to the anode and its capability of direct electron transfer. Along with simple substrate (acetate), G. acetivorans effectively utilized dark fermentation effluent for hydrogen production in MECs.

The Effect of Anode Material on the Performance of a Hydrogen Producing Microbial Electrolysis Cell, Operating with Synthetic and Real Wastewaters

The aim of the study was to assess the effect of anode materials, namely a carbon nanotube (CNT)-buckypaper and a commercial carbon paper (CP) on the performance of a two-chamber microbial electrolysis cell (MEC), in terms of hydrogen production and main electrochemical characteristics. The experiments were performed using both acetate-based synthetic wastewater and real wastewater, specifically the effluent of a dark fermentative hydrogenogenic reactor (fermentation effluent), using cheese whey (CW) as substrate. The results showed that CP led to higher hydrogen production efficiency and current density compared to the CNT-buckypaper anode, which was attributed to the better colonization of the CP electrode with electroactive microorganisms, due to the negative effects of CNT-based materials on the bacteria metabolism. By using the fermentation effluent as substrate, a two-stage process is developed, where dark fermentation (DF) of CW for hydrogen production occurs in the first step, while the DF effluent is used as substrate in the MEC, in the second step, to further increase hydrogen production. By coupling DF-MEC, a dual environmental benefit is provided, combining sustainable bioenergy generation together with wastewater treatment, a fact that is also reinforced by the toxicity data of the current study.

Dark fermentative biohydrogen production using pretreated Scenedesmus obliquus biomass under an integrated paradigm of biorefinery

In recent times, biohydrogen production from microalgal feedstock has garnered considerable research interests to sustainably replace the fossil fuels. The present work adapted an integrated approach of utilizing deoiled Scenedesmus obliquus biomass as feedstock for biohydrogen production and valorization of dark fermentation (DF) effluent via biomethanation. The microalgae was cultivated under different CO2 concentration. CO2-air sparging of 5% v/v supported maximum microalgal growth and carbohydrate production with CO2 fixation ability of 727.7 mg L−1 d−1. Thereafter, lipid present in microalgae was extracted for biodiesel production and the deoiled microalgal biomass (DMB) was subjected to different pretreatment techniques to maximize the carbohydrate recovery and biohydrogen yield. Steam heating (121 °C) in coherence with H2SO4 (0.5 N) documented highest carbohydrate recovery of 87.5%. DF of acid-thermal pretreated DMB resulted in maximum H2 yield of 97.6 mL g−1 VS which was almost 10 times higher as compared to untreated DMB (9.8 mL g−1 VS). Subsequent utilization of DF effluent in biomethanation process resulted in cumulative methane production of 1060 mL L−1. The total substrate energy recovered from integrated biofuel production system was 30%. The present study envisages a microalgal biorefinery to produce biohydrogen via DF coupled with concomitant CO2 sequestration.

Enhancing photo fermentative hydrogen production using ethanol rich dark fermentation effluents

The present study demonstrates the feasibility of a two-phase biorefinery process applied to waste substrates producing ethanol rich effluents. The process includes a dark fermentation step followed by photo fermentation and it is able to optimize hydrogen production from waste biomass. The study was conducted using winery wastewater as feedstock. The results indicate that no additional treatments are required when an appropriate dilution of the initial waste is applied. Microbial consortia contained in the winery wastewater promoted a fermentative ethanol pathway. The ethanol rich effluent was converted into hydrogen by phototrophic microorganisms. Despite the presence of inhibiting compounds, the adoption of a mixed phototrophic culture allowed to obtain good results in terms of hydrogen production. Specifically, up to 310 mLH2 gCODconsumed−1 were obtained in the photo fermentative stage. The effectiveness of ethanol rich dark fermentation effluents for hydrogen production enhancement was demonstrated. Noteworthy, polyhydroxybutyrate was also produced during the experiments. The work faces two of the major challenges in the sequential dark fermentation and photo fermentation technology applied to real waste substrates: the minimization of pre-treatments and the enhancement of the hydrogen production yields using ethanol rich DFEs.

Mixotrophic Growth of Chlorella sorokiniana on Acetate and Butyrate: Interplay Between Substrate, C:N Ratio and pH.

Microalgae can be cultivated on waste dark fermentation effluents containing volatile fatty acids (VFA) such as acetate or butyrate. These VFA can however inhibit microalgae growth at concentrations above 0.5-1 gC.L–1. This study used the model strain Chlorella sorokiniana to investigate the effects of acetate or butyrate concentration on biomass growth rates and yields alongside C:N:P ratios and pH control. Decreasing undissociated acid levels by raising the initial pH to 8.0 allowed growth without inhibition up to 5 gC.L–1 VFAs. However, VFA concentration strongly affected biomass yields irrespective of pH control or C:N:P ratios. Biomass yields on 1.0 gC.L–1 acetate were around 1.3-1.5 gC.gC–1 but decreased by 26-48% when increasing initial acetate to 2.0 gC.L–1. This was also observed for butyrate with yields decreasing up to 25%. This decrease in yield in suggested to be due to the prevalence of heterotrophic metabolism at high organic acid concentration, which reduced the amount of carbon fixed by autotrophy. Finally, the effects of C:N:P on biomass, lipids and carbohydrates production dynamics were assessed using a mixture of both substrates. In nutrient replete conditions, C. sorokiniana accumulated up to 20.5% carbohydrates and 16.4% lipids while nutrient limitation triggered carbohydrates accumulation up to 45.3%.