Bioreactor For Production Research Articles

Microbial Protein and Metabolite Profiles of Klebsiella oxytoca M5A1 in a Bubble Column Bioreactor.

The production of microbial proteins (MPs) has emerged as a critical focus in biotechnology, driven by the need for sustainable and scalable alternatives to traditional protein sources. This study investigates the efficacy of two experimental setups in producing MPs using the nitrogen-fixing bacterium Klebsiella oxytoca M5A1. K. oxytoca M5A1, known for its facultative anaerobic growth and capability to fix atmospheric nitrogen, offers a promising avenue for environmentally friendly protein production. This research compares the performance of a simple bubble column (BC) bioreactor, which promotes efficient mixing and cross-membrane gas transfer, with static fermentation, a traditional method lacking agitation and aeration. The study involved the parallel cultivation of K. oxytoca M5A1 in both systems, with key parameters such as microbial growth, glucose utilization, protein concentration, and metabolite profiles monitored over a 48 h period. The results indicate that the BC bioreactor consistently outperformed static fermentation regarding the growth rate, protein yield, and glucose utilization efficiency. The BC exhibited a significant increase in protein production, reaching 299.90 µg/mL at 48 h, compared to 219.44 µg/mL in static fermentation. The organic acid profile reveals both synthesis and utilization regimes of varying patterns. These findings highlight the advantages of the BC bioreactor for MP production, particularly its ability to maintain aerobic conditions that support higher growth and yield.

Plasma-Functionalized Liquids for Decontamination of Viable Tissues: A Comparative Approach

Plasma-functionalized liquids (PFLs) are rich in chemical species, such as ozone, hydrogen peroxide, singlet oxygen, hydroxyl radical and nitrogen oxides, commonly referred to as reactive oxygen and nitrogen species (RONS). Therefore, manifold applications are being investigated for their use in medicine, agriculture, and the environment. Depending on the goal, a suitable plasma source concept for the generation of PFLs has to be determined because the plasma generation setup determines the composition of reactive species. This study investigates three PFL-generating plasma sources—two spark discharges and a flow dielectric barrier discharge (DBD) system—for their efficacy in eliminating microbial contaminants from tissue samples aiming to replace antibiotics in the rinsing process. The final goal is to use these tissues as a cell source for cell-based meat production in bioreactors and thereby completely avoid antibiotics. Initially, a physicochemical characterization was conducted to better understand the decontamination capabilities of PFLs and their potential impact on tissue viability. The results indicate that the flow DBD system demonstrated the highest antimicrobial efficacy due to its elevated reactive species output and the possibility of direct treatment of tissues while tissue integrity remained. Achieving a balance between effective large-scale decontamination and the biocompatibility of PFLs remains a critical challenge.

Unlocking 3D printing technology for microalgal production and application

AbstractMicroalgae offer a promising alternative for sustainable nutritional supplements and functional food ingredients and hold potential to meet the growing demand for nutritious and eco-friendly food alternatives. With the escalating impacts of global climate change and increasing human activities, microalgal production must be enhanced by reducing freshwater and land use and minimizing carbon emissions. The advent of 3D printing offers novel opportunities for optimizing microalgae production, though it faces challenges such as high production costs and scalability concerns. This work aims to provide a comprehensive overview of recent advancements in 3D-printed bioreactors for microalgal production, focusing on 3D printing techniques, bio-ink types, and their applications across environmental, food, and medical fields. This review highlights the benefits of 3D-printed bioreactors, including improved mass transfer, optimized light exposure, enhanced biomass yield, and augmented photosynthesis. Current challenges and future directions of 3D printing in microalgal production are also discussed to offer new insights into boosting microalgal cultivation efficiency for expanded applications.

Development, physico-chemical characterization, and in vivo stability of a novel aglycosylated monoclonal antibody targeting FAM19A5

AbstractIntroducing aglycosylation into therapeutic monoclonal antibodies (mAbs) can prevent side effects associated with fragment crystallizable (Fc)-mediated effector functions. This modification induces structural changes in the heavy chain constant domain 2-constant domain 3 within Fc regions, which decreases antibody stability at acidic pH and high temperature. In this study, NS101, a novel aglycosylated mAb targeting family with sequence similarity 19, A5 (FAM19A5) for neurological diseases was evaluated with respect to its developability and in vivo stability as therapeutics. When recombinant CHO cells producing NS101 were cultivated using a fed-batch mode in a 500 L bioreactor, cell growth and mAb production profiles were consistent across three consecutive runs. NS101, thus produced, features an additional intra-disulfide bond in the heavy chain complementarity-determining region 3, contributing strong and sophisticated binding to the cryptic epitope. The melting temperature (Tm) of NS101 was lower than that of commercial glycosylated therapeutic mAbs, but NS101 showed better stability at 4 °C for 36 months. The binding affinity of NS101 to FAM19A5 and neonatal Fc receptor were comparable to those of glycosylated NS101. In addition, in three human cohort groups receiving 6, 12, and 24 mg/kg of NS101, the mean half-life was 22 days, and NS101 exhibited in vivo stability, considering that the half-lives of commercialized therapeutic mAbs and endogenous IgGs are 2–4 weeks and 21 days, respectively. Taken together, the results obtained here demonstrate that NS101, a novel aglycosylated mAb, has potential as a therapeutic agent for neurological diseases.

In Silico Optimization of a Fed-Batch Bioreactor for Tryptophan Production Using a Structured Hybrid Model and Several Algorithms Including a Pareto-Optimal Front

In Silico Optimization of a Fed-Batch Bioreactor for Tryptophan Production Using a Structured Hybrid Model and Several Algorithms Including a Pareto-Optimal Front

Recombinant laccase biosynthesis for efficient polydopamine coating

Laccases are versatile biocatalysts with interest for various industrial applications. This study reports the expression of Trametes versicolor laccase in Komagataella phaffii X33. The cultivation parameters (methanol and CuSO4 concentration, and temperature) for recombinant laccase production were studied in an orbital shaker. Enhanced laccase production was achieved by adding 1 % (v/v) methanol daily, supplementing 0.1 mM CuSO4 and incubating at 25 °C. Under these conditions, laccase production was scaled-up in a 4 L stirred tank bioreactor. Subsequently, laccase was concentrated and purified using a combined protocol of ultrafiltration and acetone precipitation, achieving a purification factor of 3.02. The laccase produced exhibited robust stability within a pH range from 4.0 to 8.0 and thermal stability up to 30 °C. Michaelis Menten kinetic revealed Michaelis constant (KM) and maximum rate of reaction (Vmax) values of 44.5 µM and 110.9 µM/min, respectively. Finally, laccase was employed as a biocatalyst to assist the polymerization of dopamine to polydopamine, allowing the one-step coating of cellulose filter paper, as confirmed by diffuse reflectance spectroscopy (UV-Vis DRS) and scanning electron microscopy (SEM). This work represents an advance in the field of laccase production in both orbital shaker and bioreactor, while demonstrating, for the first time, the laccase-assisted polymerization of dopamine for the coating of filter paper with polydopamine.

Bioaugmentation with Clostridium pasteurianum for high-yield continuous bio-hydrogen production in a dynamic membrane bioreactor

This study demonstrated the feasibility of achieving high-yield continuous bio-H2 production through bioaugmentation with Clostridium pasteurianum. C. pasteurianum was introduced during the initial granulation stage in a dynamic membrane bioreactor (DMBR) inoculated with heat-treated digester sludge. Following bioaugmentation, the average hydrogen yield (HY) reached 3.07 ± 0.13 mol H2/mol hexoseconsumed, with a hydraulic retention time (HRT) of 2 h. This high-yield and high-rate hydrogen production performance was achieved alongside a high concentration of suspended and granular biomass as 21.02 ± 1.01 g VSS/L. The DM played a crucial role in prolonging the solid retention time of the suspended biomass to 12.15 h. C. pasteurianum emerged as the predominant species, constituting 92.3 % of the total bacterial population. This dominance significantly contributed to achieving the high HY, reaching 77 % of the theoretical maximum HY (4 mol H2/mol hexoseconsumed) by shifting the metabolic pathway from non-H2 production pathways to hydrogen-producing acetate. However, during further continuous operation, the hydrogen production pathway was gradually shifted towards the butyrate pathway, resulting in a decreased HY of 2.26 mol H2/mol hexoseconsumed along with the decreased population of C. pasteurianum. Additional bioaugmentation of C. pasteurianum during the maturational granulation stage did not impact the metabolic pathways or microbiome significantly, as the added strain was supposed to be washed out. This study provides insights into achieving high-yield continuous bio-H2 production through bioaugmentation with C. pasteurianum during the initial granulation stage. Further studies are necessary to sustain C. pasteurianum-dominated high HY with the acetate pathway during prolonged operation.

A high-titer scalable Chinese hamster ovary transient expression platform for production of biotherapeutics.

Transient gene expression (TGE) in Chinese hamster ovary (CHO) cells offers a route to accelerate biologics development by delivering material weeks to months earlier than what is possible with conventional cell line development. However, low productivity, inconsistent product quality profiles, and scalability challenges have prevented its broader adoption. In this study, we develop a scalable CHO-based TGE system achieving 1.9 g/L of monoclonal antibody in an unmodified host. We integrated continuous flow-electroporation and alternate tangential flow (ATF) perfusion to enable an end-to-end closed system from N-1 perfusion to fed-batch 50-L bioreactor production. Optimization of both the ATF operation for three-in-one application-cell growth, buffer exchange, and cell mass concentration-and the flow-electroporation process, led to a platform for producing biotherapeutics using transiently transfected cells. We demonstrate scalability up to 50-L bioreactor, maintaining a titer over 1 g/L. We also show comparable quality between both transiently and stably produced material, and consistency across batches. The results confirm that purity, charge variants and N-glycan profiles are similar. Our study demonstrates the potential of CHO-based TGE platforms to accelerate biologics process development timelines and contributes evidence supporting its feasibility for manufacturing early clinical material, aiming to strengthen endorsement for TGE's wider implementation.

Assessing the environmental risk potential of transformation byproducts formed during fungal enzymatic treatment of a pharmaceutical mixture

The present study advances our understanding of the ecological risk potential associated with pharmaceutical mixtures treated with fungal enzymes. We optimized a submerged bioreactor for fungal enzyme production by testing three fungal species and adjusting the organic loading rate and retention time, yielding enzyme activities exceeding 20 U/L. These fungal enzymes effectively reduced the half-lives of tetracycline and sulfamethoxazole to less than 3 h in a mixture of tetracycline and sulfamethoxazole, using syringaldehyde as an optimized mediator. Analytical chemistry assessments identified transformation products (TPs) generated in situ from the mixture, revealing three novel transformation pathways. Quantitative structure–activity relationship analysis highlighted two TPs with heightened toxicity and prolonged persistence compared to their parent compound. Furthermore, these TPs exhibited distinct environmental mobility characteristics at the liquid–solid interface. Our combined experimental and computational framework allowed for a systematic screening of pharmaceutical residues, considering aspects such as toxicity, mobility, persistence, bioaccumulation, and removal. This approach has practical implications for prioritizing target pollutants in subsequent monitoring and environmental risk assessments.

Identification of infectious viruses for risk-based virus testing of CHO unprocessed bulk using next-generation sequencing.

It is important to increase manufacturing speed to make medicines more widely available. One bottleneck for CHO-based drug substance release is the in vitro viral (IVV) cell-based assay on unprocessed bulk. To increase process speed, we evaluate the suitability of replacing the IVV cell-based assay with next-generation sequencing (NGS). First, we outline how NGS is currently used in the pharmaceutical industry, and how it may apply to CHO virus testing. Second, we examine CHO virus contamination history. Since prior virus contaminants can replicate in the production bioreactor, we perform a literature search and classify 159 viruses as high, medium, low, or unknown risk based on their ability to infect CHO cells. Overall, the risk of virus contamination during the CHO manufacturing process is low. Only six viruses were reported to have contaminated CHO bioprocesses over the past several decades, and were primarily caused by fetal bovine serum or cell culture components. These virus contamination events can be mitigated through limitation and control of raw materials, combined with virus testing and virus clearance technologies. The list of CHO infectious viruses provides a starting framework for virus safety risk assessment and NGS development. Furthermore, ICH Q5A (R2) includes NGS as a molecular method for adventitious agent testing, paving a path forward for modernizing CHO virus testing.

Continuous biogas production and ex-situ biomethanation in a trickling bed bioreactor under mesophilic and thermophilic conditions

The utilization of biogas as a renewable energy source necessitates a reduction in its CO2 content. "Ex-situ biomethanation" is employed to convert CO2 in biogas to CH4 through hydrogenotrophic methanogens. In this study, a biogas stream (32–36 % CO2) originating from cow manure digestion was subjected to treatment in a trickle bed bioreactor (4 liters). The presence of microorganisms on the packing material was confirmed by using a 16 S rRNA test and SEM images. This study stands out by considering all four combinations of thermophilic and mesophilic conditions, monitoring the transition and steady-state phases in a two-reactor series setup. The mesophilic-thermophilic mode yielded the highest purity (92 %) with an 86 % CO2 conversion rate and an energy density of 13,870 kJ/m3. Additionally, the performance consistency was also assessed using a larger TBB (8 liters). Extending the residence time, the thermophilic-thermophilic mode yielded the highest CH4 concentration at 98 %. The 16 S rRNA results revealed that, influenced by the designated growth conditions encompassing culture media, pH, temperature, and reactor retention time, an enrichment of hydrogenotrophic methanogens occurred. Furthermore, promising results in terms of CH4 concentration and process efficiency were demonstrated by the trickled bed bioreactor employing liquid-in-gas dispersion.

Optimization of methane gas-liquid mass transfer during biogas-based ectoine production in bubble column bioreactors

Nowadays, the utilization of biogas for energy generation is hindered by the declining production costs of solar and wind power. A shift towards the valorization of biogas into ectoine, a highly valuable bioproduct priced at 1000 €⸱kg−1, offers a novel approach to fostering a more competitive biogas market while contributing to carbon neutrality. This study evaluated the optimization of CH4 gas-liquid mass transfer in 10 L bubble column bioreactors for CH4 conversion into ectoine and hydroxyectoine using a mixed methanotrophic culture. The influence of the empty bed residence time (EBRTs of 27, 54, and 104 min) at different membrane diffuser pore sizes (0.3 and 0.6 mm) was investigated. Despite achieving CH4 elimination capacities (CH4-ECs) of 10–12 g⸱m−3⸱h−1, an EBRT of 104 min mediated CH4 limitation within the cultivation broth, resulting in a negligible biomass growth. Reducing the EBRT to 54 min entailed CH4-ECs of 21–24 g⸱m−3⸱h−1, concomitant to a significant increase in biomass growth (up to 0.17 g⸱L⸱d−1) and reaching maximum ectoine and hydroxyectoine accumulation of 79 and 13 mg⸱gVSS−1, respectively. Conversely, process operation at an EBRT of 27 min lead to microbial inhibition, resulting in a reduced biomass growth of 0.09 g⸱L⸱d−1 and an ectoine content of 47 mg⸱gVSS−1. While the influence of diffuser pore size was less pronounced compared to EBRT, the optimal process performance was observed with a diffuser pore size of 0.6 mm.

Nano zero valent iron stimulated acetaldehyde oxidation, electron transfer, and RBO pathway for enhanced medium-chain carboxylic acids production

Nano zero-valent iron (NZVI) has been shown to effectively enhance the chain elongation (CE) process, addressing the issue of limited yield of medium-chain carboxylic acids (MCCA) from organic wastewater. However, the specific impact of NZVI on the metabolism of CE bacteria (CEB) is not well understood. In this study, it was aimed to investigate the mechanism by which an optimal concentration of NZVI influences CE metabolism, particularly in relation to ethanol oxidation, electron transfer, and MCCA synthesis. This was achieved through single-factor influence experiments and metagenomic analysis. The results showed that the addition of 1 g/gVSS NZVI achieved the highest MCCA yield (n-caproic acid + n-octanoic acid) at 2.02 g COD/L, which was 4.9 times higher than the control. This improvement in MCCA production induced by NZVI was attributed to several factors. Firstly, NZVI facilitated the oxidation of acetaldehyde, leading to its reduced accumulation in the system (from 18.4 % to 5.8 %), due to the optimized chemical environment created by NZVI corrosion, including near-neutral pH and a more reductive oxidation–reduction potential (ORP). Additionally, the inherent conductivity property of NZVI and the additional Fe ions released during corrosion improved the electron transfer efficiency between CEB. Lastly, both the composition of microbial communities and the abundance of unique enzyme genes confirmed the selective stimulation of NZVI on the reverse β-oxidation (RBO) pathway. These findings provide valuable insights into the role of NZVI in CEB metabolism and its potential application for enhancing MCCA production in CE bioreactors.

Semi-commercial scale fermentation of prawn waste by Exiguobacterium sp. GM010 for bioactive pigment production: Safety and efficacy assessment in Wistar rats

Pigment producing Exiguobacterium sp. GM010 demonstrates utilization of prawn carapace hydrolysate. Accordingly, our investigation was focused to produce cost effective bioactive pigment in 15 L semi-commercial scale. Further acute and subacute toxicities were evaluated as per OECD guidelines for application as nutraceutical. The biomass and pigment extract yield were 28.76 ± 1.78 and 0.82 ± 0.04 g/L, respectively, at 1.5 L of aeration. The UV–visible spectrum of pigment showed absorption peaks at 357, 466, and 491 nm indicating carotenoid like pigment. This was correlated with colour distribution in yellowish-orange region of CIE Lab spectrum. The qualitative and quantitative characteristics of pigment were similar between lab-scale and bioreactor production. The acute toxic dose (2000 mg/kg bw) exhibited the toxicity as determined by haematological analysis. While subacute toxic dose (250, 500, 1000 mg/kg bw) demonstrated no adverse effects on parameters such as food intake, body weight gain, vital organ weight, haematological and histopathological examination. Serum biochemistry results showed significant increase in HDL and decrease in SGPT values in treated groups compared to control. The results revealed that, Exiguobacterium sp. GM010 has efficiently utilised prawn waste to produce bioactive pigment. The dose of Exiguobacterium sp. GM010 pigment lower than 1000 mg/kg bw can be regarded as safe.

Estimation of some AM2 model parameters from a derived empirical logistic function of methane production

Because of its capability to convert organic wastes into renewable energy and into some components useful for agriculture, the anaerobic digestion technology can reduce greenhouse gas emissions in the atmosphere and the pollution. Thus, anaerobic digestion can contribute to achieving some of sustainable development goals. Consequently, many theoretical and empirical approaches are proposed for estimating, predicting and optimizing the methane produced by anaerobic digestion. In this context, the logistic function is a mathematical model that can be used to approximate empirical data of the temporal methane production in anaerobic digestion. In a previous paper, under some appropriate approximations, we have derived from AM2 model a single analytical expression in a form of a logistic function for describing the evolution of methane production in batch bioreactors. In the present paper, by comparing the three standard parameters associated with the classical empirical logistic function with that of the derived one from AM2 model; some relationships between them have been established. These relations are exploited for estimating some coefficients and parameters of AM2 model with respect to empiric logistic function parameters and vice-versa. Moreover, this possibility enables more qualitative insight about the evolution of the methane production and the influence of AM2 parameters and coefficients as well as their interaction over its processes.

Reinforcement learning based temperature control of a fermentation bioreactor for ethanol production.

Ethanol production is a significant industrial bioprocess for energy. The primary objective of this study is to control the process reactor temperature to get the desired product, that is, ethanol. Advanced model-based control systems face challenges due to model-process mismatch, but Reinforcement Learning (RL) is a class of machine learning which can help by allowing agents to learn policies directly from the environment. Hence a RL algorithm called twin delayed deep deterministic policy gradient (TD3) is employed. The control of reactor temperature is categorized into two categories namely unconstrained and constrained control approaches. The TD3 with various reward functions are tested on a nonlinear bioreactor model. The results are compared with existing popular RL algorithm, namely, deep deterministic policy gradient (DDPG) algorithm with a performance measure such as mean squared error (MSE). In the unconstrained control of the bioreactor, the TD3 based controller designed with the integral absolute error (IAE) reward yields a lower MSE of 0.22, whereas the DDPG produces an MSE of 0.29. Similarly, in the case of constrained controller, TD3 based controller designed with the IAE reward yields a lower MSE of 0.38, whereas DDPG produces an MSE of 0.48. In addition, the TD3 trained agent successfully rejects the disturbances, namely, input flow rate and inlet temperature in addition to a setpoint change with better performance metrics.

Primary clarification of mammalian cell culture fluid using enhanced sedimentation on inclined surfaces inside the single-use disposable Sudhin BioSettler150.

The first downstream processing step in the purification of a biopharmaceutical protein secreted into mammalian cell culture fluid is the primary clarification of the culture fluid. As cell densities in the fed-batch and increasingly more common perfusion bioreactors have increased over last two decades through intensified upstream bioreactor production processes, the traditional primary clarification unit operations of centrifugation and/or microfiltration become more challenging with issues like frequent desludging, cell disruption due to shear damage and quick fouling of membranes. We have developed a novel compact cell settler device exploiting the enhanced sedimentation on inclined surfaces and demonstrated that this settler device can be adapted easily to remove and contain cells or cell clumps from the clarified supernatant collected via the top effluent of the settler. In this work, we present high product recovery results during primary clarification of mammalian cell culture supernatant using our novel single-use disposable BioSettler150 while processing about 10 L of cell culture broth within short processing times of about 4 h.

Production of biologically active human basic fibroblast growth factor (hFGFb) using Nicotiana tabacum transplastomic plants.

We generated transplastomic tobacco lines that stably express a human Basic Fibroblast Growth Factor (hFGFb) in their chloroplasts stroma and purified a biologically active recombinant hFGFb. MAIN: The use of plants as biofactories presents as an attractive technology with the potential to efficiently produce high-value human recombinant proteins in a cost-effective manner. Plastid genome transformation stands out for its possibility to accumulate recombinant proteins at elevated levels. Of particular interest are recombinant growth factors, given their applications in animal cell culture and regenerative medicine. In this study, we produced recombinant human Fibroblast Growth Factor (rhFGFb), a crucial protein required for animal cell culture, in tobacco chloroplasts. We successfully generated two independent transplastomic lines that are homoplasmic and accumulate rhFGFb in their leaves. Furthermore, the produced rhFGFb demonstrated its biological activity by inducing proliferation in HEK293T cell lines. These results collectively underscore plastid genome transformation as a promising plant-based bioreactor for rhFGFb production.

Bi-directionalized promoter systems allow methanol-free production of hard-to-express peroxygenases with Komagataella Phaffii

BackgroundHeme-incorporating peroxygenases are responsible for electron transport in a multitude of organisms. Yet their application in biocatalysis is hindered due to their challenging recombinant production. Previous studies suggest Komagataella phaffi to be a suitable production host for heme-containing enzymes. In addition, co-expression of helper proteins has been shown to aid protein folding in yeast. In order to facilitate recombinant protein expression for an unspecific peroxygenase (AnoUPO), we aimed to apply a bi-directionalized expression strategy with Komagataella phaffii.ResultsIn initial screenings, co-expression of protein disulfide isomerase was found to aid the correct folding of the expressed unspecific peroxygenase in K. phaffi. A multitude of different bi-directionalized promoter combinations was screened. The clone with the most promising promoter combination was scaled up to bioreactor cultivations and compared to a mono-directional construct (expressing only the peroxygenase). The strains were screened for the target enzyme productivity in a dynamic matter, investigating both derepression and mixed feeding (methanol-glycerol) for induction. Set-points from bioreactor screenings, resulting in the highest peroxygenase productivity, for derepressed and methanol-based induction were chosen to conduct dedicated peroxygenase production runs and were analyzed with RT-qPCR. Results demonstrated that methanol-free cultivation is superior over mixed feeding in regard to cell-specific enzyme productivity. RT-qPCR analysis confirmed that mixed feeding resulted in high stress for the host cells, impeding high productivity. Moreover, the bi-directionalized construct resulted in a much higher specific enzymatic activity over the mono-directional expression system.ConclusionsIn this study, we demonstrate a methanol-free bioreactor production strategy for an unspecific peroxygenase, yet not shown in literature. Hence, bi-directionalized assisted protein expression in K. phaffii, cultivated under derepressed conditions, is indicated to be an effective production strategy for heme-containing oxidoreductases. This very production strategy might be opening up further opportunities for biocatalysis.

Effect of microalgae and bacteria inoculation on the startup of bioreactors for paper pulp wastewater and biofuel production

The use of microalgae and bacteria as a strategy for the startup of bioreactors for the treatment of industrial wastewater can be a sustainable and economically viable alternative. This technology model provides satisfactory results in the nitrification and denitrification process for nitrogen removal, organic matter removal, biomass growth, sedimentation, and byproducts recovery for added-value product production. The objective of this work was to evaluate the performance of microalgae and bacteria in their symbiotic process when used in the treatment of paper pulp industry wastewater. The experiment, lasting fourteen days, utilized four bioreactors with varying concentrations in mgVSS/L of microalgae to bacteria ratio (R1-100:100, R2-100:300, R3-100:500, R4-300:100) in the startup process. Regarding the sludge volumetric index (SVI), the results show that the R1 and R2 reactors developed SVI30/SVI10 biomass in the range of 85.57 ± 7.33% and 84.72 ± 8.19%, respectively. The lipid content in the biomass of reactors R1, R2, R3 e R4 was 13%, 7%, 19%, and 22%, respectively. This high oil content at the end of the batch, may be related to the nutritional stress that the species underwent during this feeding regime. In terms of chlorophyll, the bioreactor with an initial inoculation of 100:100 showed better symbiotic growth of microalgae and bacteria, allowing exponential growth of microalgae. The total chlorophyll value for this bioreactor was 801.46 ± 196.96 μg/L. Biological removal of nitrogen from wastewater from the paper pulp industry is a challenge due to the characteristics of the effluent, but the four reactors operated in a single batch obtained good nitrogen removal. Ammonia nitrogen removal performances were 91.55 ± 9.99%, 72.13 ± 19.18%, 64.04 ± 21.34%, and 86.15 ± 30.10% in R1, R2, R3, and R4, respectively.