**Background:** Ciliated protozoa commonly inhabit the gastrointestinal tracts of herbivorous animals, including the hindgut of millipedes. Nyctotherus species are considered commensal ciliates found in cockroaches, millipedes, reptiles, amphibians, and fish. While previous research focused on the energetic metabolism of these ciliates in hydrogenosomes and their relationships with methanogenic archaea, little was known about their ability to ferment different polysaccharide substrates and their role in hindgut fermentation of millipedes. Millipedes are litter transformers that feed on decomposing plant material and play an important role in nutrient cycling. This study aimed to investigate the growth requirements, fermentation patterns, and hydrolytic enzymatic activities of Nyctotherus ciliates obtained from the hindgut of the tropical African millipede Archispirostreptus gigas and cultivated in vitro.
**Methods:** Ciliates were isolated from the hindgut contents of three A. gigas individuals and cultivated anaerobically in a mineral medium at 30°C. The culture was fed daily with rice starch covered with 13% β-sitosterol. Species identification was performed using single-cell PCR of 18S rDNA genes, with phylogenetic trees constructed using Bayesian Inference, Maximum Likelihood, and Maximum Parsimony. Growth experiments examined the effects of temperature (23, 30, 35, and 39°C), soluble nutrients, and different polysaccharide substrates (rice starch, xylan, carboxymethyl cellulose [CMC], crystalline cellulose [CC], and inulin) on ciliate and bacterial counts. Fermentation activity was measured using the in vitro gas technique, assessing dry matter digestibility (IVDMD), gas volume, methane concentration, ammonium nitrogen, and short-chain fatty acids (SCFA) after 96 hours. Hydrolytic enzymatic activities (α-amylase, CM-cellulase, xylanase, and inulinase) were measured in crude protein extracts of harvested ciliates using the dinitrosalicylic acid method.
**Key Results:** The best growth of N. velox was observed at 30°C in the complex medium with rice starch (1420 ± 81 trophozoites/mL, 560 ± 36 cysts/mL). Ciliates died within 24 hours at 39°C and could not grow under aerobic conditions for more than 20 days. Among polysaccharide substrates, rice starch stimulated the best growth, while ciliates could survive without insoluble polysaccharides for about 30 days. The highest IVDMD was observed for rice starch (92.46 g/kg DM substrate) followed by inulin (88.3), CMC (59.3), CC (50.6), and xylan (48.1). Rice starch and inulin supported the best gas production (174 and 150 mL/g DM substrate, respectively). Methane concentration was highest for xylan (17.9% vol/vol) compared to other substrates (CMC: 4.11%, CC: 8.77%, rice starch: 5.29%, inulin: 10.2%, NoPOS: 3.85%). The highest SCFA concentrations were observed after fermentation of rice starch (45.5 mmol/L) and inulin (40.6 mmol/L), with acetate (63–68 mol%), propionate (22–30 mol%), and n-butyrate (6–9 mol%) as dominant. Hydrogen recovery was highest for xylan (80%) and lowest for CMC (28%). Enzyme assays revealed the highest catalytic activity for xylanase (91,930 nkat/L), followed by inulinase (87,830 nkat/L), α-amylase (59,830 nkat/L), and CM-cellulase (14,900 nkat/L). Specific catalytic activities were highest for α-amylase (300 nkat/g protein), xylanase (290 nkat/g), CM-cellulase (190 nkat/g), and inulinase (170 nkat/g). Molecular analysis confirmed the presence of N. velox in the culture and identified a new species, Nyctotherus archispirostreptae n. sp., from the hindgut contents.
**Clinical Implications:** This study provides the first detailed physiological characterization of N. velox from millipedes, demonstrating its role as a facultative anaerobic ciliate involved in plant polysaccharide fermentation in the hindgut. The successful long-term in vitro cultivation (1.5 years) enables future research on the complex relationships within the millipede hindgut microbiome. The observed methanogenic fermentation patterns and hydrolytic enzymatic activities indicate that N. velox is well-adapted to the diverse feeding behavior of A. gigas, contributing to the degradation of storage polysaccharides (starch, inulin) and structural polysaccharides (xylan, cellulose). The findings suggest that hindgut ciliates may play a significant role in nutrient cycling and energy production for their millipede hosts through the production of SCFA, though further studies are needed to confirm the mutualistic or commensal nature of this relationship.