Fermentation-Based Oxalic Acid Production for Sustainable Gallium Recovery from Electronic Waste

Aylin NurErkmen1✉Emailaylin.erkmen@fh-muenster.de

RolandUlber2Emailroland.ulber@mv.rptu.de

ThomasJuestel1Emailjuestel@fh-muenster.de

MirjamAltendorfner1Emailm.altendorfner@fh-muenster.de

Department of Chemical EngineeringFH Münster University of Applied SciencesStegerwaldstrasse 3948565SteinfurtGermany

2Department of Bioprocess EngineeringRPTU University of Kaiserslautern-LandauGottlieb Daimler Strasse 4967663KaiserslauternGermany

Abstract

$(\pm)$

27.73 mM OA in 5 days, which later was enhanced to 90.37

$(\pm)$

5.8 mM through intermittent pH adjustment above 4. Glucose was reported as the optimal carbon source, yielding Y\textsubscript{P/S}

$(\approx)$

0.4 (g/g). Scale-up in a 10 L bioreactor using fed-batch fermentation with pulsed feeding and pH control achieved 260.1

$(\pm)$

4.8 mM OA over 14 days. Co-production of gluconic acid and acidogenesis inhibition posed a challenge in attaining higher product yields. Collectively, these findings establish fed-batch fermentation with robust pH control as a viable strategy for sustainable OA production, enabling targeted Ga recovery from waste streams.\\Keywords: oxalic acid, ATCC1015, submerged fermentation, fed-batch, particle, morphology, bioleaching\\Key points\begin{itemize}\item A.niger strain ATCC1015 is the superior strain for biogenic oxalic acid production\\\item Fed-batch with pulsed feeding maximizes oxalic acid titer in scale-up. \\\item Gluconic acid and phosphate excess hinder selective OA production.\end{itemize}

Roland Ulber , Thomas Juestel and Mirjam Altendorfner: These authors contributed equally to this work.

Introduction

linenumbers

Oxalic acid, (sum formula: H\textsubscript{2}C\textsubscript{2}O\textsubscript{4}) is a dicarboxylic organic acid widely used in various industrial branches, among which pharmaceutical industry, agriculture, textile and leather, chemical and food industries can be counted schulerSustainableOxalicAcid2021. First synthesis of oxalic acid was established by Scheele in 1776 through sugar oxidization utilizing nitric acid, followed by an alternative protocol developed by Wöhler using cyanogen hydrolysis in 1824 vermaMetalRecoveryUsing2019. At present, oxalic acid is predominantly produced through chemical synthesis routes such as propylene, ethylene oxidation and dialkyl oxalate processes, while Rhône-Poulenc is the largest oxalic acid manufacturer on a global scale schulerSustainableOxalicAcid2021.

Oxalic acid is classified as a strong organic acid (pK\textsubscript{a1}= 1.27 ; pK\textsubscript{a2}= 4.27), dissolving mineral ores and elements through deprotonation step "acidolysis"vakilchapExploringUntappedPractices2024. According to the Hard-Soft-Acid-Base (HSAB) theory, oxalic acid establishes robust complexes with hard metals via "complexolysis", i.e. formation of octahedral coordination compounds \citep{weber2023coordination}. The dual function of oxalic acid as a reductant for metals such as for Fe(III), Co(III), and strong ligand for rare earth elements (REE) makes a promising candidate in the selective precipitation processes vermaMetalRecoveryUsing2019. A key applications of this chemistry is synthesizing REE-derivative luminescent materials, such as the red fluorescent lamp phosphor Y\textsubscript{2}O\textsubscript{3}:Eu^{3 +}, in which yttrium and europium are selectively co-precipitated and decomposed into crystalline monomodal red phosphor form at elevated temperatures at 900-980 K dupont2015rare,mesaros2015insights. The speciation of oxalic acid under varying pH, and ability to selectively extract and dissolve metals, whilst performing a precipitation for others, highlights the potential of oxalic acid in metal recovery and extraction processes from ore deposits, or electronic waste (e-waste) nadiSustainableBioextractionRare2025,vermaMetalRecoveryUsing2019,gaddFungalProductionCitric1999,gerold2024bio.

E-waste, a consequence of built-in obsolescence in electronic products, is rapidly accumulating in landfills, posing serious environmental and health hazards \citep{xavier2023comprehensive}. Yet, e-waste represents a valuable secondary source of base, precious and critical metals \citep{erkmen2025towards}. Among the critical elements identified by the EU Commission, gallium (Ga) is particularly notable due to its increasing demand in electronics and its limited primary production erkmen2025towards,ueberschaar2017challenges. Gallium, commonly recovered as a by-product of aluminum and zinc refining, presents significant challenges for recovery from secondary sources such as e-waste due to its highly dispersed nature, particularly within components like LED chip semiconductors ILLES2023110391,zhengRecoveryGalliumIndium2024,de2021review. This bottleneck in Ga supply is becoming increasingly critical, as the global transition towards alternative clean energy resources and the mass-production of next-generation electronics and semiconductors are anticipated to drive demand for gallium and its compounds \citep{sverdrup2025gallium}.

Current recycling technologies are inefficient and non-selective, often resulting in gallium being lost in slag or dominated by other metals de2021review,erkmen2025towards,reuter2012opportunities. With no established secondary production routes, gallium ends up entirely in the waste stream, highlighting the urgent need for selective and sustainable recovery methodsueberschaar2017challenges,reuter2012opportunities. Therefore, in order to secure a continuous supply line aligning with the EU's sustainability goals, the conceptualization of strategic approaches in gallium recycling from e-waste is of paramount importance.

In this context, oxalic acid presents a promising solution. A dual functionality of oxalic acid comprising acidolysis and complexolysis, enabling a dissolution and robust formation of complexesvermaMetalRecoveryUsing2019,erkmen2025leaching,gerold2024bio. As an example, Zhou et al.(2019) demonstrated a 90.36% of Ga recovery from waste surface-mounted light-emitting diodes (SMD-LEDs) under optimized conditions at 90 °C, 10 g/L solid loading, and 700 mM acid concentration \citep{zhou2019recovery}. Supporting these findings, Erkmen et al.(2025) identified oxalic acid as a selective reagent for Ga among other organic acids such as maleic, malonic, and itaconic acids \citep{erkmen2025leaching}. Accordingly, by implementing oxalic acid in Ga recovery processes, Erkmen et al. (2025) optimized the recovery process at 84.5 °C, 50 g/L solid loading, and 710.55 mM acid concentration, thereby achieving 37% gallium leaching in 3.2 hours (h) \citep{erkmen2025leaching}.

Nonetheless, despite offering a promising alternative compared to conventional harsh reagents, Choi et al. (2022) pinpointed that oxalic acid failed to meet the classification criteria of green chemicals \citep{choi2022organic}. Their assessment considered both a metal recovery (%) and toxicity (LD\textsubscript{50}), revealing oxalic acid exhibiting a higher LD\textsubscript{50} than sulfuric acid (H\textsubscript{2}SO\textsubscript{4}), raising concerns about its environmental impact \citep{choi2022organic}. This conundrum is rooted in the entire life-cycle of oxalic acid, as it is primarily derived from petrochemical processes, which are associated with volatile intermediates and significant CO\textsubscript{2} emissions schulerSustainableOxalicAcid2021.

Aligning with the aims of the European Union (EU) to achieve climate neutrality by year 2050 by prioritizing renewable energies and transitioning from fossil feed-stocks into the sustainable alternatives for material production gives an incentive for biogenic oxalic acid production as well as sustainable metal recovery from waste resources schulerSustainableOxalicAcid2021. In this regard, fermentation processes involving filamentous fungi emerges as a viable and sustainable strategy to address both 350,000-ton annual oxalic acid demand and the critical shortage in Ga supply, promoting selective secondary production based on a "key-lock" principle schulerSustainableOxalicAcid2021,erkmen2025leaching.

Filamentous fungi, specifically Aspergillus sp. and Penicillium sp. are widely used as microbial cell factories for the secretion of organic acids such as citric, oxalic, and gluconic acid through their metabolic processes gaddFungalProductionCitric1999. Notably, owing to its high productivity and industrial viability, Aspergillus niger is extensively used for citric acid fermentation \citep{ksikazek2023citric}. Early observations by Wehmer (1905) laid the foundation by distinguishing Aspergillus niger as an oxalic acid accumulator compared to Penicillium glaucum, which was later broadened by Currie and Thom (1915) to include Penicillium oxalicumcurrieOXALICACIDPRODUCING1915.

Further research into the metabolism of Aspergillus niger revealed a cytoplasmic, TCA-independent pathway for oxalate formation mediated by the enzyme oxaloacetate hydrolase (OAH)kubicekEvidenceCytoplasmicPathway1988. Kubicek et al. (1988) pinpointed the C-C splitting of oxaloacetate through OAH enzyme at pH levels above 4 kubicekEvidenceCytoplasmicPathway1988. In the same vein, Ruijter et al. (1999) demonstrated a citric acid production by A.niger mutant lacking OAH at pH 5 in the presence of manganese ions, conditions that are typically inhibitory to the wild-type ruijterOxalicAcidProduction1999.

While the oxalic acid secretion by Aspergillus niger is well-documented, the primary industrial application of A. niger has been for citric acid fermentation, where research efforts are often directed toward the suppression of oxalic acid as a by-product to maximize target yields gaddFungalProductionCitric1999, ksikazek2023citric. However, in the light of these findings regarding the cytoplasmic pathway driven by OAH enzyme, several studies have tackled the optimization of oxalic acid secretion by A. niger at different scales, adopting various methodologies with a primary focus on carbon source and pH regulation strasserHighyieldProductionOxalic1994, Cameselle_1998. The key findings from these fermentation studies are outlined in Table \ref{tab:lit_summary}.

pagebreak\newcolumntype{Y}{>{\raggedright\arraybackslash}X}{\renewcommand{\arraystretch}{2}\setlength{\tabcolsep}{5pt}\begin{table}[ht]\caption{Summary of Literature Studies}\label{tab:lit_summary}\scriptsize\begin{tabularx}{\linewidth}{l c l X >{\hspace{3em}} c c l}\topruleStrain ID & Vessel & C-source & Mode & Strategy & OA [mM] & Reference \\\midruleWU-2223 & Shake-flask & Glucose & Batch & pH

$(>)$

4 & 321 & kobayashiOxalicAcidProduction2014 \\B60 & Shake-flask & Glucose & Batch & pH

$(>)$

4 & 88.86 & kubicekEvidenceCytoplasmicPathway1988 \\ATCC 6275 & Shake-flask & Whey & Batch & C (%)^a & 4 & \citep{brown2018production} \\ATCC 9029 & Shake-flask & Whey & Batch & C (%)^a & 7 & \citep{brown2018production} \\CMB 120.49 & STR (2L) & Sucrose & Fed-Batch & pH

$(>)$

4 & 427 & strasserHighyieldProductionOxalic1994 \\1120 & Shake-flask & Whey & Batch & pH

$(\approx)$

6 & 410.97 & \citep{santoro1999influence} \\1120 & Biostat E (10L) & Whey & Batch & pH

$(\approx)$

6, kLa^b & 459.85 & bohlmannOxalicAcidProduction1998 \\1120 & Biostat E (10L) & Whey & Batch & pH

$(\approx)$

6 & 375.43 & \citep{Cameselle_1998} \\XP^c & STR (2L) & Rapeseed oil & Batch & pH

$(\approx)$

5 & 764.19 & \citep{rymowicz2003oxalic} \\W78C & Biostat A (6L) & Sucrose & Batch & pH

$(>)$

6 & 714.21 & walaszczykEffectMediumPH2018 \\F22 & BioCnS (50L) & Fructose & Batch & kLa & 83.1 & leeProcessDevelopmentOxalic2018a \\NJDL-12 & Shake-flask & Glucose & Batch & pH = 6.5 & 26.1 & liStudyOrganicAcid2016 \\NJDL-03^d & Shake-flask & Glucose & Batch & pH = 6.5 & 10.02 & liStudyOrganicAcid2016 \\\bottomrule\end{tabularx}\vspace{0.3em}\parbox{0.95\linewidth}{\footnotesize ^aOA [mM]: Adjustment in the concentration of the carbon source ^bkLa: Optimization through the kLa value by adjustng the stirrer speed (rpm) and areation (vvm) ^cXP: Modified A.niger strain through UV-mutagenesis ^dNJDL-03: Penicillium oxalicum strain}\end{table}}

The studies outlined in Table \ref{tab:lit_summary} reveal that disparate methodologies and scales complicate efforts to draw clear distinctions regarding the influence of critical parameters such as strain type, fermentation mode, and scale.

To bridge this gap, this research addresses aforementioned limitations by establishing a comprehensive framework for submerged oxalic acid fermentation by Aspergillus niger. Through a systematic study including strain comparison, pH regulation, multi-scale translation, and fermentation modes, this study establishes fundamentals for an industrially viable oxalic acid production. Developed process is designed to yield a biogenic agent rich in oxalic acid, specifically conceptualized for the recovery of gallium from e-waste and other secondary waste streams, leveraging the enhanced performance often attributed to synergistic secondary metabolites in fungal fluids maneesuwannaratPossibleMechanismGallium2016. Consequently, by transitioning away from petrochemical routes, this study positions fungal fermentation as a stand-alone green technology, contributing to sustainable bioprocess development for strategic metal recovery in alignment with the European Union’s 2050 circular economy goals.

Materials and Methods

Strain, Subculture and Inoculum Preparation

Aspergillus niger strains used in this study included ATCC 1015 (LGC Standards) and CECT 2807 (University of Valencia culture repository). Accordingly, strains were subcultured periodically (5-7 days) on potato-dextrose agar (PDA) plates under sterile conditions and incubated at 30 °C.

For spore inoculum preparation, cultures were aseptically scraped from PDA plates using sterile disposable scalpels into a solution of 0.9 N KCl and 10 µL/L Triton-X. Spore concentrations were determined using a Neubauer-improved hemocytometer via serial dilution, in which inoculum concentrations were adjusted to 10⁶-10⁷ spores/ml for shake-flask experiments and 10⁸ spores/mL for fermenter experiments. Pre-cultures in glycerol solutions were stored in cryo-vials at -20 °C.

Fermentation experiments

Shake-Flask Experiments

The nutrient media used in the shake-flask experiments were adapted from Bosshard et al. (1996) and contained (g/L): Glucose (30), NaNO\textsubscript{3} (1.5), KH\textsubscript{2}PO\textsubscript{4} (0.5), MgSO\textsubscript{4}.7H\textsubscript{2}O (0.025), KCl (0.025), yeast extract (1.6) bosshardMetalLeachingFly1996. This medium was supplemented with 100 mM 2-(N-Morpholino)ethanesulfonic acid (MES) or phosphate buffering, and the pH was adjusted to 6

$\pm$

0.2 with concentrated (3 N) NaOH. All reagents were of analytical grade. Sterilization of nutrient and agar media was performed in closed sterile vessels at 120 °C for 20 min under 1.5 psi pressure.

Sampling was conducted aseptically on sporadic intervals spanning over 240 h using sterile pipette tips and sample holders to prevent cross-contamination. pH fluctuations and total acid concentration were measured throughout the experiment. In buffering tests, pH was maintained at 6

$\pm$

0.2 using 3 N NaOH. At each interval, the

$\text{pH}$

of the culture medium was monitored using a digital

$\text{pH}$

meter with a DO-sensor, calibrated prior to use with

$\text{pH}$

$4.0$

and

$7.0$

standard buffers.

Following fermentation, fungal pellets were separated from the biogenic supernatant solutions using Nalgene vacuum filters (0.2

$\mu$

m). Biomass from the shake-flask cultures was quantified gravimetrically. Culture samples were collected in triplicate and filtered to separate the mycelia from the broth. The collected biomass was then washed twice with deionized (DI) water to remove residual media components and subsequently dried in an oven at 70

$^{\circ}$

C until a constant dry weight was achieved. The cell-free supernatant was stored at 4

$^{\circ}$

C for subsequent glucose and organic acid analysis.

Fermenter (10 L) Experiments

A Biostat-C (Sartorius AG, Göttingen, Germany) bioreactor with a 15-L total volume (10-L working volume) and an H:D ratio of 1:3 was used for all large-scale fermentation experiments. The nutrient medium, adapted from Bosshard et al. (1996), contained (g/L): Glucose (150), NaNO\textsubscript{3} (1.5), KH\textsubscript{2}PO\textsubscript{4} (0.5), MgSO\textsubscript{4}·7H\textsubscript{2}O (0.025), KCl (0.025), and yeast extract (1.6) bosshardMetalLeachingFly1996. This was further supplemented with 100 mM phosphate buffering, and the pH was maintained at 6

$\pm$

0.2 using concentrated (3 N) NaOH. All reagents used were of analytical grade.

Mixing was provided by a triple six-blade Rushton turbine impeller, and aeration was supplied through a ring sparger. The reactor temperature was controlled via a cooling jacket. Process parameters including temperature, volumetric airflow (L/min), dissolved oxygen (DO (%)), and pH were continuously monitored and controlled using an integrated amplifier system. The DO level was maintained using a cascade control system that adjusted the stirrer speed and/or aeration rate.

The pH was measured and controlled using a Mettler-Toledo S7 pH sensor, pre-calibrated using analytical pH 4 and 7 buffer solutions. Dissolved oxygen was measured with a Hamilton OxyFerm (pO\textsubscript{2}) sensor, calibrated with a two-point method (0% after nitrogen sparging and 100% after full aeration).

Sterilization of the fermenter was performed in three stages (heating, sterilization, cooling) using the sterilization mode. The sparging was adjusted to sterilization mode, and the aeration valve and cooling water were turned off. Sterilization occurred at 120

$^{\circ}$

C and up to 1.5 atm of gauge pressure for 25 min. During the cooling step, cooling water was turned on once the reactor temperature reached 85

$^{\circ}$

C to expedite the process.

For fermentation experiments, the vessel was initially filled with 8.8 L of nutrient medium, accounting for

$\approx$

0.8 L loss during sterilization. After sterilization, 0.2 v/v % pluronic F68 (foam-suppressing agent) and 0.1 N buffer solution were added through a syringe equipped with a 0.2 µm membrane filter. An inoculum of 1%, prepared from pre-grown pellet cultures (72-96 h exponential phase), was transferred aseptically from sterile culture flasks to the fermenter via a peristaltic pump. To enhance oxalic acid production, vacuum-sterilized (0.2

$\mu$

m) 3 N NaOH was integrated via peristaltic pumps after the initial lag and growth phase (48 h) to maintain the fermentation medium pH at 6

$\pm$

0.2.Following operational modes were investigated:

Batch: The fermentation was performed with an initial glucose concentration of 150 g/L without further feeding.

Fed-Batch: Based on preliminary data, a pulsed feeding strategy was employed. After an initial batch phase, pulses of a concentrated glucose solution (30 g/L) were added at 72-hour intervals to maintain a target substrate level, with the total glucose added over the 15-day fermentation for constituting the total sum of 150 g/L.

Samples (20–25 mL) were withdrawn aseptically at 24-hour intervals via a steam-sterilized port at the base of the bioreactor to monitor residual glucose and organic acid concentrations. Unlike shake-flask experiments, biomass was not quantified due to the dispersed mycelial morphology and the difficulty in accurately subtracting the pre-grown inoculum mass.

Glucose and Organic Acid Determination

Collected samples were syringe-filtered (

$0.45 \mu$

m) directly and analyzed for residual glucose using the dinitrosalicylic acid (DNS) assay \citep{miller1959use}. The DNS reagent was prepared by dissolving 10 g of DNS and 300 g of Rochelle salt in 800 mL of 0.5 N NaOH, with the final volume complemented to 1 L with double-distilled H\textsubscript{2}O. For the DNS assay, 100

$\mu$

L of the diluted sample was mixed with 400

$\mu$

L of the prepared DNS reagent. The mixture was heated in a heating block for 5 minutes at 95

$^{\circ}$

C. Subsequently, samples were promptly placed in an ice-bath. Next, the prepared samples for glucose quantification were pipetted into sterile F-type 96-well plate, in which the absorbance at 570 nm using a microplate reader was measured.

Oxalic acid concentration in the filtrate was determined via Capel-105M fully automated capillary electrophoresis instrument (Lumex Instruments, Canada). Direct samples from the fermentation liquid were syringe-filtered (

$0.45 \mu$

m) and serially diluted with ddH\textsubscript{2}O. Before sample analysis, daily conditioning on Capel-105M was performed, which comprised the serial flushing of capillaries with 1 N NaOH, ddH\textsubscript{2}O, and a buffer solution consisting of chromium (VI) oxide (10 mM), diethanolamine solution (30 mM), and Hexadecyltrimethylammonium hydroxide (CTA-OH) (3 mM). Subsequently, the samples were loaded into autosampling unit and hydrodynamically injected into the capillary at 30 mbars for 5 s for analysis.Samples were then hydrodynamically injected (30 mbars for 5 s) and analyzed at -25 kV for 8-10 minutes, with indirect UV detection at 254 nm. Analyte concentrations were calculated via peak area against a linear regression of analytical standards (oxalic, citric, glucinic acids and nitrate, phosphate, chloride, sulfate ions). All measurements were performed in triplicate (95% CI).

Results

Comparative Study of Two Strains for Biogenic Oxalic Acid Production

To differentiate the oxalic acid production capabilities between the selected A.niger fungal strains, small-scale batch fermentation experiments were conducted and monitored over a 240-hour period. Fig. 1 presents the comparative analysis of oxalic acid secretion (mM) and pH change (

$\bigtriangleup$

pH) between ATCC1015 and CECT2807.

Fig. 1

Oxalic secretion and

$(\bigtriangleup)$

pH during comparative strain test in small-scale shake fermentation experiments. In the bar chart stacked with line plot, bars correspond to the secreted oxalic acid amount (mM), and lines to the

$(\bigtriangleup)$

pH in the secondary y-axis (pH\textsubscript{initial} = 6

$(\pm)$

0.2). The data represents the mean of biological replication experiments (n=3). Error bars on both the bar and line plot denote the standard deviation of the biological triplicates.

As outlined in Fig. 1, ATCC1015 showed a notably higher mean oxalic acid secretion compared to CECT2807 throughout the fermentation spanning over 240 h. While no clear distinction could have been made in terms of acid production in the initial phase (24h), ATCC1015 exhibited rapid oxalic acid secretion in the latter sampling times, reaching a peak at 71

$\pm$

27.73 at 240 h. The trend in acid peak was corroborated by the change in pH, in which ATCC1015 exhibited sharp

$\bigtriangleup$

pH of 2.24 at 72 h, surpassing the

$\bigtriangleup$

pH of 1.5 measured for CECT2807. After sampling at 72h, pH of the fermentation medium was adjusted to 6

$\pm$

0.2 to prevent the suppression of OAH enzyme, leading to a progress in the oxalic acid secretion in latter stages of the fermentation.

Despite having higher productivity for oxalic acid, the high variation stemming from the biological or technical variability in the replicates caused a large spread, as evidence by the large standard errors, specifically in ATCC1015 cultures. Nevertheless, taken into consideration of overall remarkable production in terms of oxalic acid production, ATCC1015 strain was selected for the remaining investigations.

pH-Regulation

As numerous studies attributed the significant biogenic oxalic acid secretion to the pH regulation by keeping pH >4, complementary to the strain comparison, sporadic pH adjustment on day 1, 4, and 7 was implemented in the buffered (100 mM MES) small-scale shake cultures. In addition, a separate fermentation batch was conducted where the culture medium's pH was maintained at a neutral state (pH 6.0

$\pm$

0.2) throughout the experiment. Owing to the prolonged lag phase in this set of experiments (

$\approx$

26-28 h), neither acid secretion nor pellet formation could be detected at 24 h (data not shown).

Fig. 2

The influence of pH regulation on oxalic acid secretion in shake-flask cultures. The bars (primary y-axis) represent the mean concentration of secreted oxalic acid (mM) in cultures with intermittent pH control ("Controlled pH") versus unregulated cultures ("Natural pH shift"). The lines (secondary y-axis) indicate the measured pH at each sampling point over the 240-hour fermentation. All data points represent the mean of three biological replicates (n=3), with error bars indicating the standard deviation.

As shown in Fig.2, following the lag phase (

$\approx$

24 h), both groups experienced a significant drop in pH to

$\approx$

2.86 and secreted comparable oxalic acid by 96 hours. After adapting the initial pH-regulation on day 4 (96 h), the pH-regulated cultures demonstrated a

$\approx$

7-fold surge in the secretion at 168 h, ultimately reaching a final titer of 90.37

$\pm$

5.8 mM acid at 240 h.

On the other hand, the control group where no pH-regulation was integrated, secreted notably lower oxalic acid. As indicated on the secondary y-axis of Fig.2, the pH of the non-regulated group remained below the critical threshold at pH 4, suppressing therefore the regulation of the OAH enzyme. As the fermentation progresses toward 168 h, the discrepancy in acid generation by both groups were doubled and persisted until the finalization of the fermentation at 240 h.

Thus, these results confirm that even sporadic pH regulation on small-scale batch cultures is a highly effective strategy for boosting oxalic acid yields.

Carbon source

To determine the impact of carbon source on the oxalic acid generation in the shake cultures of A.niger, the media described in the methods and materials were adjusted by implementing glucose, sucrose and lactose at 30 g/L for each. As given in Fig 3, the choice of substrate influenced the oxalic acid generation and the overall product yield per g substrate.

Fig. 3

Comparison of carbon sources for oxalic acid secretion. The primary y-axis (bars) corresponds to the secreted oxalic acid concentration (mM), while the secondary y-axis (lines) represents the product yield on substrate (Y\textsubscript{P/S}, (g/g)). All data points represent the mean of three biological replicates (n=3), with error bars indicating the standard deviation.

The choice of carbon source significantly influenced both the final oxalic acid titer and the product yield. As shown in Fig 3, glucose proved to be the most effective substrate over the 240-hour fermentation, resulting in the highest titer (136.88

$\pm$

69.35 mM) and a product yield (Y\textsubscript{P/S}) of 0.4 (g/g). Fermentation on sucrose resulted in a final yield of 82.8

$\pm$

55.03 mM (Y\textsubscript{P/S}=

$\approx$

0.25), which was notably lower than that of glucose. In contrast, lactose was the least effective substrate, with production peaking at approximately 72.1

$\pm$

14.6 mM (Y\textsubscript{P/S}=

$\approx$

0.2) by 72 hours before a subsequent decline.

These results in Fig 3 suggest that while ATCC1015 can metabolize all three sugars, its metabolic pathways most efficiently channel the monosaccharide glucose towards oxalic acid synthesis over the full course of the fermentation. In contrast, the conversion of sucrose and lactose appeared to be less efficient or become inhibited as the fermentation progressed.

Operational Mode Comparison for Scale-Up: Batch vs. Fed-Batch

Building on the preliminary shake-flask experiments, the 10 L scale-up fermentation were conducted to compare oxalic acid production in batch and fed-bath operational modes. The performance of each strategy was assessed in terms of substrate consumption (g/L), the secreted oxalic acid amount (mM), product formation rate (rP) and attained theoretical yield (Y\textsubscript{P/S}). Figure 4 present the 384-h fermentation profiles in both processes, allowing a direct comparison of the different fermentation strategies.

Fig. 4

Comparative performance of different fermentation strategies. The panels illustrate key process parameters over time for Batch, Fed-Batch, and Repeated Fed-Batch operations. a. Glucose consumption profile. b. Oxalic acid production profile. c. Instantaneous product yield (

$(Y_{\text{inst}})$

). Data are presented as the mean

$(\pm)$

standard deviation (

$(n=3)$

)

As shown in Fig. 4, oxalic acid fermentation in batch mode exhibited distinct phases over the 384-h process. Following an initial lag phase until

$\approx$

72 h, after which oxalic acid was gradually secreted. A transition to an accelerated production phase occurred only after 312 h, which coincided with the residual glucose concentration falling below

$\approx$

20 g/L. By the end of fermentation (384 h), the final oxalic acid titer reached 155.2 mM, corresponding to an overall volumetric product formation rate (r\textsubscript{P}) of 0.036 g.L^.1.h^-1.

The fed-batch strategy, utilizing intermittent pulsed feeding, effectively promoted acid secretion following the initial lag phase (

$\approx$

28-30 h). This approach resulted in a cyclical production pattern comprising a gradual and consistent surge in the acid secretion, corresponding to 72 h intervals of pulse feeding. By the end of the second cycle at 144 h, the titer has already reached

$\approx$

100 mM (r\textsubscript{P}

$\approx$

0.06 g.L^-1.h^-1).

In the final exponential production phase (312-384 h), glucose consumption plateaued, and r\textsubscript{P} the volumetric production rate signaled a stagnation, indicating the end of the productive period.

begin{table}[h]\centering\caption{Comparison of yields and oxalic acid titers in different fermentation modes.}\label{tab1:fermenter_comparison}\begin{tabular}{@{}lccc@{}}\topruleMode & Y\textsubscript{P/S}^a & r\textsubscript{P}^b & OA [mM]^c \\\midruleBatch & 0.122 & 0.036 & 155.2

$(\pm)$

3.1 \\Fed-batch & 0.194 & 0.061 & 260.1

$(\pm)$

4.8 \\\bottomrule\end{tabular}\vspace{0.5em}\parbox{0.95\linewidth}{\centering\footnotesize^a Theoretical yields attained at 384 h \\^b Volumetric Product Formation Rate (g.L^-1.h^-1)\\^c Secreted OA amount at 384 h.\\}\end{table}

Overall, the fed-batch strategy significantly outperformed batch fermentation, achieving a final oxalic acid titer of 260.1

$\pm$

4.8 mM, a theoretical yield of 0.194 g/g and r\textsubscript{P} of 0.061 g.L^-1.h^-1. A detailed comparison of the final product titers (mM), theoretical yields (Y\textsubscript{P/S}) and product formation rates (r\textsubscript{P}) for both operation modes is presented in Table 1.

Discussion

This study systematically investigated biogenic oxalic acid production using submerged fermentation by filamentous fungi species, in particular Aspergillus niger, focusing on the interplay between strain selection, pH control, carbon source, and fermentation strategy. The findings of this study confirm that these factors are critically interconnected and must be co-optimized to maximize product yield. By integrating these variables, this work establishes a more comprehensive framework for oxalic acid fermentation than previously reported in disparate studies.

Comparative study of strains confirmed that biogenic oxalic acid production is strain-dependent, as previous investigations also indicated considerable disparities in theoretical yields across various strains in organic acid fermentation ruijterOxalicAcidProduction1999,andersen2011comparative]. Similarly, ATCC1015 exhibited a superior oxalic acid secretion (71

$\pm$

27.73 mM) compared to CECT2807 (37.9

$\pm$

7.1 mM). This findings also aligns with the preliminary studies, including Mendes et al. (2022), who reported 150 mM oxalate from ATCC1015 within 7 days, 158% higher than an engineered mutant lacking OafA (oxalic acid repression factor) \citep{mendes2022rock}.

Nevertheless, large deviations across biological replicates (n=3) were observed, particularly for ATCC1015, highlighting inherent biological variability of filametous fungi fermentation bauer2022online,coban2020organic. Variations in spore germination, pellet formation, hyphal branching, and oxygen diffusion gradients all contribute to inconsistencies cairns2023regression,veiter2018filamentous,amenaghawon2024comprehensive. This, hence, accounts for the discrepancies in the reported oxalic acid yields for ATCC1015 ranging from 2 mM to over 150 mM brisson2016bioleaching,castroBiorecoveryRareEarth2023,mendes2022rock. Therefore, these aforementioned deviations in the biological replicates of filamentous fungi species in strain comparison are attributed to inherent variability of strains, as often addresses in preliminary studies, as well as exterior parameters, sampling and experimental handling, which they all cause alterations in the oxalic acid fermentation.

Our findings on pH regulation unequivocally demonstrated the decisive role of pH in the oxalic acid secretion. As shown in Fig 2, in cultures, where intermittent pH regulation was implemented to maintain the medium above pH 4, the final oxalic acid concentration attained 90.37

$\pm$

5.8 mM. Conversely, the cultures without any pH control experienced a pH drop to approximately to 3, yielding a substantially lower concentration of 61.65

$\pm$

1.55 mM.

Aligning with the findings of Kubicek et al (1988), the discrepancy between the controlled and uncontrolled group is attributed primarily to the pH-dependent enzyme activity of oxaloacetate hydrolase (OAH), with optimal activity at pH values above 4 kubicekEvidenceCytoplasmicPathway1988. The metabolic activity of OAH is severely inhibited at acidic conditions (i.e. pH < 3), channeling carbon flux towards other metabolic pathways, thereby reducing oxalate yield kubicekEvidenceCytoplasmicPathway1988,ruijterOxalicAcidProduction1999. Thus, our findings corroborate that pH maintenance above 4 is not merely beneficial but essential for maximizing oxalic acid yields, even when implemented sporadically in small-scale batch processes.

The results suggested in Fig 3 that, the choice of a carbon source significantly influenced the oxalic acid secretion. Accordingly, glucose proved to be the most effective substrate, yielding 1.6-fold and 4.8-fold more oxalic acid than sucrose and lactose, respectively. This can be associated to the direct entry of simple sugars such as glucose into the glycolytic pathway, providing, hence, an efficient and forthright flux of carbon towards oxaloacetate, the immediate precursor of oxalic acid strasserHighyieldProductionOxalic1994,walaszczykEffectMediumPH2018. Used as a primary substrate, glucose can attain

$\approx$

75% of theoretical oxalic acid yield \citep{handayani2009production}. In contrast, more complex sugars such as sucrose and lactose require preceding enzymatic steps for break-down, thus diverting the metabolic flux in favor for the formation of other metabolites kubicekEvidenceCytoplasmicPathway1988, Cameselle_1998.

The scale-up comparison between batch and fed-batch operations provided critical insights into operational determinants in the large-scale submerged fermentation of oxalic acid. As the results in Fig 4.a. suggested, fed-batch strategy proved to be the most effective, achieving the highest final oxalic acid concentration (260.1

$\pm$

4.8 mM) and theoretical yield (0.195 g/g). By intermittently feeding of the substrate through pulsed-feeding in 72 h of cycles, the residual glucose concentration was maintained below the inhibitory threshold of 20 g/L. This approach prevented carbon catabolite repression, mitigated severe pH fluctuations, and sustained a prolonged exponential production phase, a trend also observed in other organic acid fermentation processes schmittEnhancedLMalicAcid2022a,demir2021high. On the other hand, batch fermentation exhibited a prolonged lag phase, with substantial oxalate secretion only commencing after

$\approx$

192 hours, which can be attributed to inhibitory influences such as drastic pH fluctuations needed constant monitoring and control and high initial glucose loading (150 g/L). What is more, high substrate loading can undermine oxygen transfer through osmotic pressure and increased viscosity, thus hindering organic acid generation xue2021improving,anastassiadisCitricAcidProduction2006. As indicated in Fig 4.a., exponential production phase commenced only after the residual glucose concentration fell below

$\approx$

20 g/L.

Overall, fed-batch suited the best for this purpose compared to the batch mode, attaining highest secreted oxalic acid amount, and thus volumetric product formation and theoretical yield. Despite its success, several challenges concerning the discrepancies in yields between different scales and similar operations need to be addressed.

begin{table}[h]\centering\caption{Comparison of volumetric product formation (r\textsubscript{P} with the preliminary findings concerning the oxalic acid generation across different scales and fermentation modes)}\label{tab2:Comparison_preliminary_studies}\begin{tabular}{@{}lccc@{}}\topruleOperation & Scale & r\textsubscript{P} (g.L^-1.h^-1) & Reference \\\midruleBatch & STR (10L) & 0.036 & This study\\Fed-batch & STR (10 L) & 0.061 & This study \\Batch & Shake Flask & 0.051 & This study \\Batch & STR (10L) & 0.283 & bohlmannOxalicAcidProduction1998\\Batch & Shake Flask& 0.104 & \citep{Cameselle_1998}\\Fed-Batch & STR (2L) & 0.26 & strasserHighyieldProductionOxalic1994\\Batch & STR (10L) & 0.375 & \citep{Cameselle_1998}\\Batch & Shake Flask & 0.142 & \citep{santoro1999influence}\\\bottomrule\end{tabular}\vspace{0.5em}\parbox{0.95\linewidth}{\centering}\end{table}

When compared with the previous reports as outlined in Table 2, the volumetric product rates (r\textsubscript{P}) obtained from various scales and mode of operations in this study were substantially lower. This discrepancy underscores a well-documented challenge in bioprocess development: the difficulty of extrapolating screening results from small-scale batch flasks to larger, geometrically complex batch or fed-batch fermenters sitanggang2010fermentation, keitelProcessAnalysisAnaerobe2024.

Furthermore, the inherent morphological plasticity of filamentous fungi such as Aspergillus niger presents reproducibility challenges \citep{coban2020organic}. According to the findings in this study, despite highlighting a certain degree of reproducibility in the initial phase, divergence issues emerged, which is attributed to cumulative morphological alterations and metabolic shifts driven by the buildup of secondary products over the prolonged fermentation processes \citep{coban2020organic}.

Another critical factor that influenced the variations in the results was attributed to the co-production of gluconic acid. While the preliminary studies in shake flasks investigating the influence of carbon source suggested that using glucose would favor oxalic acid synthesis, the fed-batch fermentation resulted in substantial co-production of gluconic acid (226.05

$\pm$

8.58 mM at 384 h). The slight-acidic to neutral nature of the fermentation medium (pH

$\approx$

6) may be an important factor in the co-production of gluconic acid, as pH

$\approx$

5-6 reported to be conducive for GOD enzyme, triggering oxidation of glucose into gluconic acid \citep{Cameselle_1998}. Due to this, the selectivity for oxalic acid was not thereby hindered, but also presented a complication pertaining to downstream processing so as to separate these two organic acids.

In addition, the buffering system employed during scale-up must be considered, as the use of phosphate buffer (0.1 M) exerted a critical, albeit paradoxical influence on oxalic acid fermentation. On one hand, phosphate buffering (0.1 M) successfully mitigates acid-induced product inhibition by neutralizing the secreted organic acids, which is a well-documented bottleneck in oxalic acid production manohar2002application,bohlmannOxalicAcidProduction1998. On the other hand, the surplus phosphate simultaneously favor biomass growth at the cost of acidogenesis brown2018production, mandalSubmergedProductionOxalic2005]. This trade-off is reflected in the fermentation profile shown in Fig. 4a, where the onset of exponential oxalic acid secretion was delayed until the later stages of cultivation (

$\approx$

312 h onwards), strongly suggesting that substantial acid production only commenced once the phosphate excess had been depleted, thereby inducing a metabolic shift consistent with the phosphate-starvation-driven acid synthesis reported by Upton et al. (2017) \citep{upton2017accurate}. Ultimately, the phosphate buffering created a trade-off between maintaining pH stability to support productivity and suppressing the very metabolic pathways required for acid generation.

While the volumetric product formation rates (r\textsubscript{P}) obtained in this study substantially lower than the reported values in the literature 2, our findings confirm the viability of a simplified, robust biogenic oxalic acid fermentation for sustainable critical metal recovery. The primary goal of this research was not to maximize absolute yield at the cost of operability, but to establish a reliable framework suitable for integration into industrial metal recycling. The fed-batch operation successfully achieved a high, consistent titer of

$260.1 \text{ mM}$

through straightforward

$\text{pH}$

control and intermittent feeding, thereby avoiding the extreme complexity and high capital expenditure of highly specialized fermentation systems. By prioritizing operational robustness, this method provides a reliable and sufficient supply of the biogenic agent necessary for Gallium (

$\text{Ga}$

) leaching, offering superior industrial relevance for real-world recycling applications.

Conclusions and Outlook

All in all, this study underscores the decisive and correlating factors governing biogenic oxalic acid generation via submerged fermentation, namely strain selection, pH regulation, carbon source and the scale and mode of operation. Among the evaluated strategies, fed-batch operation in the scale-up system, combined with intermittent pulsed feeding and robust pH control above 4, proved most effective in achieving high titers (

$260.1 \pm 4.8$

mM) with Aspergillus niger strain ATCC1015.

With this robust production framework established, the next critical step is to validate the application of the biogenic oxalic acid in its intended use case: critical metal recovery, specifically gallium (Ga) from electronic waste. Future investigations should focus on integrating the optimized fermentation process with bioleaching operations to develop a viable two-stage indirect recovery system. This will involve assessing the efficiency and selectivity of the cell-free supernatant against various Ga-containing precursors, thereby completing the proof-of-concept for a green, integrated supply chain for strategic metal recycling.Future research should gravitate towards these key aspects:

Leaching Validation and Selectivity: Evaluating the efficiency of the cell-free supernatant against various Ga precursors (e.g., Al, Ga, In, GaN, waste LEDs) and investigating the performance of the complex biogenic fluid in selectively recovering Ga in the presence of competing metals (Fe, Al).

Tailoring the Leaching Agent: Refining the fermentation to optimize the supernatant composition for specific leaching tasks, including:\begin{itemize}\item Morphology Control: Implementing advanced agitation or particle-enhanced strategies to stabilize pellet morphology, ensuring consistent leaching agent quality across industrial batches coban2020organic, mandalSubmergedProductionOxalic2005].\item Metabolite Selectivity: Fine-tuning

$(\text{pH})$

or utilizing selective pressure to suppress gluconic acid co-production, thereby creating a purer

$(\text{OA})$

leaching agent to simplify downstream metal purification.\item Buffering and phosphate limitation: Despite ensuring stability, phosphate buffering systems may inadvertently suppress oxalate formation. Alternative buffering strategies, evaluated also for techno-economic viability, will be crucial to balance pH stability with metabolic control.\end{itemize}

By addressing these aspects, the fermentation process can be further optimized toward a robust, selective, and economically viable platform technology for stand-alone large-scale oxalic acid production that can be implemented into integrated supply chain for critical metal recycling, moving the technology from a standalone fermentation process to a complete circular economy solution.

nolinenumbers

Supplementary material is available.

pagebreak\bibliography{ferm_bio_1.bib}

Data Availability

All data generated or analyzed during this study are included in this published article and its supplementary information files.

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$(_{\textrm{2}})$

and {Biomass}. ChemSusChem 14(18) https://doi.org/10.1002/cssc.202101272, en, https://chemistry-europe.onlinelibrary.wiley.com/doi/10.1002/cssc.202101272, 1864-5631, 1864-564X

Fory, Ewa {APPLICATION} {OF} {REPLICATED} 23 {FULL} {FACTORIAL} {CENTRAL} {COMPOSITE} {CIRCUMSCRIBED} {DESIGN} {OF} {EXPERIMENT} ({CCC} {DOE}) {FOR} {OPTIMIZATION} {OF} {OXALATE} {BIOSYNTHESIS} {BY} {ASPERGILLUS} {NIGER} {W78C}. Fory - APPLICATION OF REPLICATED 23 FULL FACTORIAL CENTRA.pdf:C\:\\Users\\Erkmen\\Zotero\\storage\\DJNS2U89\\Fory - APPLICATION OF REPLICATED 23 FULL FACTORIAL CENTRA.pdf:application/pdf, en, Statistically based design of experiment (DOE) was applied to the optimization of synthetic medium composition for oxalic acid (OA) production by Aspergillus niger in shake-flask cultures. The Box-Wilson Central Composite Circumscribed quadratic model (CCC) was used to search the optimum levels of macroelements influencing mycelia growth and OA production. Among these variables, N, P, Mg, were found to be significant factors that affected oxalic acid production. The optimum medium composition was found to be as follows: sucrose 150 g dm-3, NH4NO3 1.3 g dm-3, KH2PO4 0.3 g dm-3, MgSO4 ·7H2O 0.64 g dm-3. Under optimal culture conditions, the maximum oxalic acid dihydrate amounted to 85.7 g dm-3 with confidence level 95%.

Verma, Ankit and Kore, Rajkumar and Corbin, David R. and Shiflett, Mark B. (2019) Metal {Recovery} {Using} {Oxalate} {Chemistry}: {A} {Technical} {Review}. Industrial & Engineering Chemistry Research 58(34): 15381--15393 https://doi.org/10.1021/acs.iecr.9b02598, Verma et al. - 2019 - Metal Recovery Using Oxalate Chemistry A Technica.pdf:C\:\\Users\\Erkmen\\Zotero\\storage\\8UW8FJ9B\\Verma et al. - 2019 - Metal Recovery Using Oxalate Chemistry A Technica.pdf:application/pdf, August, 2025-05-26, en, Energy-e ﬃcient metal recovery and separation processes from a mixture of valuable metals are vital to the metallurgy and recycling industries. Oxalate has been identi ﬁed as a sustainable reagent that can provide both the desired selectivity and e ﬃcient leaching capabilities for a variety of mixed metals under mild reaction conditions. The oxalate process has a great potential to replace many of the existing metal recovery processes that use inorganic acids such as sulfuric, hydrochloric, and nitric acids. In this Review, the use of oxalate chemistry in four major metal recovery applications is discussed, namely, spent lithium-ion batteries, spent catalysts, valuable ores, and contaminated and unwanted waste streams. Recycling of critical and precious metals from spent lithium-ion batteries and catalysts has signi ﬁcant economic opportunities. For e ﬃcient metals recovery, reaction conditions (e.g., temperature, pH, time, and concentration), metal −oxalate complex formation, oxidation and reduction, and metal precipitation must all be well-understood. This Review provides an overview from articles and patents for a variety of metal recovery processes along with insights into future process development., https://pubs.acs.org/doi/10.1021/acs.iecr.9b02598, Metal {Recovery} {Using} {Oxalate} {Chemistry}, 0888-5885, 1520-5045, https://doi.org/10.15223/policy-029

Torma, Arpad E. and Singh, Ashok K. (1993) Acidolysis of coal fly ash by {Aspergillus} niger. Fuel 72(12): 1625--1630 https://doi.org/10.1016/0016-2361(93)90346-4, Torma und Singh - 1993 - Acidolysis of coal fly ash by Aspergillus niger.pdf:C\:\\Users\\Erkmen\\Zotero\\storage\\KEQD8YA6\\Torma und Singh - 1993 - Acidolysis of coal fly ash by Aspergillus niger.pdf:application/pdf, December, 2025-06-02, en, https://linkinghub.elsevier.com/retrieve/pii/0016236193903464, 00162361, https://www.elsevier.com/tdm/userlicense/1.0/

Zhan, Lu and Zhang, Yongliang and Ahmad, Zahoor and Xu, Zhenming (2020) Novel {Recycle} {Technology} for {Recovering} {Gallium} {Arsenide} from {Scraped} {Integrated} {Circuits}. ACS Sustainable Chemistry & Engineering 8(7): 2874--2882 https://doi.org/10.1021/acssuschemeng.9b07006, Zhan et al. - 2020 - Novel Recycle Technology for Recovering Gallium Ar.pdf:C\:\\Users\\Erkmen\\Zotero\\storage\\7K22MR7K\\Zhan et al. - 2020 - Novel Recycle Technology for Recovering Gallium Ar.pdf:application/pdf, February, 2025-07-03, en, From the environmental safety and resource recovery viewpoint, a novel method of recycling gallium arsenide (GaAs) from scrapped GaAs-based integrated circuits (ICs) was proposed in this study. A hydrothermal-bu ﬀering method (HBM) has been developed in which an oxidant (H2O2) in the presence of phosphate bu ﬀer solution reacted with ICs under hydrothermal conditions. The results have shown that the packaging material was e ﬀectively decomposed without any signi ﬁcant loss of GaAs under the HBM condition. Gallium and arsenic recovery rates were 99.9 and 95.5%, respectively. In addition, thermodynamically stable regions of Ga and As species in aqueous systems were identi ﬁed by an Eh −pH diagram. The function of HBM has been studied and discussed in detail. Results have shown that Na + and OH − ions provided by a bu ﬀer system accelerate the degradation of packaging materials. Meanwhile, the OH − plays an irreplaceable role in the recovery of GaAs. The states of As on the surface of the recovered GaAs were mainly positively trivalent and pentavalent, and the corresponding compounds were As2O3 and Na2HAsO4, respectively. Under optimum conditions, without releasing any toxic gases to the environment, 91.2% of the packaging materials were decomposed, probably the free radical reaction being the main mechanism; the probable decomposition pathways of packaging materials were investigated. The study provides an e ﬃcient and environmentally friendly process to treat the IC packaging material and recover maximum GaAs for further use., https://pubs.acs.org/doi/10.1021/acssuschemeng.9b07006, 2168-0485, 2168-0485, https://doi.org/10.15223/policy-029

Rosa, Ester and Di Piazza, Simone and Cecchi, Grazia and Mazzoccoli, Michela and Zerbini, Micol and Cardinale, Anna Maria and Zotti, Mirca (2022) Applied {Tests} to {Select} the {Most} {Suitable} {Fungal} {Strain} for the {Recovery} of {Critical} {Raw} {Materials} from {Electronic} {Waste} {Powder}. Recycling 7(5): 72 https://doi.org/10.3390/recycling7050072, Rosa et al. - 2022 - Applied Tests to Select the Most Suitable Fungal S.pdf:C\:\\Users\\Erkmen\\Zotero\\storage\\C5J4DCAJ\\Rosa et al. - 2022 - Applied Tests to Select the Most Suitable Fungal S.pdf:application/pdf, October, 2025-07-03, en, Electrical and electronic wastes (WEEEs) are a potential source of raw materials. The main challenge for scientists is to set up a reliable and eco-friendly process to recycle raw materials and precious elements from WEEEs. Today, we know that fungi could play an active role in green technologies aimed at recycling valuable elements. The bioaccumulation mechanism and bioleaching activity of ﬁlamentous fungal species have already been exploited fruitfully in extraction processes. However, not all fungal strains possess the same characteristics, and it is crucial to choose the right strains to use. In this work, we show a method to assess the precious elements ’ recovery ef ﬁciency from WEEE using fungal strains. A CAS agar screening test for siderophore detection was carried out with three strains. The following plate accumulation test performed on a medium added with 120 ppm of electronic waste powder highlighted the differences in accumulation capability, growth rate, and biomass production. Among the elements in tested waste, yttrium, copper, and palladium show the highest bioconcentration factor. The results con ﬁrm the biotechnological potential of fungi to recover valuable elements at the bench scale, highlighting the importance of effective screening tests to assess the most ef ﬁcient strain for each kind of waste., https://www.mdpi.com/2313-4321/7/5/72, 2313-4321, https://creativecommons.org/licenses/by/4.0/

Varia, Jeet C. and Snellings, Ruben and Hennebel, Tom (2021) Sustainable {Metal} {Recovery} from {Secondary} {Resources}: {Screening} and {Kinetic} {Studies} {Using} {Analogue} {Heterotrophic} {Metabolites}. Waste and Biomass Valorization 12(5): 2703--2721 https://doi.org/10.1007/s12649-020-01161-8, Varia et al. - 2021 - Sustainable Metal Recovery from Secondary Resource.pdf:C\:\\Users\\Erkmen\\Zotero\\storage\\5SB6SDI7\\Varia et al. - 2021 - Sustainable Metal Recovery from Secondary Resource.pdf:application/pdf, May, 2025-07-03, en, This study presents a sustainable approach for metal extraction from a range of secondary resources. Organic acids analogue to those commonly produced by heterotrophic microbes are applied via a two-step heterotrophic bioleaching approach. New insights into leaching mechanisms are developed and the potential use of the leaching residues as cementitious component is evaluated. Tested materials include a jarosite sludge from Zn refining, four ferrous metallurgy residue sludges and two dry automotive recycling shredder residues. Screening experiments tested three organic acids (i.e. citric, gluconic, oxalic acid) and their combinations. Four physicochemical factors were varied, i.e. the solid/liquid ratio between 5 and 15%, total organic acid concentration between 20 and 200 mM, leaching times of 2 to 4 h and horizontal stirring reactor mixing rate. The highest extraction capacity was achieved for a Cr/Ni rich sludges from steel treatment with up to 96% Ni and 91% Cr extraction at a 5% S/L and a mixture of 100 mM citric and 100 mM oxalic acid. Further scale-up kinetic studies on this Cr/ Ni sludge validated the optimal leaching conditions. Modelling of kinetic leaching data indicated pore diffusion rate as a bottleneck of the overall leaching process. Screening tests of the leached residue indicated potential use as sulfate-rich addition to cement, or as part of low-performance binders for large scale civil works., https://link.springer.com/10.1007/s12649-020-01161-8, Sustainable {Metal} {Recovery} from {Secondary} {Resources}, 1877-2641, 1877-265X

Pourhossein, Fatemeh and Mousavi, Seyyed Mohammad (2019) A novel step-wise indirect bioleaching using biogenic ferric agent for enhancement recovery of valuable metals from waste light emitting diode ({WLED}). Journal of Hazardous Materials 378: 120648 https://doi.org/10.1016/j.jhazmat.2019.05.041, Pourhossein und Mousavi - 2019 - A novel step-wise indirect bioleaching using bioge.pdf:C\:\\Users\\Erkmen\\Zotero\\storage\\A22N4QYU\\Pourhossein und Mousavi - 2019 - A novel step-wise indirect bioleaching using bioge.pdf:application/pdf, October, 2025-07-03, en, Waste light-emitting diodes (WLED) are of major interest as they are a considered secondary source of valuable metals with a high potential for polluting the environment. To recover the valuable metals from WLEDs, various methods have been applied such as direct and indirect bioleaching. A novel step-wise indirect bioleaching process has been developed in this study for recycling valuable metals from WLEDs using adapted Acidithiobacillus ferrooxidans. The ferric ion concentration was controlled at 4 –5 g/L with step-wise addition of biogenic ferric for faster bioleaching rate. The results indicated the negative effect of bacterial attachment in bioleaching of WLEDs. A direct bioleaching offers low copper, nickel, and gallium leach yields, while all metals ’ recovery improved with step-wise indirect bioleaching. At a pulp density of 20 g/L, the copper, nickel, and gallium recovery efficiency was 83%, 97%, 84%, respectively. In addition, leaching time was reduced to 15 days from 30 days. From a technological perspective, the study proved that step-wise indirect bioleaching by biogenic ferric resulted in maximum valuable metal recovery from WLEDs at a low cost and via a short, simple and environmentally-friendly process., https://linkinghub.elsevier.com/retrieve/pii/S0304389419305758, 03043894

Pourhossein, Fatemeh and Mousavi, Seyyed Mohammad (2018) Enhancement of copper, nickel, and gallium recovery from {LED} waste by adaptation of {Acidithiobacillus} ferrooxidans. Waste Management 79: 98--108 https://doi.org/10.1016/j.wasman.2018.07.010, Pourhossein und Mousavi - 2018 - Enhancement of copper, nickel, and gallium recover.pdf:C\:\\Users\\Erkmen\\Zotero\\storage\\TIPQKJRT\\Pourhossein und Mousavi - 2018 - Enhancement of copper, nickel, and gallium recover.pdf:application/pdf, September, 2025-07-03, en, This paper is the ﬁrst study on the extraction of Cu, Ni, and Ga from Light Emitting Diode (LED) waste by bio-hydrometallurgy technology. LEDs have a high concentration of metals and various types of brominated ﬂame retardants (BFRs). This study demonstrates the need for strains with resistance to high concentrations of LED powder. The adaptation of Acidithiobacillus ferrooxidans to LED powder was done through a serial acclimatisation procedure in ﬁve steps of 5, 10, 15, 20, and 25 g/l. The results indicated that the heavy metals tolerance of Acidithiobacillus ferrooxidans decreased as the pulp density increased from 5 to 20 g/l. The pulp density {\textgreater} 20 g/l of LED powder caused a toxic response resulting in an evident inhibitory effect on bacterial activity. In the presence of 20 g/l of LED powder, adapted Acidithiobacillus ferrooxidans exhibits higher Fe3 + level, cell amount, ORP, and lower pH than the non-adapted cells. The recovery of copper, nickel, and gallium were higher by adapted bacteria compared to non-adapted bacteria. The adapted A. ferrooxidans leached approximately 84%, 96%, and 60%, copper, nickel, and gallium, respectively. It could be concluded that adaptation can be an effective tool for enhancement of copper, nickel, and gallium bioleaching from LED powder and adapted Acidithiobacillus ferrooxidans would be a suitable strain in LED waste bioleaching., https://linkinghub.elsevier.com/retrieve/pii/S0956053X1830432X, 0956053X

Pourhossein, Fatemeh and Mousavi, Seyyed Mohammad and Beolchini, Francesca (2022) Innovative bio-acid leaching method for high recovery of critical metals from end-of-life light emitting diodes. Resources, Conservation and Recycling 182: 106306 https://doi.org/10.1016/j.resconrec.2022.106306, Pourhossein et al. - 2022 - Innovative bio-acid leaching method for high recov.pdf:C\:\\Users\\Erkmen\\Zotero\\storage\\TL4NUPZT\\Pourhossein et al. - 2022 - Innovative bio-acid leaching method for high recov.pdf:application/pdf, July, 2025-07-03, en, The main objective of this work is to introduce a new green recycling process for recovering valuable metals from electronic waste. One of the fastest-growing electronic waste streams is end-of-life light emitting diodes (LEDs). Due to environmental risk and a worldwide shortage of metals, recycling and recovering their valuable metal is an urgent task. In this study, an innovative bio-hydrometallurgical method for the extraction of valuable metals from end-of-life LED lamps is proposed. The present study evaluated a direct multi-step regulation strategy of waste content to enhance the bio-acid leaching of LED at a high pulp density (40 g/L) using a culture of sulfuroxidizing bacteria Acidithiobacillus thiooxidans (A. thiooxidans). Through a step-wise feeding strategy (10 g/d), the extraction of the metal was further improved due to balancing of A. thiooxidans population and stabilization of pH, ORP, and sulfuric acid concentration in the bioleaching solution. By comparing the results of multi-step and one-stage contact bioleaching, with multi-step contact bioleaching, Cu, Ni, and Ga extraction yields increased from 67%, 92%, and 39% to 100%, 100%, and 75%. Furthermore, this study demonstrates that the presence/ addition of EPS did not only adsorb metal ions but also increased diffusion barriers, thus diminishing metal ion transfer from LED into bioleaching solution for an extended period of time. The multi-step non-contact bio-acid leaching method enhanced strategic critical element leaching yields to 100% from end-of-life LEDs. The final product, enriched in Au and Ag, could be used as a secondary source of precious metals., https://linkinghub.elsevier.com/retrieve/pii/S0921344922001549, 09213449

Fritze, Mareike and Hedrich, Sabrina (2024) Bioleaching of Gallium from Metal Hydroxide Sludge Produced as a Waste Product During GaAs Wafer Production. Springer, 1013--1015, Conference of Metallurgists

Schmitt, Vanessa and Derenbach, Laura and Ochsenreither, Katrin (2022) Enhanced l-{Malic} {Acid} {Production} by {Aspergillus} oryzae {DSM} 1863 {Using} {Repeated}-{Batch} {Cultivation}. Frontiers in Bioengineering and Biotechnology 9: 760500 https://doi.org/10.3389/fbioe.2021.760500, Schmitt et al. - 2022 - Enhanced l-Malic Acid Production by Aspergillus or.pdf:C\:\\Users\\Erkmen\\Zotero\\storage\\GXRW26G6\\Schmitt et al. - 2022 - Enhanced l-Malic Acid Production by Aspergillus or.pdf:application/pdf, January, 2025-07-03, en, L-Malic acid is a C4-dicarboxylic acid and a potential key building block for a bio-based economy. At present, malic acid is synthesized petrochemically and its major market is the food and beverages industry. In future, malic acid might also serve as a building block for biopolymers or even replace the commodity chemical maleic anhydride. For a sustainable production of L-malic acid from renewable resources, the microbial synthesis by the mold Aspergillus oryzae is one possible route. As CO2 ﬁxation is involved in the biosynthesis, high yields are possible, and at the same time greenhouse gases can be reduced. In order to enhance the production potential of the wild-type strain Aspergillus oryzae DSM 1863, process characteristics were studied in shake ﬂasks, comparing batch, fed-batch, and repeated-batch cultivations. In the batch process, a prolonged cultivation time led to malic acid consumption. Keeping carbon source concentration on a high level by pulsed feeding could prolong cell viability and cultivation time, however, did not result in signi ﬁcant higher product levels. In contrast, continuous malic acid production could be achieved over six exchange cycles and a total fermentation time of 19 days in repeated-batch cultivations. Up to 178 g/L L-malic acid was produced. The maximum productivity (0.90 ± 0.05 g/L/h) achieved in the repeated-batch cultivation had more than doubled than that achieved in the batch process and also the average productivity (0.42 ± 0.03 g/L/h for ﬁve exchange cycles and 16 days) was increased considerably. Further repeated-batch experiments con ﬁrmed a positive effect of regular calcium carbonate additions on pH stability and malic acid synthesis. Besides calcium carbonate, nitrogen supplementation proved to be essential for the prolonged malic acid production in repeated-batch. As prolonged malic acid production was only observed in cultivations with product removal, product inhibition seems to be the major limiting factor for malic acid production by the wild-type strain. This study provides a systematic comparison of different process strategies under consideration of major in ﬂuencing factors and thereby delivers important insights into natural L-malic acid production., https://www.frontiersin.org/articles/10.3389/fbioe.2021.760500/full, 2296-4185

Lee, Sang Il and Lee, Keon Jin and Chun, Ho Hyun and Ha, Sanghyun and Gwak, Hyun Jung and Kim, Ho Myeong and Lee, Jong-Hee and Choi, Hak-Jong and Kim, Hyeong Hwan and Shin, Teak Soo and Park, Hae Woong and Kim, Jin-Cheol (2018) Process development of oxalic acid production in submerged culture of {Aspergillus} niger {F22} and its biocontrol efficacy against the root-knot nematode {Meloidogyne} incognita. Bioprocess and Biosystems Engineering 41(3): 345--352 https://doi.org/10.1007/s00449-017-1867-y, Lee et al. - 2018 - Process development of oxalic acid production in s.pdf:C\:\\Users\\Erkmen\\Zotero\\storage\\M7E97VJP\\Lee et al. - 2018 - Process development of oxalic acid production in s.pdf:application/pdf, March, 2025-07-03, en, Oxalic acid has potent nematicidal activity against the root-knot nematode Meloidogyne incognita. In this study, fermentation parameters for oxalic acid production in submerged culture of Aspergillus niger F22 at 23, 25, and 30 °C were optimized in 5-L jar fermenters. The viscosity of the culture broth increased with increasing temperature. There was a negative correlation between oxalic acid production and the apparent viscosity; high volumetric productivity of oxalic acid was obtained at low apparent viscosity (less than 1000 cP), with a productivity of more than 100 mg/L h. When the apparent viscosity was over 2500 cP, the volumetric productivity decreased below 50 mg/L h. In addition, the volumetric mass transfer coefficient, KLa, positively correlated with volumetric productivity. When the KLa value increased from 0.0 to 0.017 /s, the volumetric productivity proportionally increased up to 176 mg/L h. When the temperature decreased, KLa increased due to the decrease in viscosity, leading to increased volumetric productivity. The highest productivity of 7453.3 mg/L was obtained at the lowest temperature, i.e., 23 °C. The nematicidal activity of culture filtrate was proportional to the content of oxalic acid. Based on a constant impeller tip speed, oxalic acid production was successfully scaled up to a 500-L pilot vessel, producing a final concentration comparable to that in the 5-L jar., http://link.springer.com/10.1007/s00449-017-1867-y, 1615-7591, 1615-7605

Currie, James N. and Thom, Charles (1915) {AN} {OXALIC} {ACID} {PRODUCING} {PENICILLIUM}. Journal of Biological Chemistry 22(2): 287--293 https://doi.org/10.1016/S0021-9258(18)87646-3, Currie und Thom - 1915 - AN OXALIC ACID PRODUCING PENICILLIUM.pdf:C\:\\Users\\Erkmen\\Zotero\\storage\\95UUB6T3\\Currie und Thom - 1915 - AN OXALIC ACID PRODUCING PENICILLIUM.pdf:application/pdf, September, 2025-07-05, en, https://linkinghub.elsevier.com/retrieve/pii/S0021925818876463, 00219258, https://www.elsevier.com/tdm/userlicense/1.0/

Zhao, Shuai and Zheng, Bo-Wen and Wang, Yu-Cang and He, Fei and Wang, Li-Juan and Lin, Xiong and Luo, Xue-Mei and Feng, Jia-Xun (2023) Environmentally-friendly biorecovery of manganese from electrolytic manganese residue using a novel {Penicillium} oxalicum strain {Z6}-5-1: {Kinetics} and mechanism. Journal of Hazardous Materials 446: 130662 https://doi.org/10.1016/j.jhazmat.2022.130662, Zhao et al. - 2023 - Environmentally-friendly biorecovery of manganese .pdf:C\:\\Users\\Erkmen\\Zotero\\storage\\J5CRK65Y\\Zhao et al. - 2023 - Environmentally-friendly biorecovery of manganese .pdf:application/pdf, March, 2025-07-05, en, https://linkinghub.elsevier.com/retrieve/pii/S030438942202458X, Environmentally-friendly biorecovery of manganese from electrolytic manganese residue using a novel {Penicillium} oxalicum strain {Z6}-5-1, 03043894

Jiang, Yifan and Tian, Jiang and Ge, Fei (2020) New {Insight} into {Carboxylic} {Acid} {Metabolisms} and {pH} {Regulations} {During} {Insoluble} {Phosphate} {Solubilisation} {Process} by {Penicillium} oxalicum {PSF}-4. Current Microbiology 77(12): 4095--4103 https://doi.org/10.1007/s00284-020-02238-2, Jiang et al. - 2020 - New Insight into Carboxylic Acid Metabolisms and p.pdf:C\:\\Users\\Erkmen\\Zotero\\storage\\JV68E8FX\\Jiang et al. - 2020 - New Insight into Carboxylic Acid Metabolisms and p.pdf:application/pdf, December, 2025-07-05, en, Phosphate solubilising fungi (PSF) play an important role in increasing phosphorus (P) bioavailability and the fertility of soil. However, the mechanism by which PSF solubilise insoluble P using carboxylic acid is not fully understood. In this study, Penicillium oxalicum PSF-4 was isolated and shown to have satisfactory solubilisation performance towards tricalcium phosphate (TP) and iron phosphate (IP). In treatments with TP or IP, the soluble P (Psol) concentrations were positively correlated with the carboxyl groups in organic acids but negatively correlated with the pKsp of insoluble P sources and pH. Additionally, the compositions of the organic acids (considering the lowest pKa value for acids with several pKas) were remarkably different between the treatments: oxalic (pKa = 1.27), malic (pKa = 3.46), and formic (pKa = 3.75) acids in TP treatments (maximum 381 mg/L); and lactic (pKa = 3.85), gluconic (pKa = 3.86), and citric (pKa = 3.12) acids in IP treatments (maximum 1634 mg/L). The addition of H + without P. oxalicum PSF-4 inoculation markedly improved Psol concentrations. The above results offer new insights that the pKsp of P sources, compositions of carboxylic acids, and pH regulation are the key factors influencing P solubilisation of different insoluble P sources., https://link.springer.com/10.1007/s00284-020-02238-2, 0343-8651, 1432-0991

Odoni, Dorett I.. Organic acid production in {Aspergillus} niger and other filamentous fungi. Odoni - 2017 - Organic acid production in Aspergillus niger and o.pdf:C\:\\Users\\Erkmen\\Zotero\\storage\\G4X5SACC\\Odoni - 2017 - Organic acid production in Aspergillus niger and o.pdf:application/pdf, 10.18174/423035, 2017, October, Wageningen University, 2025-07-05, en, https://research.wur.nl/en/publications/f5cdab36-26b2-4af8-8e84-65f77a30b7eb

Odoni, Dorett I and Laothanachareon, Thanaporn and Vazquez-Vilar, Marta and van, Merlijn P and Schonewille, Tom and Bruinsma, Lyon and Suarez-Diez, Maria and Schaap, Peter J Aspergillus niger citrate exporter revealed by comparison of two alternative citrate producing conditions. Odoni et al. - Aspergillus niger citrate exporter revealed by com.pdf:C\:\\Users\\Erkmen\\Zotero\\storage\\9ZAQ7IRK\\Odoni et al. - Aspergillus niger citrate exporter revealed by com.pdf:application/pdf, en

Li, Zhen and Bai, Tongshuo and Dai, Letian and Wang, Fuwei and Tao, Jinjin and Meng, Shiting and Hu, Yunxiao and Wang, Shimei and Hu, Shuijin (2016) A study of organic acid production in contrasts between two phosphate solubilizing fungi: {Penicillium} oxalicum and {Aspergillus} niger. Scientific Reports 6: 25313 https://doi.org/10.1038/srep25313, April, 2025-07-05, https://www.nature.com/articles/srep25313, A study of organic acid production in contrasts between two phosphate solubilizing fungi, 2045-2322

Zhang, Yifan and Gao, Hui and Zhao, Dongliang and Chen, Xueming and Zhu, Feng and Li, Yinsheng and Xue, Shengguo (2024) Microbially-driven alkaline regulation: {Organic} acid secretion behavior of {Penicillium} oxalicum and charge neutralization in bauxite residue. Environmental Research 240: 117489 https://doi.org/10.1016/j.envres.2023.117489, Zhang et al. - 2024 - Microbially-driven alkaline regulation Organic ac.pdf:C\:\\Users\\Erkmen\\Zotero\\storage\\8NLFQ2HW\\Zhang et al. - 2024 - Microbially-driven alkaline regulation Organic ac.pdf:application/pdf, January, 2025-07-05, en, Microbially-driven alkaline neutralization in bauxite residue by functional microorganisms is a promising approach for the ecological rehabilitation on alkaline disposal areas. However, the alkali resistance and acid secretion mechanism of functional microorganisms are still unknown, which limits their application. Here, saline-alkaline resistance, acid production performance, and differentially expressed genes of Penicillium oxalicum (P. oxalicum, a functional fungus screened from a typical disposal area) were investigated and its bioneutralization efficiency was evaluated. This fungus exhibited high tolerance to alkalinity (pH 12), and salinity (NaCl 2.0 M), and produced a large amount of oxalic acid to reduce the medium pH to 2.0. Transcriptome showed that alkali stress induced the overexpression of genes related to antioxidant and stress-resistant enzymes (GST, KatE) and glycolytic pathway rate-limiting enzymes (HK). The rate of glycolysis and other organic acid metabolism processes was increased with higher stress resistance of P. oxalicum. The integrated application of P. oxalicum and maize straw accelerated the dissolved organic carbon content and stabilized the leachate pH of bauxite residue at about 7.4. 3DEEM and BIOSEM analysis indicated that P. oxalicum maintained high activity in the residue leachate and continuously decomposed the maize straw for their metabolism. P. oxalicum showed strong alkaline resistance, biomass degradation capacity, and alkaline regulation potential, which should be beneficial for microbial-driven alkaline regulation in bauxite residue., https://linkinghub.elsevier.com/retrieve/pii/S0013935123022934, Microbially-driven alkaline regulation, 00139351

Nadi, Sahba and Pourhossein, Fatemeh and Mousavi, Seyyed Mohammad (2025) Sustainable bio-extraction of rare earth elements from discarded {LED} lamps. Scientific Reports 15(1): 21655 https://doi.org/10.1038/s41598-025-04937-x, Nadi et al. - 2025 - Sustainable bio-extraction of rare earth elements .pdf:C\:\\Users\\Erkmen\\Zotero\\storage\\CFX9EX9A\\Nadi et al. - 2025 - Sustainable bio-extraction of rare earth elements .pdf:application/pdf, July, 2025-07-05, en, https://www.nature.com/articles/s41598-025-04937-x, 2045-2322

Rohr, Katja and Gremm, Lisa and Geinitz, Bertram and Jourdier, Etienne and Wiechert, Wolfgang and Ben Chaabane, Fadhel and Oldiges, Marco (2024) Optimizing microbioreactor cultivation strategies for {Trichoderma} reesei: from batch to fed-batch operations. Microbial Cell Factories 23(1): 112 https://doi.org/10.1186/s12934-024-02371-8, Rohr et al. - 2024 - Optimizing microbioreactor cultivation strategies .pdf:C\:\\Users\\Erkmen\\Zotero\\storage\\UUUK8NRK\\Rohr et al. - 2024 - Optimizing microbioreactor cultivation strategies .pdf:application/pdf, April, 2025-07-05, en, Background Filamentous fungi have long been recognized for their exceptional enzyme production capabilities. Among these, Trichoderma reesei has emerged as a key producer of various industrially relevant enzymes and is particularly known for the production of cellulases. Despite the availability of advanced gene editing techniques for T. reesei, the cultivation and characterization of resulting strain libraries remain challenging, necessitating welldefined and controlled conditions with higher throughput. Small-scale cultivation devices are popular for screening bacterial strain libraries. However, their current use for filamentous fungi is limited due to their complex morphology. Results This study addresses this research gap through the development of a batch cultivation protocol using a microbioreactor for cellulase-producing T. reesei strains (wild type, RutC30 and RutC30 TR3158) with offline cellulase activity analysis. Additionally, the feasibility of a microscale fed-batch cultivation workflow is explored, crucial for mimicking industrial cellulase production conditions. A batch cultivation protocol was developed and validated using the BioLector microbioreactor, a Round Well Plate, adapted medium and a shaking frequency of 1000 rpm. A strong correlation between scattered light intensity and cell dry weight underscores the reliability of this method in reflecting fungal biomass formation, even in the context of complex fungal morphology. Building on the batch results, a fed-batch strategy was established for T. reesei RutC30. Starting with a glucose concentration of 2.5 g l −1in the batch phase, we introduced a dual-purpose lactose feed to induce cellulase production and prevent carbon catabolite repression. Investigating lactose feeding rates from 0.3 to 0.75 g (l h) −1 , the lowest rate of 0.3 g (l h) −1revealed a threefold increase in cellobiohydrolase and a fivefold increase in β-glucosidase activity compared to batch processes using the same type and amount of carbon sources. Conclusion We successfully established a robust microbioreactor batch cultivation protocol for T. reesei wild type, RutC30 and RutC30 TR3158, overcoming challenges associated with complex fungal morphologies. The study highlights the effectiveness of microbioreactor workflows in optimizing cellulase production with T. reesei, providing a valuable tool for simultaneous assessment of critical bioprocess parameters and facilitating efficient strain screening. The findings underscore the potential of microscale fed-batch strategies for enhancing enzyme production capabilities, revealing insights for future industrial applications in biotechnology., https://microbialcellfactories.biomedcentral.com/articles/10.1186/s12934-024-02371-8, Optimizing microbioreactor cultivation strategies for {Trichoderma} reesei, 1475-2859

Nielsen, Jens (1996) Modelling the morphology of filamentous microorganisms. Trends in Biotechnology 14(11): 438--443 https://doi.org/10.1016/0167-7799(96)10055-X, Nielsen - 1996 - Modelling the morphology of filamentous microorgan.pdf:C\:\\Users\\Erkmen\\Zotero\\storage\\G6798FXG\\Nielsen - 1996 - Modelling the morphology of filamentous microorgan.pdf:application/pdf, November, 2025-07-05, en, https://linkinghub.elsevier.com/retrieve/pii/016777999610055X, 01677799, https://www.elsevier.com/tdm/userlicense/1.0/

Umar, Aisha and Abid, Islem and Elshikh, Mohamed S and Dufoss é, Laurent and Abdel-Azeem, Ahmed M. and Ali, Iftikhar (2023) Agitation role ({Dissolved} {Oxygen}) in production of laccase from newly identified {Ganoderma} multistipitatum sp. nov. and its effect on mycelium morphology. BMC Microbiology 23: 280 https://doi.org/10.1186/s12866-023-03009-2, October, 2025-07-05, https://bmcmicrobiol.biomedcentral.com/articles/10.1186/s12866-023-03009-2, 1471-2180

Schrader, Marcel and Schrinner, Kathrin and Polomsky, Laura and Ivanov, Dimitri and Kampen, Ingo and Schilde, Carsten and Krull, Rainer and Kwade, Arno (2023) Quantification and modeling of macroparticle-induced mechanical stress for varying shake flask cultivation conditions. Frontiers in Bioengineering and Biotechnology 11: 1254136 https://doi.org/10.3389/fbioe.2023.1254136, September, 2025-07-05, en, https://www.frontiersin.org/articles/10.3389/fbioe.2023.1254136/full, 2296-4185

B öl, Markus and Schrinner, Kathrin and Tesche, Sebastian and Krull, Rainer (2021) Challenges of influencing cellular morphology by morphology engineering techniques and mechanical induced stress on filamentous pellet systems —{A} critical review. Engineering in Life Sciences 21(3-4): 51--67 https://doi.org/10.1002/elsc.202000060, March, 2025-07-05, en, https://onlinelibrary.wiley.com/doi/10.1002/elsc.202000060, 1618-0240, 1618-2863

Karaffa, Levente and Kubicek, Christian P. (2019) Citric acid and itaconic acid accumulation: variations of the same story?. Applied Microbiology and Biotechnology 103(7): 2889--2902 https://doi.org/10.1007/s00253-018-09607-9, April, 2025-07-05, en, http://link.springer.com/10.1007/s00253-018-09607-9, Citric acid and itaconic acid accumulation, 0175-7598, 1432-0614

Lv, Jun and Zhang, Bo-Bo and Liu, Xiao-Dong and Zhang, Chan and Chen, Lei and Xu, Gan-Rong and Cheung, Peter Chi Keung (2017) Enhanced production of natural yellow pigments from {Monascus} purpureus by liquid culture: {The} relationship between fermentation conditions and mycelial morphology. Journal of Bioscience and Bioengineering 124(4): 452--458 https://doi.org/10.1016/j.jbiosc.2017.05.010, Lv et al. - 2017 - Enhanced production of natural yellow pigments fro.pdf:C\:\\Users\\Erkmen\\Zotero\\storage\\XJQL54WM\\Lv et al. - 2017 - Enhanced production of natural yellow pigments fro.pdf:application/pdf, October, 2025-07-05, en, https://linkinghub.elsevier.com/retrieve/pii/S1389172317302360, Enhanced production of natural yellow pigments from {Monascus} purpureus by liquid culture, 13891723, https://www.elsevier.com/tdm/userlicense/1.0/

Walisko, Robert and Moench-Tegeder, Judith and Blotenberg, Jana and Wucherpfennig, Thomas and Krull, Rainer The {Taming} of the {Shrew} - {Controlling} the {Morphology} of {Filamentous} {Eukaryotic} and {Prokaryotic} {Microorganisms}. In: Krull, Rainer and Bley, Thomas (Eds.) Filaments in {Bioprocesses}, Walisko et al. - 2015 - The Taming of the Shrew - Controlling the Morpholo.pdf:C\:\\Users\\Erkmen\\Zotero\\storage\\PH5L8FEZ\\Walisko et al. - 2015 - The Taming of the Shrew - Controlling the Morpholo.pdf:application/pdf, 1--27, Series Title: Advances in Biochemical Engineering/Biotechnology, 10.1007/10_2015_322, 2015, Springer International Publishing, 2025-07-05, en, https://link.springer.com/10.1007/10_2015_322, 978-3-319-20510-6 978-3-319-20511-3, 149, Cham

Olmos, E. and Mehmood, N. and Haj Husein, L. and Goergen, J.-L. and Fick, M. and Delaunay, S. (2013) Effects of bioreactor hydrodynamics on the physiology of {Streptomyces}. Bioprocess and Biosystems Engineering 36(3): 259-272 https://doi.org/10.1007/s00449-012-0794-1, March, 2025-07-05, en, http://link.springer.com/10.1007/s00449-012-0794-1, 1615-7591, 1615-7605, http://www.springer.com/tdm

Sitanggang, A B and Wu, H S and Wang, S S and Lan, J C W Fermentation {Strategies}: {Nutritional} {Requirements}. en

Suresh, S. and Srivastava, V.C. and Mishra, I.M. (2009) Critical analysis of engineering aspects of shaken flask bioreactors. Critical Reviews in Biotechnology 29(4): 255--278 https://doi.org/10.3109/07388550903062314, Suresh et al. - 2009 - Critical analysis of engineering aspects of shaken.pdf:C\:\\Users\\Erkmen\\Zotero\\storage\\DV3GUQE9\\Suresh et al. - 2009 - Critical analysis of engineering aspects of shaken.pdf:application/pdf, December, 2025-07-05, en, http://www.tandfonline.com/doi/full/10.3109/07388550903062314, 0738-8551, 1549-7801

Papagianni, Maria (2007) Advances in citric acid fermentation by {Aspergillus} niger: {Biochemical} aspects, membrane transport and modeling. Biotechnology Advances 25(3): 244--263 https://doi.org/10.1016/j.biotechadv.2007.01.002, Papagianni - 2007 - Advances in citric acid fermentation by Aspergillu.pdf:C\:\\Users\\Erkmen\\Zotero\\storage\\NXUR52VY\\Papagianni - 2007 - Advances in citric acid fermentation by Aspergillu.pdf:application/pdf, May, 2025-07-05, en, Citric acid is regarded as a metabolite of energy metabolism, of which the concentration will rise to appreciable amounts only under conditions of substantive metabolic imbalances. Citric acid fermentation conditions were established during the 1930s and 1940s, when the effects of various medium components were evaluated. The biochemical mechanism by which Aspergillus niger accumulates citric acid has continued to attract interest even though its commercial production by fermentation has been established for decades. Although extensive basic biochemical research has been carried out with A. niger, the understanding of the events relevant for citric acid accumulation is not completely understood. This review is focused on citric acid fermentation by A. niger. Emphasis is given to aspects of fermentation biochemistry, membrane transport in A. niger and modeling of the production process. © 2007 Elsevier Inc. All rights reserved., https://linkinghub.elsevier.com/retrieve/pii/S0734975007000183, Advances in citric acid fermentation by {Aspergillus} niger, 07349750, https://www.elsevier.com/tdm/userlicense/1.0/

Rikmanis, Maris and Berzin š, Andrejs and Viesturs, Uldis (2007) Excess turbulence as a cause of turbohypobiosis in cultivation of microorganisms. Open Life Sciences 2(4): 481--501 https://doi.org/10.2478/s11535-007-0038-6, Rikmanis et al. - 2007 - Excess turbulence as a cause of turbohypobiosis in.pdf:C\:\\Users\\Erkmen\\Zotero\\storage\\DGQNHNZM\\Rikmanis et al. - 2007 - Excess turbulence as a cause of turbohypobiosis in.pdf:application/pdf, December, 2025-07-05, en, The present review describes the in ﬂuence of di ﬀerent types of mixing systems under excess turbulence conditions on microorganisms. Turbohypobiosis phenomena were described by applying a method for measurement of the kinetic energy of ﬂow ﬂuctuations based on the piezoe ﬀect. It can be assumed that the shear stress e ﬀect (the state of turbohypobiosis) plays a role mainly when alternative mechanisms in cells cannot ensure a normal physiological state under stress conditions. Practically any system (inner construction of a bioreactor, culture and cultivation conditions, including mixing) requires its own optimisation to achieve the ﬁnal goal, namely, the maximum product and/or biomass yields from substrate (YP/S or/and YX/S ), respectively. Data on the biotechnological performance of cultivation as well as power input, kinetic energy (e) of ﬂow ﬂuctuations, air consumption rate, rotational speed, tip speed, etc. do not correlate directly if the mixing systems (impellers-ba ﬄes) are dissimilar. Even the widely used speci ﬁc power consumption cannot be relied upon for scaling up the cultivation performance using dissimilar mixing systems. A biochemical explanation for substrate and product transport via cell walls, carbon pathways, energy generation and utilisation, etc. furnishes insight into cellular interactions with turbulence of di ﬀerent origin for di ﬀerent types of microorganisms (single cells, mycelia forming cells, etc.)., https://www.degruyter.com/document/doi/10.2478/s11535-007-0038-6/html, 2391-5412, http://creativecommons.org/licenses/by-nc-nd/3.0/

Papagianni, Maria and Mattey, Michael (2006) Morphological development of {Aspergillus} niger in submerged citric acid fermentation as a function of the spore inoculum level. {Application} of neural network and cluster analysis for characterization of mycelial morphology. Microbial Cell Factories 5(1): 3 https://doi.org/10.1186/1475-2859-5-3, Papagianni und Mattey - 2006 - Morphological development of Aspergillus niger in .pdf:C\:\\Users\\Erkmen\\Zotero\\storage\\TY95LLYU\\Papagianni und Mattey - 2006 - Morphological development of Aspergillus niger in .pdf:application/pdf, December, 2025-07-05, en, Background: Although the citric acid fermentation by Aspergillus niger is one of the most important industrial microbial processes and various aspects of the fermentation appear in a very large number of publications since the 1950s, the effect of the spore inoculum level on fungal morphology is a rather neglected area. The aim of the presented investigations was to quantify the effects of changing spore inoculum level on the resulting mycelial morphology and to investigate the physiology that underlines the phenomena. Batch fermentations were carried out in a stirred tank bioreactor, which were inoculated directly with spores in concentrations ranging from 104 to 109 spores per ml. Morphological features, evaluated by digital image analysis, were classified using an artificial neural network (ANN), which considered four main object types: globular and elongated pellets, clumps and free mycelial trees. The significance of the particular morphological features and their combination was determined by cluster analysis. Results: Cell volume fraction analysis for the various inoculum levels tested revealed that by rising the spore inoculum level from 104 to 109 spores per ml, a clear transition from pelleted to dispersed forms occurs. Glucosamine formation and release by the mycelium appears to be related to spore inoculum level. Maximum concentrations detected in fermentations inoculated with 104 and 105 spores/ml, where pellets predominated. At much higher inoculum levels (108, 109 spores/ml), lower dissolved oxygen levels during the early fermentation phase were associated with slower ammonium ions uptakes and significantly lower glucosamine concentrations while the mycelium developed in dispersed morphologies. A big increase in the main and total hyphal lengths and branching frequency was observed in mycelial trees as inoculum levels rise from 104 to 109 spores/ml, while in aggregated forms particle sizes and their compactness decreased. Conclusion: The methods used in this study, allowed for the detailed quantification of the transition between the two extreme morphological forms. The impact of spore inoculum level on the detailed characteristics of the particular morphological forms produced was high. Control of mycelial morphology is often regarded as a prerequisite to ensure increased productivities in industrial applications. The research described here demonstrates that adjusting the spore inoculum level controls effectively mycelial morphology., https://microbialcellfactories.biomedcentral.com/articles/10.1186/1475-2859-5-3, 1475-2859

Papagianni, Maria (2004) Fungal morphology and metabolite production in submerged mycelial processes. Biotechnology Advances 22(3): 189--259 https://doi.org/10.1016/j.biotechadv.2003.09.005, Papagianni - 2004 - Fungal morphology and metabolite production in sub.pdf:C\:\\Users\\Erkmen\\Zotero\\storage\\4M399CMQ\\Papagianni - 2004 - Fungal morphology and metabolite production in sub.pdf:application/pdf, January, 2025-07-05, en, The use of fungi for the production of commercial products is ancient, but it has increased rapidly over the last 50 years. Fungi are morphologically complex organisms, differing in structure at different times in their life cycle, differing in form between surface and submerged growth, differing also with the nature of the growth medium and physical environment. Many genes and physiological mechanisms are involved in the process of morphogenesis. In submerged culture, a large number of factors contribute to the development of any particular morphological form. Factors affecting morphology include the type and concentration of carbon substrate, levels of nitrogen and phosphate, trace minerals, dissolved oxygen and carbon dioxide, pH and temperature. Physical factors affecting morphology include fermenter geometry, agitation systems, rheology and the culture modes, whether batch, fed-batch or continuous. In many cases, particular morphological forms achieve maximum performance. It is a very difficult task to deduce unequivocal general relationships between process variables, product formation and fungal morphology since too many parameters influence these interrelationships and the role of many of them is still not fully understood., https://linkinghub.elsevier.com/retrieve/pii/S073497500300168X, 07349750, https://www.elsevier.com/tdm/userlicense/1.0/

Cui, Y.Q and van der Lans, R.G.J.M and Giuseppin, M.L.F and Luyben, K.C.A.M (1998) Influence of fermentation conditions and scale on the submerged fermentation of {Aspergillus} awamori. Enzyme and Microbial Technology 23(1-2): 157--167 https://doi.org/10.1016/S0141-0229(98)00041-6, Cui et al. - 1998 - Influence of fermentation conditions and scale on .pdf:C\:\\Users\\Erkmen\\Zotero\\storage\\8T46P5B9\\Cui et al. - 1998 - Influence of fermentation conditions and scale on .pdf:application/pdf, July, 2025-07-05, en, https://linkinghub.elsevier.com/retrieve/pii/S0141022998000416, 01410229, https://www.elsevier.com/tdm/userlicense/1.0/

Prosser, J. I. and Tough, A. J. (1991) Growth {Mechanisms} and {Growth} {Kinetics} of {Filamentous} {Microorganisms}. Critical Reviews in Biotechnology 10(4): 253--274 https://doi.org/10.3109/07388559109038211, Prosser und Tough - 1991 - Growth Mechanisms and Growth Kinetics of Filamento.pdf:C\:\\Users\\Erkmen\\Zotero\\storage\\FBWXC5LW\\Prosser und Tough - 1991 - Growth Mechanisms and Growth Kinetics of Filamento.pdf:application/pdf, January, 2025-07-05, en, Filamentous microorganisms are of major biotechnological importance, being responsible for production of the majority of secondary metabolites, particularly antibiotics. Two main groups are involved, filamentous fungi and filamentous actinomycetes, particularly the streptomycetes. In terms of cellular growth mechanisms, these groups differ greatly. Eukaryotic fungi possess subcellular organelles and cytoskeletal structures directing growth while prokaryotic streptomycetes have no such cellular organization. Despite these fundamental differences, both groups exhibit similar morphologies, growth patterns, growth forms, and hyphal and mycelial growth kinetics on solid media and in liquid culture both grow as dispersed mycelia and pellets. The article therefore discusses the relationship between cellular growth mechanisms and vegetative growth in both filamentous fungi and actinomycetes, the conceptual and theoretical models applicable to both groups, and the significance of such models in industrial fermentation processes., http://www.tandfonline.com/doi/full/10.3109/07388559109038211, 0738-8551, 1549-7801

Metz, B. and Kossen, N. W. F. (1977) The growth of molds in the form of pellets –a literature review. Biotechnology and Bioengineering 19(6): 781--799 https://doi.org/10.1002/bit.260190602, Metz und Kossen - 1977 - The growth of molds in the form of pellets –a liter.pdf:C\:\\Users\\Erkmen\\Zotero\\storage\\WTKHKLQZ\\Metz und Kossen - 1977 - The growth of molds in the form of pellets –a liter.pdf:application/pdf, June, 2025-07-05, en, https://onlinelibrary.wiley.com/doi/10.1002/bit.260190602, 0006-3592, 1097-0290, http://onlinelibrary.wiley.com/termsAndConditions\#vor

Modeling, {Simulation} and {Experimental} {Characterization} of {Mechanical} and {Surface}-{Induced} {Stresses} on {Pellets} of the {Filamentous} {Microorganism} {Lentzea} aerocolonigenes. Dispersity, {Structure} and {Phase} {Changes} of {Proteins} and {Bio} {Agglomerates} in {Biotechnological} {Processes}, 10.1007/978-3-031-63164-1_17, 2024, Dinius, Anna and Schrader, Marcel and Schrinner, Kathrin and Kampen, Ingo and Krull, Rainer and Kwade, Arno, Springer Nature Switzerland, 2025-07-07, en, https://link.springer.com/10.1007/978-3-031-63164-1_17, 978-3-031-63163-4 978-3-031-63164-1, https://www.springernature.com/gp/researchers/text-and-data-mining, Cham

Santoro, R and Cameselle, C and Rodriguez-Couto, S and Sanroman, A (1999) Influence of milk whey, nitrogen and phosphorus concentration on oxalic acid production by Aspergillus niger. Bioprocess Engineering 20(1): 1--5 https://doi.org/10.1007/PL00009032, Springer

Zheng, Kun and Benedetti, Marc F. and Jain, Rohan and Pollmann, Katrin and {van Hullebusch}, Eric D. (2024) Recovery of Gallium (and Indium) from Spent {{LEDs}}: {{Strong}} Acids Leaching versus Selective Leaching by Siderophore Desferrioxamine {{E}}. Separation and Purification Technology 338: 126566 https://doi.org/10.1016/j.seppur.2024.126566, 2024-02-12, 1383-5866, June, Recovery of Gallium (and Indium) from Spent {{LEDs}}

Ksi{\k{a}}{\.z}ek, Ewelina (2023) Citric acid: properties, microbial production, and applications in industries. Molecules 29(1): 22 https://doi.org/10.3390/molecules29010022, MDPI

Maneesuwannarat, Sirikan and Kudpeng, Kanjana and Yingchutrakul, Yodying and Roytrakul, Sittiruk and Vangnai, Alisa S and Yamashita, Mitsuo and Thiravetyan, Paitip (2019) A possible protein model involved in gallium arsenide leaching by Cellulosimicrobium funkei. Minerals Engineering 137 https://doi.org/10.1016/j.mineng.2019.04.002, Elsevier

Erkmen, Aylin Nur and Ulber , Roland and J{\"u}stel, Thomas and Altendorfner, Mirjam (2025) Towards sustainable recycling of critical metals from e-waste: Bioleaching and phytomining. Resources, Conservation and Recycling 215: 108057 https://doi.org/10.1016/j.hazadv.2023.100397, Elsevier

Chowdhury, Dilder (2020) Power Gallium Nitride Technology: The Need for Efficient Power Conversion. IEEE Electrification Magazine 8(2): 6-10 https://doi.org/10.1109/MELE.2020.2985480, Gallium nitride;Silicon;Logic gates;Power conversion;HEMTs;Inverters

Akyildiz, Kubra and Kim, Ji-Hye and So, Ju-Hee and Koo, Hyung-Jun (2022) Recent progress on micro-and nanoparticles of gallium-based liquid metals: From preparation to applications. Journal of Industrial and Engineering Chemistry 116: 120--141 Elsevier

Vinhal, Jonathan Ten{\'o}rio and de Oliveira, Rafael Piumatti and Coleti, Jorge Luis and Espinosa, Denise Crocce Romano (2022) Characterization of end-of-life LEDs: Mapping critical, valuable and hazardous elements in different devices. Waste Management 151: 113--122 Elsevier

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Cenci, Marcelo Pilotto and Dal Berto, Frederico Christ and Camargo, Priscila Silva Silveira and Veit, Hugo Marcelo (2021) Separation and concentration of valuable and critical materials from wasted LEDs by physical processes. Waste Management 120: 136--145 Elsevier

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Acknowledgement

The authors wish to thank Prof.Dr. Thomas Schupp and Sebastian Makran for providing tools and assistance with sugar and acid quantification. We extend our gratitude to Dr. Vadim Okum for providing technical support on the Capel-105 M system, for insights into its fine-tuning, and for input that improved the manuscript's technical details on capillary electrophoresis.

bmhead{Declarations}Nothing to declare

Funding

Not applicable

Competing interests

The authors declare no competing interests.

Ethics approval

This article does not contain any studies with humanparticipants or animals performed by any of the authors.

bmhead{Consent to participate}Not applicable.

bmhead{Data availability}All data generated or analyzed during this study areincluded in this published article and its supplementary informationfiles.

Consent to publish

All authors agreed on the publication.

Author Contribution

AE conceived, designed and conducted the experiments. MA and RU supervised the research. AE performed sample measurements and data analysis, data visualization. AE wrote the original manuscript. TJ, MA and RU reviewed and revised the manuscript. All authors read and approved the manuscript.

Yes

Abstract

Oxalic acid (OA) is a versatile reagent ubiquitously used in pharmaceutical, agricultural, and chemical industries. Aligning with the European Union's 2050 circular economy agenda, its potential application in ore processing, and particularly metal extraction from electronic waste (e-waste) has garnered significant attention. Gallium (Ga) is a technologically strategic metal at high supply risk, driven by high demand from the electronics industry and limited primary production. Oxalic acid (OA), known for superior dissolution and complexation, is a highly selective metal recovery reagent that has been reported to outperform conventional reagents in gallium (Ga) recovery. However, current petrochemical OA production opposes global sustainability goals. To address this, submerged fermentation using filamentous fungi, notably Aspergillus niger offers a promising OA production route, that goes hand-in-hand with selective metal recovery. This study investigates OA generation through submerged fermentation, beginning with shake-flask experiments that identified ATCC1015 as the suitable candidate, achieving 71 ± 27.73 mM OA in 5 days, which later was enhanced to 90.37 ± 5.8 mM through intermittent pH adjustment above 4. Glucose was reported as the optimal carbon source, yielding YP/S ≈ 0.4 (g/g). Scale-up in a 10L bioreactor using fed-batch fermentation with pulsed feeding and pH control achieved 260.1 ± 4.8 mM OA over 14 days. Co-production of gluconic acid and acidogenesis inhibition posed a challenge in attaining higher product yields. Collectively, these findings establish fed-batch fermentation with robust pH control as a viable strategy for sustainable OA production, enabling targeted Ga recovery from waste streams.