The freshly developed MICROBI carriers, significantly smaller than conventional media used in Moving Bed Biofilm Reactor (MBBR) wastewater treatment, present both opportunities and challenges-particularly in biomass measurement and calculation. Drawing on data from prior studies, spanning four months and two seasons of containerized technology trials, alongside three months of laboratory experiments, we systematically analyzed the colonized, TTCstained media. Using light microscopy, we measured media diameter and surface area, examining approximately 8,500 microscopic images to calculate biofilm thickness, media size, and surface area. By integrating these findings with literaturederived estimations of biofilm density across developmental stages, we evaluated the differences in biomass dynamics between cleaning organism types and seasonal variations within containerized systems. Our results revealed a steep growth rate for heterotrophs and notable, albeit slower, growth for autotrophs. Remarkably, both organism types yielded biomass levels significantly exceeding those of conventional wastewater cleaning methods. However, advancements in precisely measuring biofilm density and improving media porosity measurements remain essential for future research.

Traditional carriers play a major role in wastewater treatment worldwide due to their reliability, ease of production, well-established analytical methods, and strong treatment performance. Recent studies indicate that polyvinyl-alcohol-based microcarriers may surpass conventional media, as their smaller size, higher porosity, and increased specific surface area enable them to retain substantially more biomass within reactors. However, their practical application remains limited because fewer analytical methods and studies exist for these materials, largely due to their small dimensions and heat sensitivity, and their behaviour under industrial conditions—including their kinetics—has yet to be fully characterised and validated. This study aims to address these gaps by reviewing existing biomass measurement standards and highlighting their limitations when applied to microcarriers and by proposing alternative experimental approaches better suited for evaluating biomass on such sensitive yet high-capacity carriers. We present a set of experimental methods (still subject to further refinement) that demonstrate reliable performance with these materials, and to validate our approach, we quantified biomass in both in vitro systems and containerised-scale technologies, reaching up to 14 kg/m³ during winter and 8.7 kg/m³ in spring. Laboratory-scale experiments showed that both heterotrophic and autotrophic cultures can achieve high biomass levels of up to 21 kg/m³ and 16 kg/m³, respectively. Heterotrophs exhibited lower growth inhibition under shear stress, while autotrophs displayed a distinct shear-force niche around 0.09 µN within the reactor.